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The molecular virology of coronaviruses

Open AccessPublished:July 13, 2020DOI:https://doi.org/10.1074/jbc.REV120.013930
      Few human pathogens have been the focus of as much concentrated worldwide attention as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment, and its multifaceted immune evasion strategies. We highlight aspects of the viral life cycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.
      The Coronaviridae family of viruses are enveloped, single-stranded positive-sense RNA viruses grouped into four genera (alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus) that primarily infect birds and mammals, including humans and bats. The seven coronaviruses known to infect humans fall within the alpha- and betacoronavirus genera, whereas gamma- and deltacoronaviruses primarily infect birds. Coronaviruses have been studied for decades using the model betacoronavirus, murine hepatