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Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition

  • Alan N. Engelman
    Correspondence
    To whom correspondence should be addressed: Dept. of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215. Tel.: 617-632-4381; Fax: 617-632-4338; E-mail: [email protected]
    Affiliations
    Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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Open AccessPublished:August 29, 2019DOI:https://doi.org/10.1074/jbc.REV119.006901
      Antiretroviral inhibitors that are used to manage HIV infection/AIDS predominantly target three enzymes required for virus replication: reverse transcriptase, protease, and integrase. Although integrase inhibitors were the last among this group to be approved for treating people living with HIV, they have since risen to the forefront of treatment options. Integrase strand transfer inhibitors (INSTIs) are now recommended components of frontline and drug-switch antiretroviral therapy formulations. Integrase catalyzes two successive magnesium-dependent polynucleotidyl transferase reactions, 3′ processing and strand transfer, and INSTIs tightly bind the divalent metal ions and viral DNA end after 3′ processing, displacing from the integrase active site the DNA 3′-hydroxyl group that is required for strand transfer activity. Although second-generation INSTIs present higher barriers to the development of viral drug resistance than first-generation compounds, the mechanisms underlying these superior barrier profiles are incompletely understood. A separate class of HIV-1 integrase inhibitors, the allosteric integrase inhibitors (ALLINIs), engage integrase distal from the enzyme active site, namely at the binding site for the cellular cofactor lens epithelium-derived growth factor (LEDGF)/p75 that helps to guide integration into host genes. ALLINIs inhibit HIV-1 replication by inducing integrase hypermultimerization, which precludes integrase binding to genomic RNA and perturbs the morphogenesis of new viral particles. Although not yet approved for human use, ALLINIs provide important probes that can be used to investigate the link between HIV-1 integrase and viral particle morphogenesis. Herein, I review the mechanisms of retroviral integration as well as the promises and challenges of using integrase inhibitors for HIV/AIDS management.

      Introduction

      Combination antiretroviral therapy (cART)
      The abbreviations used are: cART
      combination antiretroviral therapy
      ALLINI
      allosteric integrase inhibitor
      BIC
      bictegravir
      CAB
      cabotegravir
      CCD
      catalytic core domain
      CIC
      conserved intasome core
      cryo-EM
      cryogenic EM
      CTD
      C-terminal domain
      CSC
      cleaved synaptic complex
      DTG
      dolutegravir
      EVG
      elvitegravir
      FDA
      Food and Drug Administration
      IBD
      integrase-binding domain
      IN
      integrase
      INSTI
      integrase strand transfer inhibitor
      KPN
      karyopherin
      LA
      long-acting
      LEDGF
      lens epithelium-derived growth factor
      LRA
      latency reversal agent
      LTR
      long terminal repeat
      MMTV
      mouse mammary tumor virus
      MVV
      Maedi-visna virus
      NNRTI
      non-nucleoside reverse transcriptase inhibitor
      NRTI
      nucleoside reverse transcriptase inhibitor
      NTD
      N-terminal domain
      PDB
      Protein Data Bank
      PFV
      prototype foamy virus
      PI
      protease inhibitor
      PIC
      preintegration complex
      PLHIV
      people living with HIV
      PPT
      polypurine tract
      PR
      protease
      PrEP
      pre-exposure prophylaxis
      RAL
      raltegravir
      RANBP
      Ran-binding protein
      RNP
      ribonucleoprotein
      RT
      reverse transcriptase
      SHIV
      simian-human immunodeficiency virus
      SSC
      stable synaptic complex
      t-BSF
      tert-butylsulfonamide
      TCC
      target capture complex
      TNPO
      transportin
      SH3
      Src homology 3.
      treats patients with a mixture of drugs to inhibit different steps of the HIV-1 replication cycle (
      • Arts E.J.
      • Hazuda D.J.
      HIV-1 antiretroviral drug therapy.
      ) (Fig. 1). Unique among animal viruses is the requirement for retroviruses to integrate their genetic information into the genome of the host cell that they infect. Integration is mediated by the viral protein integrase (IN), which is incorporated into fledgling viral particles alongside the other viral enzymes reverse transcriptase (RT) and protease (PR). PR initiates virus particle maturation by cleaving viral Gag and Gag-Pol polyprotein precursors into separate viral structural proteins and enzymes, which is required to form the viral core (reviewed in Ref.
      • Sundquist W.I.
      • Kräusslich H.-G.
      HIV-1 assembly, budding, and maturation.
      ). The core consists of the viral ribonucleoprotein (RNP) complex, which contains two copies of the RNA genome bound by viral nucleocapsid, IN, and RT proteins, encased within a fullerene shell composed of the viral capsid protein (reviewed in Ref.
      • Yamashita M.
      • Engelman A.N.
      Capsid-dependent host factors in HIV-1 infection.
      ). RT converts retroviral RNA into a single molecule of linear DNA containing a copy of the viral long terminal repeat (LTR) at each end (Figure 1, Figure 2) (reviewed in Ref.
      • Engelman A.
      ). The linear DNA, comprised of U3 and U5 terminal sequences in respective upstream and downstream LTRs, is the substrate for IN-mediated viral DNA insertion into chromosomal DNA (
      • Brown P.O.
      • Bowerman B.
      • Varmus H.E.
      • Bishop J.M.
      Correct integration of retroviral DNA in vitro.
      ,
      • Fujiwara T.
      • Mizuuchi K.
      Retroviral DNA integration: structure of an integration intermediate.
      • Brown P.O.
      • Bowerman B.
      • Varmus H.E.
      • Bishop J.M.
      Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein.
      ).
      Figure thumbnail gr1
      Figure 1HIV replication cycle. After entry into a susceptible target cell, RT converts genomic RNA into linear DNA within the confines of the reverse transcription complex (RTC) (
      • Fassati A.
      • Goff S.P.
      Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1.
      ). Processing of the viral DNA ends by IN yields the PIC (
      • Miller M.D.
      • Farnet C.M.
      • Bushman F.D.
      Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition.
      ), which can integrate the endogenous DNA made by reverse transcription into recombinant target DNA in vitro (
      • Brown P.O.
      • Bowerman B.
      • Varmus H.E.
      • Bishop J.M.
      Correct integration of retroviral DNA in vitro.
      ). Following nuclear import and integration, the provirus (flanked by composite cyan/yellow/magenta LTRs) serves as a transcriptional template to produce viral mRNAs for translation of viral proteins as well as nascent viral genomes that co-assemble with viral proteins to form immature virions that bud out from the infected cell (
      • Sundquist W.I.
      • Kräusslich H.-G.
      HIV-1 assembly, budding, and maturation.
      ). Shown is a generalized scheme that depicts the major steps of HIV-1 replication, although it is important to note that deviations from this plan exist throughout Retroviridae. Most notably, spumavirus reverse transcription occurs during the second half of the infectious cycle (after integration), and spumaviral particles accordingly predominantly contain dsDNA (
      • Moebes A.
      • Enssle J.
      • Bieniasz P.D.
      • Heinkelein M.
      • Lindemann D.
      • Bock M.
      • McClure M.O.
      • Rethwilm A.
      Human foamy virus reverse transcription that occurs late in the viral replication cycle.
      ,
      • Yu S.F.
      • Baldwin D.N.
      • Gwynn S.R.
      • Yendapalli S.
      • Linial M.L.
      Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses.
      ). The primary steps in the HIV-1 replication cycle that are inhibited by the two major classes of IN inhibitors discussed herein are indicated.
      Figure thumbnail gr2
      Figure 2DNA cutting and joining steps of retroviral integration. The linear viral reverse transcript (lavender lines; plus-strands shaded more darkly than same-colored minus-strands throughout the cartoon) contains a copy of the LTR at each end composed of cyan U3, yellow R repeat, and magenta U5 sequences. The upstream LTR is abutted by the primer-binding site (PBS; purple box), whereas the downstream element is abutted by the polypurine tract (PPT; lavender box). During 3′ processing, IN hydrolyzes the DNA adjacent to invariant CA dinucleotides, which for HIV-1 liberates the pGTOH dinucleotide from each end. After nuclear localization, the intasome interacts with host target DNA (gray lines with targeted green sequence) to promote DNA strand transfer. The DNA gaps that persist after strand transfer are repaired by host cell machinery to yield a target site duplication (thin green lines) flanking the integrated provirus.
      Four classes of antiretroviral drugs, nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), PR inhibitors (PIs), and IN strand transfer inhibitors (INSTIs) have in recent years comprised frontline cART formulations (
      • Cihlar T.
      • Fordyce M.
      Current status and prospects of HIV treatment.
      ). Highlighting the success of the INSTI drug class, current guidelines recommend the use of a second-generation INSTI (dolutegravir (DTG) or bictegravir (BIC)) co-formulated with two NRTIs to treat most people living with HIV (PLHIV) who have not previously failed an INSTI-containing regimen (
      • Saag M.S.
      • Benson C.A.
      • Gandhi R.T.
      • Hoy J.F.
      • Landovitz R.J.
      • Mugavero M.J.
      • Sax P.E.
      • Smith D.M.
      • Thompson M.A.
      • Buchbinder S.P.
      • Del Rio C.
      • Eron Jr., J.J.
      • Fätkenheuer G.
      • Günthard H.F.
      • Molina J.-M.
      • Jacobsen D.M.
      • Volberding P.A.
      Antiretroviral drugs for treatment and prevention of HIV infection in adults: 2018 recommendations of the International Antiviral Society–U.S.A. panel.
      ,
      Panel on Antiretroviral Guidelines for Adults and Adolescents
      Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV.
      ). INSTIs inhibit IN strand transfer activity and thus specifically block the integration step within the HIV-1 life cycle (
      • Hazuda D.J.
      • Felock P.
      • Witmer M.
      • Wolfe A.
      • Stillmock K.
      • Grobler J.A.
      • Espeseth A.
      • Gabryelski L.
      • Schleif W.
      • Blau C.
      • Miller M.D.
      Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.
      ) (Fig. 1). A separate class of inhibitors, the allosteric IN inhibitors (ALLINIs), by contrast inhibit particle maturation (
      • Jurado K.A.
      • Wang H.
      • Slaughter A.
      • Feng L.
      • Kessl J.J.
      • Koh Y.
      • Wang W.
      • Ballandras-Colas A.
      • Patel P.A.
      • Fuchs J.R.
      • Kvaratskhelia M.
      • Engelman A.
      Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.
      ,
      • Fontana J.
      • Jurado K.A.
      • Cheng N.
      • Ly N.L.
      • Fuchs J.R.
      • Gorelick R.J.
      • Engelman A.N.
      • Steven A.C.
      Distribution and redistribution of HIV-1 nucleocapsid protein in immature, mature, and integrase-inhibited virions: a role for integrase in maturation.
      ) (see below). To fully understand the nature of these different types of inhibitors, it is important to appreciate the different steps of HIV-1 replication (Fig. 1) as well as the mechanistic and structural bases of retroviral DNA integration.

      Mechanism of retroviral integration

      IN is a polynucleotidyl transferase composed of three conserved protein domains: the N-terminal domain (NTD) with conserved His and Cys residues (HHCC motif) that coordinate Zn2+ binding for 3-helix bundle formation; the catalytic core domain (CCD), which adopts an RNase H fold and harbors the enzyme active site composed of invariant carboxylate residues (DDE motif); and the C-terminal domain (CTD), which adopts an SH3 fold (reviewed in Ref.
      • Lesbats P.
      • Engelman A.N.
      • Cherepanov P.
      Retroviral DNA integration.
      ). The role of the DDE residues in catalysis is to coordinate the positions of two divalent cations, which under physiological conditions are almost certainly magnesium, to deprotonate attacking oxygen nucleophiles and destabilize scissile phosphodiester bonds for one-step transesterification chemistry (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.
      ,
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ). Similar functionalities exist across a large superfamily of polynucleotidyl transferases that includes related enzymes such as transposase proteins and RNase H (reviewed in Ref.
      • Nowotny M.
      Retroviral integrase superfamily: the structural perspective.
      ).
      Two different IN activities, 3′ processing and strand transfer, are required for integration (Fig. 2). During 3′ processing, IN prepares the linear reverse transcript for integration by hydrolyzing the DNA ends 3′ of conserved CA dinucleotides, which most often liberates a dinucleotide from each end (
      • Roth M.J.
      • Schwartzberg P.L.
      • Goff S.P.
      Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence.
      ,
      • Katzman M.
      • Katz R.A.
      • Skalka A.M.
      • Leis J.
      The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.
      • Sherman P.A.
      • Fyfe J.A.
      Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.
      ). However, symmetrical DNA processing is not required for integration; the upstream terminus of spumaviral DNA is 5′-TG, obfuscating the need for U3 end processing by IN (
      • Juretzek T.
      • Holm T.
      • Gärtner K.
      • Kanzler S.
      • Lindemann D.
      • Herchenröder O.
      • Picard-Maureau M.
      • Rammling M.
      • Heinkelein M.
      • Rethwilm A.
      Foamy virus integration.
      ), whereas a trinucleotide is processed from the U5 end of some primate lentiviruses (
      • Randolph C.A.
      • Champoux J.J.
      The majority of simian immunodeficiency virus/Mne circle junctions result from ligation of unintegrated viral DNA ends that are aberrant for integration.
      ,
      • Du Z.
      • Ilyinskii P.O.
      • Lally K.
      • Desrosiers R.C.
      • Engelman A.
      A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.
      ), including HIV-2 (
      • Whitcomb J.M.
      • Hughes S.H.
      The sequence of human immunodeficiency virus type 2 circle junction suggests that integration protein cleaves the ends of linear DNA asymmetrically.
      ). During strand transfer, IN uses the CAOH-3′ hydroxyl groups to cut chromosomal DNA in a staggered fashion, which, due to the nature of SN2 chemistry, simultaneously joins the viral DNA ends to the 5′-phosphate groups of the dsDNA cut (
      • Fujiwara T.
      • Mizuuchi K.
      Retroviral DNA integration: structure of an integration intermediate.
      ,
      • Brown P.O.
      • Bowerman B.
      • Varmus H.E.
      • Bishop J.M.
      Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein.
      ,
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.
      ). The resulting gapped DNA intermediate with unjoined viral DNA 5′ ends is repaired by host cell machineries to yield the integrated provirus flanked by the sequence duplication of the host DNA cut, which for HIV-1 is most often 5 bp (
      • Vincent K.A.
      • York-Higgins D.
      • Quiroga M.
      • Brown P.O.
      Host sequences flanking the HIV provirus.
      ,
      • Vink C.
      • Groenink M.
      • Elgersma Y.
      • Fouchier R.A.
      • Tersmette M.
      • Plasterk R.H.
      Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA.
      ) (Fig. 2).

      Intasome structure and function

      Integration in cells is mediated by the preintegration complex (PIC), which is a large nucleoprotein complex derived from the core of the infecting virion (
      • Bowerman B.
      • Brown P.O.
      • Bishop J.M.
      • Varmus H.E.
      A nucleoprotein complex mediates the integration of retroviral DNA.
      ,
      • Farnet C.M.
      • Haseltine W.A.
      Integration of human immunodeficiency virus type 1 DNA in vitro.
      ). Within the confines of the PIC, IN functions as part of the intasome nucleoprotein complex, which is comprised of a multimer of IN and the viral DNA ends (
      • Murphy J.E.
      • Goff S.P.
      A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo.
      • Miller M.D.
      • Farnet C.M.
      • Bushman F.D.
      Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition.
      ,
      • Wei S.-Q.
      • Mizuuchi K.
      • Craigie R.
      A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration.
      ,
      • Chen H.
      • Wei S.-Q.
      • Engelman A.
      Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome.
      ,
      • McCord M.
      • Chiu R.
      • Vora A.C.
      • Grandgenett D.P.
      Retrovirus DNA termini bound by integrase communicate in trans for full-site integration in vitro.
      ,
      • Li M.
      • Mizuuchi M.
      • Burke Jr., T.R.
      • Craigie R.
      Retroviral DNA integration: reaction pathway and critical intermediates.
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ) (Figure 1, Figure 3). A series of X-ray crystallographic and single-particle cryogenic electron microscopy (cryo-EM) structures determined over the past decade has clarified that the number of IN molecules required to build the intasome differs depending on the type of retrovirus (reviewed in Ref.
      • Engelman A.N.
      • Cherepanov P.
      Retroviral intasomes arising.
      ). Seven retroviral genera are grouped into two subfamilies of Retroviridae: Spumavirinae, solely harboring the spumaviruses, and Orthoretrovirinae, which encompass the lentiviruses, such as HIV-1, as well as α-, β-, δ-, ϵ-, and γ-retroviruses. X-ray crystal structures of the spumavirus prototype foamy virus (PFV) intasome provided initial high-resolution views of the functional IN-viral DNA architecture as well as critical insight into the mechanism of INSTI action (see below) (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ,
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration through X-ray structures of its key intermediates.
      ,
      • Hare S.
      • Vos A.M.
      • Clayton R.F.
      • Thuring J.W.
      • Cummings M.D.
      • Cherepanov P.
      Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.
      ).
      Figure thumbnail gr3
      Figure 3Retroviral intasome structures. A–C, representative intasomes from the spumavirus PFV (A; protein database (PDB) accession code 3OY9), β-retrovirus MMTV (B; PDB code 3JCA), and lentivirus MVV (C; PDB code 5M0Q) are color-coded to highlight the CIC. Green and blue, catalytically active IN protomers; cyan, supporting IN CCDs; black, DNA strands. Whereas four PFV IN molecules suffice to form the CIC, both MMTV and MVV require six IN protomers. For MMTV, critical CTDs (magenta) are donated by flanking IN dimers, leading to an overall IN octamer. In MVV, flanking IN tetramers provide the critical CTDs, resulting in an overall IN hexadecamer. Gray coloring in B and C deemphasizes IN elements that do not compose the CIC. D–F, resected CCD and CTD domains from above green IN protomers, oriented to highlight the different CCD-CTD linker regions (dark gray). Associated magenta CTDs from separate IN protomers in E and F assume similar positions as the green CTD in D. Red sticks, DDE catalytic triad residues.
      The PFV intasome is composed of an IN tetramer with the following division of labor. Two extended, intertwined IN molecules (Fig. 3A, blue and green) harbor operational active sites and thus catalyze 3′ processing and strand transfer activities, whereas the other two IN molecules (Fig. 3A, cyan) with nonoperational active sites serve as bookends to truss the DNA-bound IN protomers together (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ). The interwoven nature of the two catalytically active IN molecules, with their NTDs mutually swapped between CCDs, was observed previously in crystal structures of two-domain lentiviral IN NTD-CCD constructs in the absence of DNA (
      • Wang J.-Y.
      • Ling H.
      • Yang W.
      • Craigie R.
      Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein.
      ,
      • Hare S.
      • Di Nunzio F.
      • Labeja A.
      • Wang J.
      • Engelman A.
      • Cherepanov P.
      Structural basis for functional tetramerization of lentiviral integrase.
      ). Prior to these structures, the NTD from one IN protomer had been shown to function in trans with the active site of a separate IN molecule within the active HIV IN multimer (
      • van Gent D.C.
      • Vink C.
      • Groeneger A.A.M.O.
      • Plasterk R.H.A.
      Complementation between HIV integrase proteins mutated in different domains.
      ,
      • Engelman A.
      • Bushman F.D.
      • Craigie R.
      Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.
      ). The interwoven NTD-CCD arrangement at the heart of the machine leverages the participation of both viral DNA ends in intasome assembly and DNA recombination.
      Four types of intasomes describe the ground states and product complexes associated with IN 3′ processing and strand transfer activities. IN processes the viral DNA ends in the context of the initial stable synaptic complex (SSC), yielding the cleaved synaptic complex (CSC) after viral DNA hydrolysis. The target capture complex (TCC) describes the CSC bound to target or host DNA, whereas strand transfer yields the strand transfer complex (
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ,
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ,
      • Engelman A.N.
      • Cherepanov P.
      Retroviral intasomes arising.
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration through X-ray structures of its key intermediates.
      ,
      • Yin Z.
      • Lapkouski M.
      • Yang W.
      • Craigie R.
      Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration.
      ). The overall conformation of the PFV intasome structure changes little as the complex morphs from the SSC to the strand transfer complex and promotes IN 3′ processing and strand transfer activities (
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ,
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ,
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration through X-ray structures of its key intermediates.
      ,
      • Yin Z.
      • Lapkouski M.
      • Yang W.
      • Craigie R.
      Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration.
      ,
      • Maskell D.P.
      • Renault L.
      • Serrao E.
      • Lesbats P.
      • Matadeen R.
      • Hare S.
      • Lindemann D.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      Structural basis for retroviral integration into nucleosomes.
      ). Although integration occurs largely throughout animal cell genomes (
      • Engelman A.
      Most of the avian genome appears available for retroviral DNA integration.
      ,
      • Carteau S.
      • Hoffmann C.
      • Bushman F.
      Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.
      ), host DNA sequences that contort to fit the target DNA-binding interface within the CSC are preferred targets (
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration through X-ray structures of its key intermediates.
      ,
      • Maskell D.P.
      • Renault L.
      • Serrao E.
      • Lesbats P.
      • Matadeen R.
      • Hare S.
      • Lindemann D.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      Structural basis for retroviral integration into nucleosomes.
      ,
      • Pryciak P.M.
      • Varmus H.E.
      Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection.
      ,
      • Serrao E.
      • Krishnan L.
      • Shun M.-C.
      • Li X.
      • Cherepanov P.
      • Engelman A.
      • Maertens G.N.
      Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding.
      ,
      • Pasi M.
      • Mornico D.
      • Volant S.
      • Juchet A.
      • Batisse J.
      • Bouchier C.
      • Parissi V.
      • Ruff M.
      • Lavery R.
      • Lavigne M.
      DNA minicircles clarify the specific role of DNA structure on retroviral integration.
      ) (for a detailed review, see Ref.
      • Engelman A.N.
      • Singh P.K.
      Cellular and molecular mechanisms of HIV-1 integration targeting.
      ). Thus, strand transfer proceeds without gross rearrangements in intasome architecture.
      Studies of additional retroviral intasomes unveiled a common structural feature at the hearts of the machines that was coined the conserved intasome core (CIC) (
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ) (Fig. 3, A–C). However, as mentioned, different viruses utilize different numbers of IN protomers to form the CIC. The tetrameric IN architecture of the PFV intasome defines the basic features of the CIC, including two active protomers with CCDs and NTDs swapped across a synaptic interface where two CTDs engage target DNA for integration (
      • Maertens G.N.
      • Hare S.
      • Cherepanov P.
      The mechanism of retroviral integration through X-ray structures of its key intermediates.
      ) and two additional IN molecules that bookend the active subunits (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ). Whereas four PFV IN molecules suffice to build the CIC, α- and β-retroviral intasomes require eight IN molecules (
      • Ballandras-Colas A.
      • Brown M.
      • Cook N.J.
      • Dewdney T.G.
      • Demeler B.
      • Cherepanov P.
      • Lyumkis D.
      • Engelman A.N.
      Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.
      ,
      • Yin Z.
      • Shi K.
      • Banerjee S.
      • Pandey K.K.
      • Bera S.
      • Grandgenett D.P.
      • Aihara H.
      Crystal structure of the Rous sarcoma virus intasome.
      ), and the lentiviruses HIV-1 and Maedi-visna virus (MVV) require 12 and 16 IN protomers, respectively (
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ,
      • Passos D.O.
      • Li M.
      • Yang R.
      • Rebensburg S.V.
      • Ghirlando R.
      • Jeon Y.
      • Shkriabai N.
      • Kvaratskhelia M.
      • Craigie R.
      • Lyumkis D.
      Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.
      ). The requirement for the different numbers of IN molecules stems from evolutionary constraints on the amino acid composition of the linker that connects the CCD and CTD (
      • Ballandras-Colas A.
      • Brown M.
      • Cook N.J.
      • Dewdney T.G.
      • Demeler B.
      • Cherepanov P.
      • Lyumkis D.
      • Engelman A.N.
      Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.
      ). The linker in PFV IN, composed of ∼50 residues, is sufficiently long to allow the CTDs of the active IN protomers to assume the positions required for host DNA binding (Fig. 3D). The analogous linker in α- and β-retroviruses, at only ∼8 residues, precludes the necessary CTD positioning (Fig. 3E). These viruses accordingly use two flanking IN dimers to position the critical CTDs, resulting in overall IN octamers. The lentiviral IN CCD-CTD linker, although composed of ∼15–20 residues, adopts α-helical conformation (
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ,
      • Chen J. C.-H.
      • Krucinski J.
      • Miercke L.J.W.
      • Finer-Moore J.S.
      • Tang A.H.
      • Leavitt A.D.
      • Stroud R.M.
      Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding.
      ) that likewise imposes a distance constraint to preclude proper IN CTD positioning from the catalytically active IN molecules (Fig. 3F). In the MVV intasome, IN tetramers donate the required CTDs, resulting in an overall IN hexadecamer (
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ). The HIV-1 intasome structure employed a fusion composed of heterologous Sso7d protein appended onto the IN N terminus, which significantly improved IN solubility and enzyme activity (
      • Passos D.O.
      • Li M.
      • Yang R.
      • Rebensburg S.V.
      • Ghirlando R.
      • Jeon Y.
      • Shkriabai N.
      • Kvaratskhelia M.
      • Craigie R.
      • Lyumkis D.
      Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.
      ,
      • Li M.
      • Jurado K.A.
      • Lin S.
      • Engelman A.
      • Craigie R.
      Engineered hyperactive integrase for concerted HIV-1 DNA integration.
      ). Flanking Sso7d-IN dimers were seen to donate the required CTDs to complete the CIC structure, resulting in an overall IN dodecamer. On the one hand, it seems likely that some flexibility is tolerated in terms of the multimeric character of the flanking IN oligomer that donates the CTD to the CIC structure, minimally requiring an IN dimer. On the other hand, it is possible that the use of the heterologous protein domain precluded high occupancy of flanking IN tetramers in the Sso7d-IN intasome structure. Additional structures derived from WT HIV-1 or related primate lentiviral IN proteins may further inform the IN-to-viral DNA stoichiometry necessarily for HIV-1 IN function.
      A key unanswered question in retroviral integration research is the mechanism of intasome assembly. DNA-based tetramerization of the predominant IN species in solution has been proposed (
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ) based on observations that PFV IN in the absence of DNA is monomeric (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ), α- and β-retroviral INs are predominantly dimeric (
      • Ballandras-Colas A.
      • Brown M.
      • Cook N.J.
      • Dewdney T.G.
      • Demeler B.
      • Cherepanov P.
      • Lyumkis D.
      • Engelman A.N.
      Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.
      ,
      • Yin Z.
      • Shi K.
      • Banerjee S.
      • Pandey K.K.
      • Bera S.
      • Grandgenett D.P.
      • Aihara H.
      Crystal structure of the Rous sarcoma virus intasome.
      ), and lentiviral INs are predominantly tetrameric although with evidence for additional lower- and higher-order forms (
      • Hare S.
      • Di Nunzio F.
      • Labeja A.
      • Wang J.
      • Engelman A.
      • Cherepanov P.
      Structural basis for functional tetramerization of lentiviral integrase.
      ,
      • Engelman A.
      • Bushman F.D.
      • Craigie R.
      Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.
      ,
      • Ballandras-Colas A.
      • Maskell D.P.
      • Serrao E.
      • Locke J.
      • Swuec P.
      • Jónsson S.R.
      • Kotecha A.
      • Cook N.J.
      • Pye V.E.
      • Taylor I.A.
      • Andrésdóttir V.
      • Engelman A.N.
      • Costa A.
      • Cherepanov P.
      A supramolecular assembly mediates lentiviral DNA integration.
      ,
      • van Gent D.C.
      • Elgersma Y.
      • Bolk M.W.J.
      • Vink C.
      • Plasterk R.H.A.
      DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2.
      • Lee S.P.
      • Xiao J.
      • Knutson J.R.
      • Lewis M.S.
      • Han M.K.
      Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro.
      ,
      • Cherepanov P.
      • Maertens G.
      • Proost P.
      • Devreese B.
      • Van Beeumen J.
      • Engelborghs Y.
      • De Clercq E.
      • Debyser Z.
      HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells.
      ,
      • Faure A.
      • Calmels C.
      • Desjobert C.
      • Castroviejo M.
      • Caumont-Sarcos A.
      • Tarrago-Litvak L.
      • Litvak S.
      • Parissi V.
      HIV-1 integrase crosslinked oligomers are active in vitro.
      ,
      • McKee C.J.
      • Kessl J.J.
      • Shkriabai N.
      • Dar M.J.
      • Engelman A.
      • Kvaratskhelia M.
      Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein.
      • Pandey K.K.
      • Bera S.
      • Grandgenett D.P.
      The HIV-1 integrase monomer induces a specific interaction with LTR DNA for concerted integration.
      ). However, the relationship between protein behavior in solution and the multimeric state of IN in virions or during reverse transcription is largely unknown. Because HIV-1 IN binds genomic RNA in virions (
      • Kessl J.J.
      • Kutluay S.B.
      • Townsend D.
      • Rebensburg S.
      • Slaughter A.
      • Larue R.C.
      • Shkriabai N.
      • Bakouche N.
      • Fuchs J.R.
      • Bieniasz P.D.
      • Kvaratskhelia M.
      HIV-1 integrase binds the viral RNA genome and is essential during virion morphogenesis.
      ), it seems possible that RNA-bound IN may transfer to the DNA ends as they form during reverse transcription to initiate SSC formation. The IN tail region, which is composed of the amino acids C-terminal from the CTD SH3 fold, varies in length from about 5 residues in the lentivirus equine infectious anemia virus to 55 residues in MMTV. The tail region in α-retroviral IN, which is 19 residues, can regulate DNA-dependent IN octamer formation (
      • Pandey K.K.
      • Bera S.
      • Shi K.
      • Aihara H.
      • Grandgenett D.P.
      A C-terminal “tail” region in the Rous sarcoma virus integrase provides high plasticity of functional integrase oligomerization during intasome assembly.
      ,
      • Bera S.
      • Pandey K.K.
      • Aihara H.
      • Grandgenett D.P.
      Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase.
      ). Although implicating a role for this region of IN in intasome assembly, tail regions are unresolved in all IN and intasome structures solved to date, limiting the interpretation of how the tail might regulate nucleoprotein complex formation.

      INSTIs

      Research in the mid-1980s first established a role for the 3′ region of the pol gene, which encodes for IN, in retroviral replication (
      • Donehower L.A.
      • Varmus H.E.
      A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration.
      • Panganiban A.T.
      • Temin H.M.
      The retrovirus pol gene encodes a product required for DNA integration: Identification of a retrovirus int locus.
      ,
      • Schwartzberg P.
      • Colicelli J.
      • Goff S.P.
      Construction and analysis of deletion mutations in the pol gene of moloney murine leukemia virus: a new viral function required for productive infection.
      • Quinn T.P.
      • Grandgenett D.P.
      Genetic evidence that the avian retrovirus DNA endonuclease domain of pol is necessary for viral integration.
      ), and the extension of this requirement to HIV-1 highlighted IN as a high-value antiviral target (
      • LaFemina R.L.
      • Schneider C.L.
      • Robbins H.L.
      • Callahan P.L.
      • LeGrow K.
      • Roth E.
      • Schleif W.A.
      • Emini E.A.
      Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.
      ). However, a scant number of promising preclinical lead compounds were known by the time RT and PR inhibitors were administered to patients in cART formulations (
      • Collier A.C.
      • Coombs R.W.
      • Schoenfeld D.A.
      • Bassett R.L.
      • Timpone J.
      • Baruch A.
      • Jones M.
      • Facey K.
      • Whitacre C.
      • McAuliffe V.J.
      • Friedman H.M.
      • Merigan T.C.
      • Reichman R.C.
      • Hooper C.
      • Corey L.
      Treatment of human immunodeficiency virus infection with saquinavir, zidovudine, and zalcitabine.
      ,
      • D'Aquila R.T.
      • Hughes M.D.
      • Johnson V.A.
      • Fischl M.A.
      • Sommadossi J.-P.
      • Liou S.-H.
      • Timpone J.
      • Myers M.
      • Basgoz N.
      • Niu M.
      • Hirsch M.S.
      Nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine in patients with HIV-1 infection: a randomized, double-blind, placebo-controlled trial.
      • Staszewski S.
      • Miller V.
      • Rehmet S.
      • Stark T.
      • De Crée J.
      • De Brabander M.
      • Peeters M.
      • Andries K.
      • Moeremans M.
      • De Raeymaeker M.
      • Pearce G.
      • Van den Broeck R.
      • Verbiest W.
      • Stoffels P.
      Virological and immunological analysis of a triple combination pilot study with loviride, lamivudine and zidovudine in HIV-1-infected patients.
      ), calling into question whether IN inhibitors would ever make it to the clinic. Indeed, around this time, I can recall one of the more prominent researchers in our field espousing the view at a national meeting that clinical IN inhibitors were unattainable. The reasoning here was based on the observation that an equal number of IN, RT, and PR molecules are packaged into each virion particle, which one can estimate as 120 based on the 20:1 synthesis ratio of Gag to Gag-Pol (
      • Jacks T.
      • Power M.D.
      • Masiarz F.R.
      • Luciw P.A.
      • Barr P.J.
      • Varmus H.E.
      Characterization of ribosomal frameshifting in HIV-1 gag-pol expression.
      ) and circa 2,400 Gag molecules per virion (
      • Carlson L.-A.
      • Briggs J.A.G.
      • Glass B.
      • Riches J.D.
      • Simon M.N.
      • Johnson M.C.
      • Müller B.
      • Grünewald K.
      • Kräusslich H.-G.
      Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis.
      ). Per replication cycle, RT and PR catalyze roughly 19,400 and 12,900 chemical reactions, respectively. However, the same population of IN molecules performs only four chemical reactions. How then could one effectively inhibit IN in the face of this seemingly large excess of available enzyme? Fortuitously, my colleague turned out to be incorrect. What was unknown at the time of our discussion was the utility of molecules designed to inhibit IN strand transfer activity. Whereas HIV-1 IN processes the viral DNA ends to yield the CSC concomitant with or soon after reverse transcription (
      • Miller M.D.
      • Farnet C.M.
      • Bushman F.D.
      Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition.
      ,
      • Munir S.
      • Thierry S.
      • Subra F.
      • Deprez E.
      • Delelis O.
      Quantitative analysis of the time-course of viral DNA forms during the HIV-1 life cycle.
      ) (Fig. 1), integration into chromosomal DNA does not occur until hours to days (
      • Munir S.
      • Thierry S.
      • Subra F.
      • Deprez E.
      • Delelis O.
      Quantitative analysis of the time-course of viral DNA forms during the HIV-1 life cycle.
      • Butler S.L.
      • Hansen M.S.
      • Bushman F.D.
      A quantitative assay for HIV DNA integration in vivo.
      ,
      • Pierson T.C.
      • Zhou Y.
      • Kieffer T.L.
      • Ruff C.T.
      • Buck C.
      • Siliciano R.F.
      Molecular characterization of preintegration latency in human immunodeficiency virus type 1 infection.
      • Mohammadi P.
      • Desfarges S.
      • Bartha I.
      • Joos B.
      • Zangger N.
      • Muñoz M.
      • Günthard H.F.
      • Beerenwinkel N.
      • Telenti A.
      • Ciuffi A.
      24 hours in the life of HIV-1 in a T cell line.
      ) or, in some extreme cases, weeks later (
      • Cardozo E.F.
      • Andrade A.
      • Mellors J.W.
      • Kuritzkes D.R.
      • Perelson A.S.
      • Ribeiro R.M.
      Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration.
      ). The comparatively long-lived CSC intasome replication intermediate is a pharmacological HIV-1 Achilles' heel that is leveraged fully by the INSTI class of antiretroviral compounds.
      Because HIV-1 IN purified from recombinant sources displayed 3′ processing and strand transfer activities in vitro (
      • Engelman A.
      • Mizuuchi K.
      • Craigie R.
      HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.
      ,
      • Sherman P.A.
      • Fyfe J.A.
      Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.
      ,
      • Bushman F.D.
      • Fujiwara T.
      • Craigie R.
      Retroviral DNA integration directed by HIV integration protein in vitro.
      ,
      • Bushman F.D.
      • Craigie R.
      Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA.
      ), systems to search for inhibitory molecules of HIV-1 IN activity were readily scalable (
      • Craigie R.
      • Mizuuchi K.
      • Bushman F.D.
      • Engelman A.
      A rapid in vitro assay for HIV DNA integration.
      ,
      • Hazuda D.J.
      • Hastings J.C.
      • Wolfe A.L.
      • Emini E.A.
      A novel assay for the DNA strand-transfer reaction of HIV-1 integrase.
      • Vink C.
      • Banks M.
      • Bethell R.
      • Plasterk R.H.A.
      A high-throughput, non-radioactive microtiter plate assay for activity of the human immunodeficiency virus integrase protein.
      ). However, due to suboptimal assay designs, few early leads turned out to specifically inhibit HIV-1 integration under physiological conditions (
      • Farnet C.M.
      • Wang B.
      • Lipford J.R.
      • Bushman F.D.
      Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.
      ,
      • Hazuda D.J.
      • Felock P.J.
      • Hastings J.C.
      • Pramanik B.
      • Wolfe A.L.
      Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase.
      ). Consider the following example. A compound such as ethidium bromide that would likely score as a hit if test compounds were comixed together with IN and viral DNA is highly unlikely to specifically inhibit integration in infected cells. Numerous early compounds accordingly lacked specificity to inhibit integration during HIV-1 infection (reviewed in Ref.
      • Pommier Y.
      • Johnson A.A.
      • Marchand C.
      Integrase inhibitors to treat HIV/Aids.
      ). A key turning point in IN inhibitor development came from reformulating the design of the in vitro assay to prebind IN to a synthetically preprocessed viral DNA end substrate (
      • Hazuda D.J.
      • Felock P.J.
      • Hastings J.C.
      • Pramanik B.
      • Wolfe A.L.
      Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase.
      ) and screen for inhibitors of strand transfer activity, which led to the discovery of first-in-class INSTIs (
      • Hazuda D.J.
      • Felock P.
      • Witmer M.
      • Wolfe A.
      • Stillmock K.
      • Grobler J.A.
      • Espeseth A.
      • Gabryelski L.
      • Schleif W.
      • Blau C.
      • Miller M.D.
      Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells.
      ). Although these diketo acid compounds were never licensed to treat PLHIV, they nevertheless served as important molecules with which to probe INSTI mechanisms of action. INSTIs harbor two commonalities across otherwise diverse pharmacophores (Figs. 4A and 5). At the hearts of the compounds are three adjacent heteroatoms (most usually oxygen; red in Fig. 5, B and C), whereas a terminal halogenated benzene ring connects to the rest of the molecule via a flexible linker (Fig. 4A; blue in Fig. 5, B and C). The compounds avidly bound IN-viral DNA complexes yet failed to appreciably bind HIV-1 IN in the absence of viral DNA (
      • Espeseth A.S.
      • Felock P.
      • Wolfe A.
      • Witmer M.
      • Grobler J.
      • Anthony N.
      • Egbertson M.
      • Melamed J.Y.
      • Young S.
      • Hamill T.
      • Cole J.L.
      • Hazuda D.J.
      HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.
      ), and subsequent work revealed the importance of the terminal deoxyadenylate residue at the 3′ end of processed viral DNA in the regulation of INSTI binding and dissociation (
      • Dicker I.B.
      • Samanta H.K.
      • Li Z.
      • Hong Y.
      • Tian Y.
      • Banville J.
      • Remillard R.R.
      • Walker M.A.
      • Langley D.R.
      • Krystal M.
      Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors.
      ,
      • Langley D.R.
      • Samanta H.K.
      • Lin Z.
      • Walker M.A.
      • Krystal M.R.
      • Dicker I.B.
      The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors.
      ). The conserved INSTI heteroatoms engage the divalent metal ions that are bound by the DDE active-site residues (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ,
      • Grobler J.A.
      • Stillmock K.
      • Hu B.
      • Witmer M.
      • Felock P.
      • Espeseth A.S.
      • Wolfe A.
      • Egbertson M.
      • Bourgeois M.
      • Melamed J.
      • Wai J.S.
      • Young S.
      • Vacca J.
      • Hazuda D.J.
      Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.
      ).
      Figure thumbnail gr4
      Figure 4INSTI structures and ALLINI chemotypes. A, diagrams of the four FDA-licensed INSTIs as well as investigational second-generation compound 6p (
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Métifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke T.R.
      Structure-guided optimization of HIV integrase strand transfer inhibitors.
      ,
      • Smith S.J.
      • Zhao X.Z.
      • Burke Jr., T.R.
      • Hughes S.H.
      HIV-1 integrase inhibitors that are broadly effective against drug-resistant mutants.
      ). B, representative ALLINI chemotypes. Asterisks mark common positions of t-butoxyacid moieties.
      Figure thumbnail gr5
      Figure 5Mechanisms of INSTI action. A, close-up view of one PFV IN active site in the CSC intasome structure (PDB code 3OY9) with IN secondary elements labeled. Additional labels highlight the conserved CA dinucleotide of the transferred DNA strand (magenta sticks) and associated G nucleotide of the nontransferred strand (orange), the 3′-OH nucleophile of the terminal deoxyadenylate used by IN to cut chromosomal DNA, as well as IN residues (in sticks) that compose the catalytic triad (Asp-128, Asp-185, and Glu-221) and that when changed confer INSTI resistance (Tyr-212 and Asn-224). Blue and red stick colors denote nitrogen and oxygen atoms, respectively. Gray spheres, divalent metal ions; α, β, and η denote α-helix, β-strand, and 310-helix, respectively. B, RAL (cyan sticks)-bound PFV intasome structure (PDB code 3OYA) oriented as in A to highlight the mode of INSTI binding. RAL binding results in a greater than 6-Å displacement of the 3′-OH of the terminal deoxyadenylate from the IN active site. The position of the RAL methyl-oxadiazole group is highlighted in the cartoon on the left as well as the chemical diagram on the right, which was reconfigured from A to accentuate the position within the crystal structure. Other colors and labeling are the same as in A or as described under “INSTIs.” C, same as in B, except with DTG bound (PDB code 3S3M). The position of the IN β4-α2 connector is noted; other labeling is the same as in A and B. D, RAL (magenta) from the drug-bound PFV CSC structure (PDB code 3OYA) is overlaid with the PFV SSC structure (green; PDB code 4E7I) to highlight mimicry between drug oxygen atoms and oxygen atoms critical for IN 3′ processing activity (red sphere, nucleophilic water (W) molecule; red bridge in viral DNA (vDNA), scissile phosphodiester bond). E, similar to D; RAL is superimposed onto the PFV TCC structure (IN and target DNA in cyan and vDNA in blue; PDB code 4E7K) to highlight similarly positioned drug oxygen atoms with the vDNA 3′-oxygen and scissile phosphodiester bond in target DNA (tDNA) critical for strand transfer activity. Other labeling is the same as in A and B.
      Raltegravir (RAL) in 2007 was the first INSTI licensed by the United States Food and Drug Administration (FDA) (
      • Summa V.
      • Petrocchi A.
      • Bonelli F.
      • Crescenzi B.
      • Donghi M.
      • Ferrara M.
      • Fiore F.
      • Gardelli C.
      • Gonzalez Paz O.
      • Hazuda D.J.
      • Jones P.
      • Kinzel O.
      • Laufer R.
      • Monteagudo E.
      • Muraglia E.
      • Nizi E.
      • Orvieto F.
      • Pace P.
      • Pescatore G.
      • Scarpelli R.
      • Stillmock K.
      • Witmer M.V.
      • Rowley M.
      Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection.
      ) and elvitegravir (EVG) in 2012 became the second licensed INSTI (
      • Sato M.
      • Motomura T.
      • Aramaki H.
      • Matsuda T.
      • Yamashita M.
      • Ito Y.
      • Kawakami H.
      • Matsuzaki Y.
      • Watanabe W.
      • Yamataka K.
      • Ikeda S.
      • Kodama E.
      • Matsuoka M.
      • Shinkai H.
      Novel HIV-1 integrase inhibitors derived from quinolone antibiotics.
      ). (Fig. 4A). Although prior work demonstrated the importance of divalent metal ion and viral DNA sequence for INSTI binding (
      • Dicker I.B.
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      • Li Z.
      • Hong Y.
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      • Banville J.
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      ,
      • Langley D.R.
      • Samanta H.K.
      • Lin Z.
      • Walker M.A.
      • Krystal M.R.
      • Dicker I.B.
      The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors.
      • Grobler J.A.
      • Stillmock K.
      • Hu B.
      • Witmer M.
      • Felock P.
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      • Wolfe A.
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      • Bourgeois M.
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      • Young S.
      • Vacca J.
      • Hazuda D.J.
      Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.
      ), the field lacked a detailed view of how INSTIs inhibited IN strand transfer activity. Fortuitously, both NRTIs and INSTIs, which target respective RT and IN active sites composed of invariant amino acid residues, inhibit a wide range of retroviruses (
      • Ruprecht R.M.
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      • Rossoni L.D.
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      • Tsai C.C.
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      ,
      • North T.W.
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      Feline immunodeficiency virus, a model for reverse transcriptase-targeted chemotherapy for acquired immune deficiency syndrome.
      ,
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      French ANRS HIV-2 Cohort (ANRS CO 05 VIH-2)
      HIV-2 integrase gene polymorphism and phenotypic susceptibility of HIV-2 clinical isolates to the integrase inhibitors raltegravir and elvitegravir in vitro.
      ,
      • Shimura K.
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      Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137).
      ,
      • Lewis M.G.
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      • Collins M.
      • Barreca M.L.
      • Iraci N.
      • Chirullo B.
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      Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy.
      ,
      • Paprotka T.
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      • Chaipan C.
      • Burdick R.
      • Delviks-Frankenberry K.A.
      • Hu W.-S.
      • Pathak V.K.
      Inhibition of xenotropic murine leukemia virus-related virus by APOBEC3 proteins and antiviral drugs.
      ,
      • Smith R.A.
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      Susceptibility of the human retrovirus XMRV to antiretroviral inhibitors.
      • Koh Y.
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      Differential sensitivities of retroviruses to integrase strand transfer inhibitors.
      ) including spumaviruses (
      • Moebes A.
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      • Heinkelein M.
      • Lindemann D.
      • Bock M.
      • McClure M.O.
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      Human foamy virus reverse transcription that occurs late in the viral replication cycle.
      ,
      • Valkov E.
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      • Hare S.
      • Helander A.
      • Roversi P.
      • McClure M.
      • Cherepanov P.
      Functional and structural characterization of the integrase from the prototype foamy virus.
      ). Thus, the PFV intasome could serve as a model system to investigate INSTI mechanism of action. Co-crystal structures with RAL or EVG revealed that the halobenzyl groups assumed the position of the purine rings of the 3′-deoxyadenylate residue, supplanting the terminal nucleoside from the IN active site (Fig. 5, A and B). INSTI binding accordingly inactivates the intasome complex by displacing from the enzyme active site the DNA 3′-OH group that is required to cut chromosomal DNA for strand transfer activity (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ).
      Second-generation INSTI compounds physically expand upon first generation scaffolds while maintaining both metal-chelating and DNA-supplanting drug functions. Such modifications include increasing the length of the linker between the metal-chelating and halobenzyl moieties (
      • Min S.
      • Song I.
      • Borland J.
      • Chen S.
      • Lou Y.
      • Fujiwara T.
      • Piscitelli S.C.
      Pharmacokinetics and safety of S/GSK1349572, a next-generation HIV integraseinhibitor, in healthy volunteers.
      ,
      • Tsiang M.
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      • Mulato A.
      • Hansen D.
      • Kan E.
      • Tsai L.
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      • Yant S.R.
      • Yu H.
      • Kukolj G.
      • Cihlar T.
      • et al.
      Antiviral activity of bictegravir (GS-9883), a novel potent HIV-1 integrase strand transfer inhibitor with an improved resistance profile.
      • Naidu B.N.
      • Walker M.A.
      • Sorenson M.E.
      • Ueda Y.
      • Matiskella J.D.
      • Connolly T.P.
      • Dicker I.B.
      • Lin Z.
      • Bollini S.
      • Terry B.J.
      • Higley H.
      • Zheng M.
      • Parker D.D.
      • Wu D.
      • Adams S.
      • Krystal M.R.
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      The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor.
      ), increasing the number of central ring moieties to three (
      • Min S.
      • Song I.
      • Borland J.
      • Chen S.
      • Lou Y.
      • Fujiwara T.
      • Piscitelli S.C.
      Pharmacokinetics and safety of S/GSK1349572, a next-generation HIV integraseinhibitor, in healthy volunteers.
      ,
      • Tsiang M.
      • Jones G.S.
      • Goldsmith J.
      • Mulato A.
      • Hansen D.
      • Kan E.
      • Tsai L.
      • Bam R.A.
      • Stepan G.
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      • Niedziela-Majka A.
      • Yant S.R.
      • Yu H.
      • Kukolj G.
      • Cihlar T.
      • et al.
      Antiviral activity of bictegravir (GS-9883), a novel potent HIV-1 integrase strand transfer inhibitor with an improved resistance profile.
      ,
      • Wiscount C.M.
      • Williams P.D.
      • Tran L.O.
      • Embrey M.W.
      • Fisher T.E.
      • Sherman V.
      • Homnick C.F.
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      • Lyle T.A.
      • Wai J.S.
      • Vacca J.P.
      • Wang Z.
      • Felock P.J.
      • Stillmock K.A.
      • et al.
      10-Hydroxy-7,8-dihydropyrazino[1′,2′:1,5]pyrrolo[2,3-d]pyridazine-1,9(2H,6H)-diones: potent, orally bioavailable HIV-1 integrase strand-transfer inhibitors with activity against integrase mutants.
      ), and, akin to EVG, derivatization of a second ring that lies distal from the halobenzyl group (
      • Egbertson M.S.
      • Wai J.S.
      • Cameron M.
      • Hoerrner R.S.
      • Raheem I.T.
      • Walji A.M.
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      Discovery of 2-pyridinone aminals: a prodrug strategy to advance a second generation of HIV-1 integrase strand transfer inhibitors.
      ,
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Metifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke Jr., T.R.
      HIV-1 integrase strand transfer inhibitors with reduced susceptibility to drug resistant mutant integrases.
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Métifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke T.R.
      Structure-guided optimization of HIV integrase strand transfer inhibitors.
      ) (Fig. 4A). Second-generation INSTIs more fully occupy the IN active-site region that spans from the DNA-binding pocket on the one side to the connector sequence that links IN secondary structural elements β4 and α2 on the other (
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Metifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke Jr., T.R.
      HIV-1 integrase strand transfer inhibitors with reduced susceptibility to drug resistant mutant integrases.
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Métifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke T.R.
      Structure-guided optimization of HIV integrase strand transfer inhibitors.
      ,
      • Hare S.
      • Smith S.J.
      • Métifiot M.
      • Jaxa-Chamiec A.
      • Pommier Y.
      • Hughes S.H.
      • Cherepanov P.
      Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572).
      • DeAnda F.
      • Hightower K.E.
      • Nolte R.T.
      • Hattori K.
      • Yoshinaga T.
      • Kawasuji T.
      • Underwood M.R.
      Dolutegravir interactions with HIV-1 integrase-DNA: structural rationale for drug resistance and dissociation kinetics.
      ) (Fig. 5C, β4-α2 connector).
      Overlaying the structures of INSTI-bound PFV intasomes to those of the SSC and TCC yielded important insight into the mechanism of drug action (
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ,
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Metifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke Jr., T.R.
      HIV-1 integrase strand transfer inhibitors with reduced susceptibility to drug resistant mutant integrases.
      ,
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Métifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke T.R.
      Structure-guided optimization of HIV integrase strand transfer inhibitors.
      ). The RAL metal-chelating oxygen atom distal from the halobenzyl group coincided with the nucleophilic water molecule for IN 3′ processing activity (red sphere in Fig. 5D), whereas the RAL-chelating oxygen proximal to the halobenzyl coincided with the scissile phosphodiester bond in viral DNA (Fig. 5D). For strand transfer, the halobenzyl-proximal oxygen coincided with the nucleophilic 3′-oxygen of processed viral DNA, whereas the distal RAL oxygen overlapped with the scissile phosphodiester bond in target DNA (
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ) (Fig. 5E). These observations first identified INSTIs as IN substrate mimics, which was subsequently expanded through the broader concept of substrate envelope. Previously espoused for HIV-1 PR and the mechanism of PI action, the substrate envelope is defined as the space occupied by the substrate (peptide in the case of PR; DNA for IN) in an enzyme active site. Because the enzyme must interact with the substrate for catalysis, drugs that interfere with enzyme–substrate interactions should be inhibitory and might impart relatively high resistance barriers (
      • King N.M.
      • Prabu-Jeyabalan M.
      • Nalivaika E.A.
      • Schiffer C.A.
      Combating susceptibility to drug resistance: lessons from HIV-1 protease.
      ,
      • Prabu-Jeyabalan M.
      • King N.M.
      • Nalivaika E.A.
      • Heilek-Snyder G.
      • Cammack N.
      • Schiffer C.A.
      Substrate envelope and drug resistance: crystal structure of RO1 in complex with wild-type human immunodeficiency virus type 1 protease.
      • Shen Y.
      • Altman M.D.
      • Ali A.
      • Nalam M.N.L.
      • Cao H.
      • Rana T.M.
      • Schiffer C.A.
      • Tidor B.
      Testing the substrate-envelope hypothesis with designed pairs of compounds.
      ). Indeed, second-generation INSTI elements distal from the halobenzyl groups coincide with the position of host DNA in PFV intasome structures (
      • Hare S.
      • Maertens G.N.
      • Cherepanov P.
      3′-Processing and strand transfer catalysed by retroviral integrase in crystallo.
      ,
      • Zhao X.Z.
      • Smith S.J.
      • Maskell D.P.
      • Metifiot M.
      • Pye V.E.
      • Fesen K.
      • Marchand C.
      • Pommier Y.
      • Cherepanov P.
      • Hughes S.H.
      • Burke Jr., T.R.
      HIV-1 integrase strand transfer inhibitors with reduced susceptibility to drug resistant mutant integrases.
      ,
      • Hare S.
      • Smith S.J.
      • Métifiot M.
      • Jaxa-Chamiec A.
      • Pommier Y.
      • Hughes S.H.
      • Cherepanov P.
      Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572).
      ) (Fig. 5E), likely accounting for the competition between target DNA and INSTIs for binding to HIV-1 IN-viral DNA complexes (
      • Espeseth A.S.
      • Felock P.
      • Wolfe A.
      • Witmer M.
      • Grobler J.
      • Anthony N.
      • Egbertson M.
      • Melamed J.Y.
      • Young S.
      • Hamill T.
      • Cole J.L.
      • Hazuda D.J.
      HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.
      ). Compound modifications that further interfere with HIV-1 IN–substrate interactions could improve INSTI potency and increase the barrier to acquire drug-resistant mutations (
      • Smith S.J.
      • Zhao X.Z.
      • Burke Jr., T.R.
      • Hughes S.H.
      HIV-1 integrase inhibitors that are broadly effective against drug-resistant mutants.
      ).
      Second-generation INSTIs are currently undergoing extensive safety evaluation due to their planned global rollout for HIV/AIDS treatment. Although DTG was initially deemed safe for pregnant women (
      • Zash R.
      • Jacobson D.L.
      • Diseko M.
      • Mayondi G.
      • Mmalane M.
      • Essex M.
      • Gaolethe T.
      • Petlo C.
      • Lockman S.
      • Holmes L.B.
      • Makhema J.
      • Shapiro R.L.
      Comparative safety of dolutegravir-based or efavirenz-based antiretroviral treatment started during pregnancy in Botswana: an observational study.
      ), follow-up work highlighted a greater frequency of neural tube defect in infants born to Botswanan mothers who were taking DTG-containing cART since the time of conception (4 of 426; 0.94%) versus frequencies observed in the general population (86 of 87,755; 0.1%) or in infants from mothers taking other cART regimens (14 of 11,300; 0.12%) (
      • Zash R.
      • Makhema J.
      • Shapiro R.L.
      Neural-tube defects with dolutegravir treatment from the time of conception.
      ). Such observations prompted several regulatory agencies including the FDA and the World Health Organization in 2018 to issue alerts regarding possible increased risk of neural tube defect in infants born to mothers taking DTG-containing cART at the time of conception (
      • Nakkazi E.
      Changes to dolutegravir policy in several African countries.
      ). Subsequent retrospective analyses have failed to detect a link between DTG usage and neural tube birth defect, although such studies were generally limited by sample size (
      • Chouchana L.
      • Beeker N.
      • Treluyer J.M.
      Is there a safety signal for dolutegravir and integrase inhibitors during pregnancy?.
      ,
      • Vannappagari V.
      • Thorne C.
      for APR EPPICC
      Pregnancy and neonatal outcomes following prenatal exposure to dolutegravir.
      ). Retrospective analysis has also failed to detect an increase in the frequency of neural tube defect in infants born to mothers on RAL-based drug regimens (
      • Shamsuddin H.
      • Raudenbush C.L.
      • Sciba B.L.
      • Zhou Y.P.
      • Mast T.C.
      • Greaves W.L.
      • Hanna G.J.
      • Leong R.
      • Straus W.
      Evaluation of neural tube defects (NTDs) after exposure to raltegravir during pregnancy.
      ). Recent follow-up work that increased the number of patients from 426 to 1,683 in the Botswanan cohort revised the neural tube defect frequency from 0.94% to 0.3%, which was still 0.2% greater than the frequencies observed in control populations (
      • Zash R.
      • Holmes L.
      • Diseko M.
      • Jacobson D.L.
      • Brummel S.
      • Mayondi G.
      • Isaacson A.
      • Davey S.
      • Mabuta J.
      • Mmalane M.
      • Gaolathe T.
      • Essex M.
      • Lockman S.
      • Makhema J.
      • Shapiro R.L.
      Neural-tube defects and antiretroviral treatment regimens in Botswana.
      ). It will be informative to ascertain whether BIC, which is the other licensed second-generation INSTI and is structurally related to DTG (Fig. 4A), also influences neural tube defect frequency versus control populations.

      Mechanisms of HIV resistance to INSTIs

      Substitutions of HIV-1 IN residues Gln-148, Asn-155, or Tyr-143 were recognized early on as separate genetic pathways to clinical RAL resistance (
      • Cooper D.A.
      • Steigbigel R.T.
      • Gatell J.M.
      • Rockstroh J.K.
      • Katlama C.
      • Yeni P.
      • Lazzarin A.
      • Clotet B.
      • Kumar P.N.
      • Eron J.E.
      • Schechter M.
      • Markowitz M.
      • Loutfy M.R.
      • Lennox J.L.
      • Zhao J.
      • et al.
      Subgroup and resistance analyses of raltegravir for resistant HIV-1 infection.
      ), and mutant viral strains harboring changes such as Q148H/G140S or N155H conveyed significant resistance to EVG as well (reviewed in Ref.
      • McColl D.J.
      • Chen X.
      Strand transfer inhibitors of HIV-1 integrase: bringing IN a new era of antiretroviral therapy.
      ). Although structure-based studies with PFV intasomes informed the mechanisms of drug resistance (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ,
      • Hare S.
      • Vos A.M.
      • Clayton R.F.
      • Thuring J.W.
      • Cummings M.D.
      • Cherepanov P.
      Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.
      ), partial amino acid identity between PFV and HIV-1 INs limits the extent of information that can be gleaned from the model system. PFV and HIV-1 IN are overall 18.4% identical, whereas their respective CCDs share 22.4% identity (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ). Fortuitously, two of the three clinically relevant amino acids, Tyr-143 and Asn-155, are conserved as Tyr-212 and Asn-224 in PFV IN (Fig. 5A). The binding mode of RAL to the PFV intasome in particular informed the Tyr-143 resistance pathway, as the methyl-oxadiazole constituent of this drug stacked against the p-cresol side chain of IN residue Tyr-212 (Fig. 5B). Changes that would reduce the aromatic nature of HIV-1 IN residue Tyr-143 would accordingly result in loss of an important RAL binding contact. The structure accounted for the relative specificity of RAL resistance to Tyr-143 changes in IN (
      • Métifiot M.
      • Vandegraaff N.
      • Maddali K.
      • Naumova A.
      • Zhang X.
      • Rhodes D.
      • Marchand C.
      • Pommier Y.
      Elvitegravir overcomes resistance to raltegravir induced by integrase mutation Y143.
      ,
      • Huang W.
      • Frantzell A.
      • Fransen S.
      • Petropoulos C.J.
      Multiple genetic pathways involving amino acid position 143 of HIV-1 integrase are preferentially associated with specific secondary amino Aacid substitutions and confer resistance to raltegravir and cross-resistance to elvitegravir.
      ), as only RAL among the clinical INSTIs harbors the methyl-oxadiazole constituent (Fig. 4A). Although Asn-224 similarly resides near the INSTI-binding pocket, it does not directly contact bound drugs. The mutant His side chain in the intasome structure derived from PFV IN N224H interacted with the 3′-deoxyadenylate–bridging phosphate, which was disrupted by second-generation INSTI MK-2048 binding (
      • Hare S.
      • Vos A.M.
      • Clayton R.F.
      • Thuring J.W.
      • Cummings M.D.
      • Cherepanov P.
      Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.
      ). Although disruption of the His–DNA interaction could contribute to the mechanism of clinical INSTI resistance to HIV-1 N155H (
      • Hare S.
      • Vos A.M.
      • Clayton R.F.
      • Thuring J.W.
      • Cummings M.D.
      • Cherepanov P.
      Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.
      ), intasome structures with INs that share greater amino acid identity to HIV-1 are expected to more completely inform INSTI resistance mechanisms outside of the Tyr-143 pathway. Of note, although MVV is a lentivirus, its IN also shares limited amino acid sequence identity with HIV-1 IN (27.4% overall; 34.3% between CCDs). Given fast-paced advancements in single-particle cryo-EM (
      • Lyumkis D.
      Challenges and opportunities in cryo-EM single-particle analysis.
      ,
      • Mitra A.K.
      Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age.
      ), one can optimistically expect comparatively high-resolution structures of INSTIs bound to the HIV-1 intasome in the not too distant future. Such structures should critically inform mechanisms of INSTI drug resistance as well as how to potentially improve INSTI potencies moving forward.
      Clinical (
      • Cahn P.
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      • Mingrone H.
      • Shuldyakov A.
      • Brites C.
      • Andrade-Villanueva J.F.
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      • Reuter T.
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      Dolutegravir versus raltegravir in antiretroviralexperienced, integrase-inhibitor-naive adults with HIV: week 48 results from the randomised, double-blind, non-inferiority SAILING study.
      ,
      • Raffi F.
      • Jaeger H.
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      • Albrecht H.
      • Belonosova E.
      • Gatell J.M.
      • Baril J.G.
      • Domingo P.
      • Brennan C.
      • Almond S.
      • Min S.
      extended SPRING-2 Study Group
      Once-daily dolutegravir versus twice-daily raltegravir in antiretroviral-naive adults with HIV-1 infection (SPRING-2 study): 96 week results from a randomised, double-blind, non-inferiority trial.
      • Raffi F.
      • Rachlis A.
      • Stellbrink H.J.
      • Hardy W.D.
      • Torti C.
      • Orkin C.
      • Bloch M.
      • Podzamczer D.
      • Pokrovsky V.
      • Pulido F.
      • Almond S.
      • Margolis D.
      • Brennan C.
      • Min S.
      SPRING-2 Study Group
      Once-daily dolutegravir versus raltegravir in antiretroviral-naive adults with HIV-1 infection: 48 week results from the randomised, double-blind, non-inferiority SPRING-2 study.
      ) as well as in vitro (
      • Kobayashi M.
      • Yoshinaga T.
      • Seki T.
      • Wakasa-Morimoto C.
      • Brown K.W.
      • Ferris R.
      • Foster S.A.
      • Hazen R.J.
      • Miki S.
      • Suyama-Kagitani A.
      • Kawauchi-Miki S.
      • Taishi T.
      • Kawasuji T.
      • Johns B.A.
      • Underwood M.R.
      • Garvey E.P.
      • Sato A.
      • Fujiwara T.
      In vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor.
      ) studies have highlighted the superior resistance profiles of second-generation INSTIs such as DTG compared with predecessor first-generation compounds. Whereas selection of HIV-1 resistance in cell culture invariably leads to changes in IN that can confer >100-fold resistance to RAL and EVG, such resistance is much harder to come by for DTG, and the selected changes in IN, such as R263K, engender just a few-fold resistance to the compound (
      • Quashie P.K.
      • Mesplède T.
      • Han Y.-S.
      • Oliveira M.
      • Singhroy D.N.
      • Fujiwara T.
      • Underwood M.R.
      • Wainberg M.A.
      Characterization of the R263K mutation in HIV-1 integrase that confers low-level resistance to the second-generation integrase strand transfer inhibitor dolutegravir.
      ). Whereas cART formulations typically comprise three distinct compounds, such observations inspired clinical evaluation of DTG as a monotherapy or as dual therapy in conjunction with an NRTI, NNRTI, or PI (
      • Katlama C.
      • Soulié C.
      • Caby F.
      • Denis A.
      • Blanc C.
      • Schneider L.
      • Valantin M.-A.
      • Tubiana R.
      • Kirstetter M.
      • Valdenassi E.
      • Nguyen T.
      • Peytavin G.
      • Calvez V.
      • Marcelin A.-G.
      Dolutegravir as monotherapy in HIV-1-infected individuals with suppressed HIV viraemia.
      • Rojas J.
      • Blanco J.L.
      • Marcos M.A.
      • Lonca M.
      • Tricas A.
      • Moreno L.
      • Gonzalez-Cordon A.
      • Torres B.
      • Mallolas J.
      • Garcia F.
      • Gatell J.M.
      • Martinez E.
      Dolutegravir monotherapy in HIV-infected patients with sustained viral suppression.
      ,
      • Oldenbuettel C.
      • Wolf E.
      • Ritter A.
      • Noe S.
      • Heldwein S.
      • Pascucci R.
      • Wiese C.
      • Von Krosigk A.
      • Jaegel-Guedes E.
      • Jaeger H.
      • Balogh A.
      • Koegl C.
      • Spinner C.D.
      Dolutegravir monotherapy as treatment de-escalation in HIV-infected adults with virological control: DoluMono cohort results.
      ,
      • Wijting I.
      • Rokx C.
      • Boucher C.
      • van Kampen J.
      • Pas S.
      • de Vries-Sluijs T.
      • Schurink C.
      • Bax H.
      • Derksen M.
      • Andrinopoulou E.R.
      • van der Ende M.
      • van Gorp E.
      • Nouwen J.
      • Verbon A.
      • Bierman W.
      • Rijnders B.
      Dolutegravir as maintenance monotherapy for HIV (DOMONO): a phase 2, randomised non-inferiority trial.
      ,
      • Blanco J.L.
      • Rojas J.
      • Paredes R.
      • Negredo E.
      • Mallolas J.
      • Casadella M.
      • Clotet B.
      • Gatell J.M.
      • de Lazzari E.
      • Martinez E.
      DOLAM Study Team
      Dolutegravir-based maintenance monotherapy versus dual therapy with lamivudine: a planned 24 week analysis of the DOLAM randomized clinical trial.
      • Aboud M.
      • Orkin C.
      • Podzamczer D.
      • Bogner J.R.
      • Baker D.
      • Khuong-Josses M.A.
      • Parks D.
      • Angelis K.
      • Kahl L.P.
      • Blair E.A.
      • Adkison K.
      • Underwood M.
      • Matthews J.E.
      • Wynne B.
      • Vandermeulen K.
      • Gartland M.
      • Smith K.
      Efficacy and safety of dolutegravir-rilpivirine for maintenance of virological suppression in adults with HIV-1: 100-week data from the randomised, open-label, phase 3 SWORD-1 and SWORD-2 studies.
      ). Based on rates of virological failure, the use of DTG as a monotherapy for PLHIV is contra-indicated, whereas the evaluation of dual therapy options is ongoing (
      • Aboud M.
      • Orkin C.
      • Podzamczer D.
      • Bogner J.R.
      • Baker D.
      • Khuong-Josses M.A.
      • Parks D.
      • Angelis K.
      • Kahl L.P.
      • Blair E.A.
      • Adkison K.
      • Underwood M.
      • Matthews J.E.
      • Wynne B.
      • Vandermeulen K.
      • Gartland M.
      • Smith K.
      Efficacy and safety of dolutegravir-rilpivirine for maintenance of virological suppression in adults with HIV-1: 100-week data from the randomised, open-label, phase 3 SWORD-1 and SWORD-2 studies.
      ) (reviewed in Ref.
      • Wandeler G.
      • Buzzi M.
      • Anderegg N.
      • Sculier D.
      • BÈguelin C.
      • Egger M.
      • Calmy A.
      Virologic failure and HIV drug resistance on simplified, dolutegravir-based maintenance therapy: systematic review and meta-analysis. Version 2.
      ). Current guidelines recommend the use of DTG, BIC, or RAL with two NRTIs for most PLHIV (
      • Saag M.S.
      • Benson C.A.
      • Gandhi R.T.
      • Hoy J.F.
      • Landovitz R.J.
      • Mugavero M.J.
      • Sax P.E.
      • Smith D.M.
      • Thompson M.A.
      • Buchbinder S.P.
      • Del Rio C.
      • Eron Jr., J.J.
      • Fätkenheuer G.
      • Günthard H.F.
      • Molina J.-M.
      • Jacobsen D.M.
      • Volberding P.A.
      Antiretroviral drugs for treatment and prevention of HIV infection in adults: 2018 recommendations of the International Antiviral Society–U.S.A. panel.
      ,
      Panel on Antiretroviral Guidelines for Adults and Adolescents
      Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV.
      ).
      The rates at which INSTIs dissociate from IN-viral DNA complexes in vitro have informed the mechanisms of drug action and drug resistance. Consistent with its comparatively high resistance barrier, the dissociative half-life of DTG, 71 h, was significantly longer than the corresponding RAL and EVG values of about 9 and 3 h, respectively (
      • Hightower K.E.
      • Wang R.
      • Deanda F.
      • Johns B.A.
      • Weaver K.
      • Shen Y.
      • Tomberlin G.H.
      • Carter 3rd, H.L.
      • Broderick T.
      • Sigethy S.
      • Seki T.
      • Kobayashi M.
      • Underwood M.R.
      Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.
      ). Although analyses of mutant IN-viral complexes failed to identify a direct correlation between drug dissociation half-life and antiviral potency and resistance, HIV-1 was generally sensitive to INSTIs when compound dissociative half-life was greater than 4 h and resistant to inhibition when half-lives were less than 1 h (
      • Hightower K.E.
      • Wang R.
      • Deanda F.
      • Johns B.A.
      • Weaver K.
      • Shen Y.
      • Tomberlin G.H.
      • Carter 3rd, H.L.
      • Broderick T.
      • Sigethy S.
      • Seki T.
      • Kobayashi M.
      • Underwood M.R.
      Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.
      ). Thus, INSTI dissociative half-life is a useful predictor of drug potency and drug resistance. Whereas some IN amino acid substitutions, such as Y143R/K/C, increase dissociation by altering a direct IN-INSTI contact (
      • Hare S.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      Retroviral intasome assembly and inhibition of DNA strand transfer.
      ), changes such as Q148H/G140S seemingly act indirectly by altering the conformation of the IN active-site region (
      • Hare S.
      • Vos A.M.
      • Clayton R.F.
      • Thuring J.W.
      • Cummings M.D.
      • Cherepanov P.
      Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.
      ).
      Alterations of viral DNA sequence, especially the terminal deoxyadenylate residue, also alter INSTI dissociative half-life (
      • Langley D.R.
      • Samanta H.K.
      • Lin Z.
      • Walker M.A.
      • Krystal M.R.
      • Dicker I.B.
      The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors.
      ), although to date, LTR sequence changes have not been implicated in INSTI resistance. Reverse transcription initiates with minus-strand DNA synthesis via a co-packaged host tRNALys3 primer that engages the primer-binding site near the 5′ end of the viral RNA genome (see Ref.
      • Engelman A.
      for review). Synthesis of the plus-strand of retroviral DNA is primed via an oligonucleotide derived from the 3′ polypurine (PPT) tract. In the RNA genome, the 3′ PPT abuts the U3 sequence that will form the upstream viral DNA terminus after reverse transcription. Selection of DTG resistance in cell culture has revealed changes in the HIV-1 3′ PPT, which was unexpected because this sequence abuts the downstream LTR distal from the viral DNA termini (Fig. 2) (
      • Malet I.
      • Subra F.
      • Charpentier C.
      • Collin G.
      • Descamps D.
      • Calvez V.
      • Marcelin A.-G.
      • Delelis O.
      Mutations located outside the integrase gene can confer resistance to HIV-1 integrase strand transfer inhibitors.
      ). One possible explanation is that the alterations lead to misprocessing of the PPT during reverse transcription and accordingly extend the U3 DNA terminus, which would be a suboptimal sequence for IN binding (
      • Das A.T.
      • Berkhout B.
      How polypurine tract changes in the HIV-1 RNA genome can cause resistance against theintegrase inhibitor dolutegravir.
      ). However, sequencing of 2-LTR circles, which form at low frequency in the cell nucleus via DNA ligation and thus provide a snapshot of viral DNA end sequences, failed to identify the hypothesized PPT extension (
      • Malet I.
      • Subra F.
      • Richetta C.
      • Charpentier C.
      • Collin G.
      • Descamps D.
      • Calvez V.
      • Marcelin A.-G.
      • Delelis O.
      Reply to Das and Berkhout, “How polypurine tract changes in the HIV-1 RNA genome can cause resistance against the integrase inhibitor dolutegravir”.
      ). PPT mutations have been recorded in one patient who received DTG monotherapy (
      • Wijting I.E.A.
      • Lungu C.
      • Rijnders B.J.A.
      • van der Ende M.E.
      • Pham H.T.
      • Mesplede T.
      • Pas S.D.
      • Voermans J.J.C.
      • Schuurman R.
      • van de Vijver D.A.M.C.
      • Boers P.H.M.
      • Gruters R.A.
      • Boucher C.A.B.
      • van Kampen J.J.A.
      HIV-1 resistance dynamics in patients with virologic failure to dolutegravir maintenance monotherapy.
      ), indicating that such changes may very well be clinically relevant. Additional work is required to more fully document the frequency of PPT changes in patients that fail DTG therapy as well as how such changes engender drug resistance.
      Other changes outside of the IN coding region, including the HIV-1 env gene, can confer resistance to DTG (
      • Van Duyne R.
      • Kuo L.S.
      • Pham P.
      • Fujii K.
      • Freed E.O.
      Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle.
      ). HIV-1 can infect cells through fusing directly with the cellular plasma membrane or through the virological synapse that forms between an infected cell and an uninfected cell (
      • McDonald D.
      • Wu L.
      • Bohks S.M.
      • KewalRamani V.N.
      • Unutmaz D.
      • Hope T.J.
      Recruitment of HIV and its receptors to dendritic cell-T cell junctions.
      ) (reviewed in Ref.
      • Law K.M.
      • Satija N.
      • Esposito A.M.
      • Chen B.K.
      Cell-to-cell spread of HIV and viral pathogenesis.
      ). The env mutations effectively increased the multiplicity of HIV-1 infection by significantly increasing the efficiency of cell-to-cell infection (
      • Van Duyne R.
      • Kuo L.S.
      • Pham P.
      • Fujii K.
      • Freed E.O.
      Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle.
      ). This mechanism of drug resistance, which is indirect because it is highly unlikely to influence the dissociative half-life of DTG from the HIV-1 intasome, is reminiscent of prior reports that HIV-1 infection through the virological synapse reduced the efficacy of certain cART compounds (
      • Sigal A.
      • Kim J.T.
      • Balazs A.B.
      • Dekel E.
      • Mayo A.
      • Milo R.
      • Baltimore D.
      Cell-to-cell spread of HIV permits ongoing replication despite antiretroviral therapy.
      ,
      • Agosto L.M.
      • Zhong P.
      • Munro J.
      • Mothes W.
      Highly active antiretroviral therapies are effective against HIV-1 cell-to-cell transmission.
      ). Additional work is required to determine whether changes in HIV-1 env can confer resistance to DTG in the clinical setting (
      • Van Duyne R.
      • Kuo L.S.
      • Pham P.
      • Fujii K.
      • Freed E.O.
      Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle.
      ).
      The investigational second-generation INSTI cabotegravir (CAB) formulated as a crystalline nanoparticle conferred long-acting (LA) protection against challenge by chimeric simian-HIV (SHIV) in the macaque model of HIV/AIDS (
      • Andrews C.D.
      • Spreen W.R.
      • Mohri H.
      • Moss L.
      • Ford S.
      • Gettie A.
      • Russell-Lodrigue K.
      • Bohm R.P.
      • Cheng-Mayer C.
      • Hong Z.
      • Markowitz M.
      • Ho D.D.
      Long-acting integrase inhibitor protects macaques from intrarectal simian/human immunodeficiency virus.
      ,
      • Andrews C.D.
      • Yueh Y.L.
      • Spreen W.R.
      • St Bernard L.
      • Boente-Carrera M.
      • Rodriguez K.
      • Gettie A.
      • Russell-Lodrigue K.
      • Blanchard J.
      • Ford S.
      • Mohri H.
      • Cheng-Mayer C.
      • Hong Z.
      • Ho D.D.
      • Markowitz M.
      A long-acting integrase inhibitor protects female macaques from repeated high-dose intravaginal SHIV challenge.
      • Radzio J.
      • Spreen W.
      • Yueh Y.L.
      • Mitchell J.
      • Jenkins L.
      • García-Lerma J.G.
      • Heneine W.
      The long-acting integrase inhibitor GSK744 protects macaques from repeated intravaginal SHIV challenge.
      ). LA-CAB administered as a monotherapy or in combination with LA-rilpivirine, which is a long-acting NNRTI, is being evaluated as a pre-exposure prophylaxis (PrEP) to prevent HIV-1 infection (reviewed in Ref.
      • Singh K.
      • Sarafianos S.G.
      • Sönnerborg A.
      Long-acting anti-HIV drugs targeting HIV-1 reverse transcriptase and integrase.
      ). One of the biggest factors contributing to the emergence of anti-HIV drug resistance is dosing protocol compliance, which for oral cART formulations is one to several pills daily. LA regimens largely obfuscate the need for end-user dose monitoring, which could increase compliance, although at the same time such regimens require regular injections to maintain plasma trough concentrations above values required to inhibit HIV-1 replication. Macaques that were positive for SHIV RNA but seronegative at the time of infection could develop resistance to LA-CAB, and the associated IN changes conferred potent cross-resistance to all licensed INSTIs (
      • Radzio-Basu J.
      • Council O.
      • Cong M.-E.
      • Ruone S.
      • Newton A.
      • Wei X.
      • Mitchell J.
      • Ellis S.
      • Petropoulos C.J.
      • Huang W.
      • Spreen W.
      • Heneine W.
      • García-Lerma J.G.
      Drug resistance emergence in macaques administered cabotegravir long-acting for pre-exposure prophylaxis during acute SHIV infection.
      ). Such observations highlight the need to carefully monitor patients to avoid initiating PrEP during unrecognized acute HIV-1 infection. CAB in cell culture is marginally less effective at inhibiting infection by certain INSTI-resistant viruses than is either DTG or BIC (
      • Smith S.J.
      • Zhao X.Z.
      • Burke Jr., T.R.
      • Hughes S.H.
      Efficacies of cabotegravir and bictegravir against drug-resistant HIV-1 integrase mutants.
      ), and both DTG (
      • Sillman B.
      • Bade A.N.
      • Dash P.K.
      • Bhargavan B.
      • Kocher T.
      • Mathews S.
      • Su H.
      • Kanmogne G.D.
      • Poluektova L.Y.
      • Gorantla S.
      • McMillan J.
      • Gautam N.
      • Alnouti Y.
      • Edagwa B.
      • Gendelman H.E.
      Creation of a long-acting nanoformulated dolutegravir.
      ) and BIC (
      • Mandal S.
      • Prathipati P.K.
      • Belshan M.
      • Destache C.J.
      A potential long-acting bictegravir loaded nano-drug delivery system for HIV-1 infection: a proof-of-concept study.
      ) have been formulated as LA compounds. Future research will evaluate the efficacy of LA INSTIs to treat at risk patients with PrEP as well as PLHIV.

      ALLINIs

      Despite relatively high barriers, second-generation INSTIs do select for resistance (
      • Wijting I.E.A.
      • Lungu C.
      • Rijnders B.J.A.
      • van der Ende M.E.
      • Pham H.T.
      • Mesplede T.
      • Pas S.D.
      • Voermans J.J.C.
      • Schuurman R.
      • van de Vijver D.A.M.C.
      • Boers P.H.M.
      • Gruters R.A.
      • Boucher C.A.B.
      • van Kampen J.J.A.
      HIV-1 resistance dynamics in patients with virologic failure to dolutegravir maintenance monotherapy.
      ,
      • Radzio-Basu J.
      • Council O.
      • Cong M.-E.
      • Ruone S.
      • Newton A.
      • Wei X.
      • Mitchell J.
      • Ellis S.
      • Petropoulos C.J.
      • Huang W.
      • Spreen W.
      • Heneine W.
      • García-Lerma J.G.
      Drug resistance emergence in macaques administered cabotegravir long-acting for pre-exposure prophylaxis during acute SHIV infection.
      ,
      • Zhang W.W.
      • Cheung P.K.
      • Oliveira N.
      • Robbins M.A.
      • Harrigan P.R.
      • Shahid A.
      Accumulation of multiple mutations in vivo confers cross-resistance to new and existing integrase inhibitors.
      ). As exemplified by the clinical successes of the NRTIs and NNRTIs, it would accordingly be highly beneficial to have additional drug classes that inhibit IN activity through novel mechanisms of action.
      Whereas a number of different types of IN-targeting molecules have been described in the literature, the class of compounds collectively known as ALLINIs has advanced the furthest. Predecessor compounds of potent ALLINIs were discovered via two different means, including a high-throughput screen for inhibitors of IN 3′ processing activity (
      • Tsantrizos Y.S.
      • Boes M.
      • Brochu C.
      • Fenwick C.
      • Malenfant E.
      • Mason S.
      • Pesant M.
      November 22, Inhibitors of human immunodeficiency virus replication.
      ,
      • Fenwick C.W.
      • Tremblay S.
      • Wardrop E.
      • Bethell R.
      • Coulomb R.
      • Elston R.
      • Faucher A.-M.
      • Mason S.
      • Simoneau B.
      • Tsantrizos Y.
      • Yoakim C.
      Resistance studies with HIV-1 non-catalytic site integrase inhibitors.
      • Fader L.D.
      • Malenfant E.
      • Parisien M.
      • Carson R.
      • Bilodeau F.
      • Landry S.
      • Pesant M.
      • Brochu C.
      • Morin S.
      • Chabot C.
      • Halmos T.
      • Bousquet Y.
      • Bailey M.D.
      • Kawai S.H.
      • Coulombe R.
      • et al.
      Discovery of BI 224436, a noncatalytic site integrase inhibitor (NCINI) of HIV-1.
      ) and structure-guided modeling of the amino acid contacts that mediate the interaction of HIV-1 IN with the host integration targeting cofactor lens epithelium-derived growth factor (LEDGF)/p75 (
      • Christ F.
      • Voet A.
      • Marchand A.
      • Nicolet S.
      • Desimmie B.A.
      • Marchand D.
      • Bardiot D.
      • Van der Veken N.J.
      • Van Remoortel B.
      • Strelkov S.V.
      • De Maeyer M.
      • Chaltin P.
      • Debyser Z.
      Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication.
      ). In addition to ALLINI (
      • Kessl J.J.
      • Jena N.
      • Koh Y.
      • Taskent-Sezgin H.
      • Slaughter A.
      • Feng L.
      • de Silva S.
      • Wu L.
      • Le Grice S.F.J.
      • Engelman A.
      • Fuchs J.R.
      • Kvaratskhelia M.
      Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors.
      ), such compounds have been referred to as LEDGIN for LEDGF-interaction site (
      • Christ F.
      • Voet A.
      • Marchand A.
      • Nicolet S.
      • Desimmie B.A.
      • Marchand D.
      • Bardiot D.
      • Van der Veken N.J.
      • Van Remoortel B.
      • Strelkov S.V.
      • De Maeyer M.
      • Chaltin P.
      • Debyser Z.
      Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication.
      ), NCINI for noncatalytic site IN inhibitor (
      • Fenwick C.W.
      • Tremblay S.
      • Wardrop E.
      • Bethell R.
      • Coulomb R.
      • Elston R.
      • Faucher A.-M.
      • Mason S.
      • Simoneau B.
      • Tsantrizos Y.
      • Yoakim C.
      Resistance studies with HIV-1 non-catalytic site integrase inhibitors.
      ,
      • Balakrishnan M.
      • Yant S.R.
      • Tsai L.
      • O'Sullivan C.
      • Bam R.A.
      • Tsai A.
      • Niedziela-Majka A.
      • Stray K.M.
      • Sakowicz R.
      • Cihlar T.
      Non-catalytic site HIV-1 integrase inhibitors disrupt core maturation and induce a reverse transcription block in target cells.
      ), IN-LAI for IN-LEDGF allosteric inhibitor (
      • Le Rouzic E.
      • Bonnard D.
      • Chasset S.
      • Bruneau J.-M.
      • Chevreuil F.
      • Le Strat F.
      • Nguyen J.
      • Beauvoir R.
      • Amadori C.
      • Brias J.
      • Vomscheid S.
      • Eiler S.
      • Lévy N.
      • Delelis O.
      • Deprez E.
      • et al.
      Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.
      ), and MINI for multimeric IN inhibitor (
      • Sharma A.
      • Slaughter A.
      • Jena N.
      • Feng L.
      • Kessl J.J.
      • Fadel H.J.
      • Malani N.
      • Male F.
      • Wu L.
      • Poeschla E.
      • Bushman F.D.
      • Fuchs J.R.
      • Kvaratskhelia M.
      A new class of multimerization selective inhibitors of HIV-1 integrase.
      ).
      Although HIV-1 in large part integrates throughout the human genome (
      • Carteau S.
      • Hoffmann C.
      • Bushman F.
      Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.
      ), it does so in nonrandom fashion, on average favoring active genes that reside within relatively gene-dense regions of chromosomes (
      • Schröder A.R.W.
      • Shinn P.
      • Chen H.
      • Berry C.
      • Ecker J.R.
      • Bushman F.
      HIV-1 integration in the human genome favors active genes and local hotspots.
      ). This targeting preference is largely dictated through specific interactions of two PIC-associated proteins, IN and capsid, with respective host factors LEDGF/p75 and cleavage and polyadenylation specificity factor 6 (
      • Sowd G.A.
      • Serrao E.
      • Wang H.
      • Wang W.
      • Fadel H.J.
      • Poeschla E.M.
      • Engelman A.N.
      A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin.
      ,
      • Achuthan V.
      • Perreira J.M.
      • Sowd G.A.
      • Puray-Chavez M.
      • McDougall W.M.
      • Paulucci-Holthauzen A.
      • Wu X.
      • Fadel H.J.
      • Poeschla E.M.
      • Multani A.S.
      • Hughes S.H.
      • Sarafianos S.G.
      • Brass A.L.
      • Engelman A.N.
      Capsid-CPSF6 interaction licenses nuclear HIV-1 trafficking to sites of viral DNA integration.
      ) (for a recent review, see Ref.
      • Engelman A.N.
      • Singh P.K.
      Cellular and molecular mechanisms of HIV-1 integration targeting.
      ). LEDGF/p75 is a chromosome-associated (
      • Cherepanov P.
      • Maertens G.
      • Proost P.
      • Devreese B.
      • Van Beeumen J.
      • Engelborghs Y.
      • De Clercq E.
      • Debyser Z.
      HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells.
      ,
      • Nishizawa Y.
      • Usukura J.
      • Singh D.P.
      • Chylack Jr., L.T.
      • Shinohara T.
      Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus.
      ,
      • Maertens G.
      • Cherepanov P.
      • Pluymers W.
      • Busschots K.
      • De Clercq E.
      • Debyser Z.
      • Engelborghs Y.
      LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells.
      ) transcriptional co-activator (
      • Ge H.
      • Si Y.
      • Roeder R.G.
      Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.
      ) that harbors two globular domains, an N-terminal PWWP (for Pro-Trp-Trp-Pro) chromatin reader with affinity for histone 3 Lys-36 trimethylation (
      • Pradeepa M.M.
      • Sutherland H.G.
      • Ule J.
      • Grimes G.R.
      • Bickmore W.A.
      Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing.
      ,
      • Eidahl J.O.
      • Crowe B.L.
      • North J.A.
      • McKee C.J.
      • Shkriabai N.
      • Feng L.
      • Plumb M.
      • Graham R.L.
      • Gorelick R.J.
      • Hess S.
      • Poirier M.G.
      • Foster M.P.
      • Kvaratskhelia M.
      Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes.
      • van Nuland R.
      • van Schaik F.M.
      • Simonis M.
      • van Heesch S.
      • Cuppen E.
      • Boelens R.
      • Timmers H.M.
      • van Ingen H.
      Nucleosomal DNA binding drives the recognition of H3K36-methylated nucleosomes by the PSIP1-PWWP domain.
      ), and a downstream region that was termed the IN-binding domain (IBD) because it mediated the binding of LEDGF/p75 to HIV-1 IN in vitro (
      • Cherepanov P.
      • Devroe E.
      • Silver P.A.
      • Engelman A.
      Identification of an evolutionarily-conserved domain in LEDGF/p75 that binds HIV-1 integrase.
      ). The LEDGF/p75 IBD is a PHAT domain (for pseudo-HEAT repeat analogous topology) composed of two helix-hairpin-helix HEAT repeats (
      • Cherepanov P.
      • Sun Z.-Y.J.
      • Rahman S.
      • Maertens G.
      • Wagner G.
      • Engelman A.
      Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75.
      ) (Fig. 6A, left). The IN CCD dimerizes via an extensive interface with DDE catalytic triads positioned at distal apices (
      • Dyda F.
      • Hickman A.B.
      • Jenkins T.M.
      • Engelman A.
      • Craigie R.
      • Davies D.R.
      Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases.
      ). LEDGF/p75 hotspot interaction residues Ile-365, Asp-366, and Phe-406 within the IBD hairpins engage both IN monomers at the CCD dimer interface (
      • Cherepanov P.
      • Sun Z.-Y.J.
      • Rahman S.
      • Maertens G.
      • Wagner G.
      • Engelman A.
      Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75.
      ,
      • Cherepanov P.
      • Ambrosio A.L.B.
      • Rahman S.
      • Ellenberger T.
      • Engelman A.
      From the cover: structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75.
      ). Whereas Asp-366 hydrogen-bonds with the backbone amides of residues Glu-170 and His-171 within one IN monomer, Ile-365 occupies a hydrophobic pocket composed of IN residues from both IN monomers (Fig. 6A, right). Electropositive residues within IBD α1 make additional contacts with electronegative residues within the HIV IN NTD (
      • Hare S.
      • Shun M.C.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.
      ). HIV-1 IN subunits undergo dynamic exchange in solution (
      • McKee C.J.
      • Kessl J.J.
      • Shkriabai N.
      • Dar M.J.
      • Engelman A.
      • Kvaratskhelia M.
      Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein.
      ), and LEDGF/p75 binding accordingly stabilized HIV-1 IN dimers and tetramers (
      • Hare S.
      • Di Nunzio F.
      • Labeja A.
      • Wang J.
      • Engelman A.
      • Cherepanov P.
      Structural basis for functional tetramerization of lentiviral integrase.
      ,
      • McKee C.J.
      • Kessl J.J.
      • Shkriabai N.
      • Dar M.J.
      • Engelman A.
      • Kvaratskhelia M.
      Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein.
      ,
      • Hayouka Z.
      • Rosenbluh J.
      • Levin A.
      • Loya S.
      • Lebendiker M.
      • Veprintsev D.
      • Kotler M.
      • Hizi A.
      • Loyter A.
      • Friedler A.
      Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium.
      ,
      • Tsiang M.
      • Jones G.S.
      • Hung M.
      • Samuel D.
      • Novikov N.
      • Mukund S.
      • Brendza K.M.
      • Niedziela-Majka A.
      • Jin D.
      • Liu X.
      • Mitchell M.
      • Sakowicz R.
      • Geleziunas R.
      Dithiothreitol causes HIV-1 integrase dimer dissociation while agents interacting with the integrase dimer interface promote dimer formation.
      ) and significantly stimulated IN catalytic activities in vitro (
      • Hare S.
      • Di Nunzio F.
      • Labeja A.
      • Wang J.
      • Engelman A.
      • Cherepanov P.
      Structural basis for functional tetramerization of lentiviral integrase.
      ,
      • Cherepanov P.
      • Maertens G.
      • Proost P.
      • Devreese B.
      • Van Beeumen J.
      • Engelborghs Y.
      • De Clercq E.
      • Debyser Z.
      HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells.
      ,
      • McKee C.J.
      • Kessl J.J.
      • Shkriabai N.
      • Dar M.J.
      • Engelman A.
      • Kvaratskhelia M.
      Dynamic modulation of HIV-1 integrase structure and function by cellular lens epithelium-derived growth factor (LEDGF) protein.
      ,
      • Hare S.
      • Shun M.C.
      • Gupta S.S.
      • Valkov E.
      • Engelman A.
      • Cherepanov P.
      A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.
      ,
      • Turlure F.
      • Maertens G.
      • Rahman S.
      • Cherepanov P.
      • Engelman A.
      A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo.
      ). The LEDGF/p75-IN interaction is specific to the lentivirus genus of Retroviridae (
      • Llano M.
      • Vanegas M.
      • Fregoso O.
      • Saenz D.
      • Chung S.
      • Peretz M.
      • Poeschla E.M.
      LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes.
      ,
      • Busschots K.
      • Vercammen J.
      • Emiliani S.
      • Benarous R.
      • Engelborghs Y.
      • Christ F.
      • Debyser Z.
      The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding.
      • Cherepanov P.
      LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro.
      ).
      Figure thumbnail gr6
      Figure 6ALLINI mimicry of LEDGF/p75 binding to the HIV-1 IN CCD dimer. A, solution structure of the LEDGF/p75 IBD (left; PDB code 1Z9E) and IBD-CCD co-crystal structure (right; PDB code 2B4J) highlight the locations of hotspot-interacting residues Ile-365 and Asp-366 in the hairpin that connects α-helices 1 and 2 and Phe-406 in the α4-α5 hairpin (left). Whereas Asp-366 interacts with the backbone amide groups of IN residues Glu-170 and His-171 of the cyan IN monomer (dashed lines), Ile-365 occupies a hydrophobic pocket composed of IN residues from each monomer (i.e. Trp-132 of the green IN monomer and Met-178 of the cyan monomer; right). Other colorings denote atoms of interacting amino acid residues: nitrogen (blue), sulfur (yellow), and oxygen (red). B, quinoline ALLINI BI 224436 (left, chemical diagram) bound to the IN CCD dimer (right, PDB code 6NUJ). The interactions between the compound carboxylic acid and backbone amides within the IN cyan monomer are analogous to those shown in A for LEDGF/p75 residue Asp-366. Thr-174 of the IN cyan monomer additionally interacts with the t-butoxy moiety of the drug. Other labeling is the same as in A. C, binding of pyridine ALLINI KF116 (left, chemical structure) to the IN CCD dimer (right, PDB code 4O55). This view, rotated down ∼90° from A and B, is shown to accentuate the drug-binding pocket. In addition to the contacts described in B, Thr-125 of the green IN monomer interacts with the benzimidazole moiety of KF116. Other labeling is as defined in A and B.
      Cellular depletion of LEDGF/p75 predominantly limits HIV-1 infection by reducing the level of integrated viral DNA (
      • Llano M.
      • Saenz D.T.
      • Meehan A.
      • Wongthida P.
      • Peretz M.
      • Walker W.H.
      • Teo W.
      • Poeschla E.M.
      An essential role for LEDGF/p75 in HIV integration.
      • Vandekerckhove L.
      • Christ F.
      • Van Maele B.
      • De Rijck J.
      • Gijsbers R.
      • Van den Haute C.
      • Witvrouw M.
      • Debyser Z.
      Transient and stable knockdown of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle of human immunodeficiency virus.
      ,
      • Marshall H.M.
      • Ronen K.
      • Berry C.
      • Llano M.
      • Sutherland H.
      • Saenz D.
      • Bickmore W.
      • Poeschla E.
      • Bushman F.D.
      Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting.
      ,
      • Shun M.-C.
      • Raghavendra N.K.
      • Vandegraaff N.
      • Daigle J.E.
      • Hughes S.
      • Kellam P.
      • Cherepanov P.
      • Engelman A.
      LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration.
      ,
      • Schrijvers R.
      • De Rijck J.
      • Demeulemeester J.
      • Adachi N.
      • Vets S.
      • Ronen K.
      • Christ F.
      • Bushman F.D.
      • Debyser Z.
      • Gijsbers R.
      LEDGF/p75-independent HIV-1 replication demonstrates a role for HRP-2 and remains sensitive to inhibition by LEDGINs.
      • Wang H.
      • Jurado K.A.
      • Wu X.
      • Shun M.C.
      • Li X.
      • Ferris A.L.
      • Smith S.J.
      • Patel P.A.
      • Fuchs J.R.
      • Cherepanov P.
      • Kvaratskhelia M.
      • Hughes S.H.
      • Engelman A.
      HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.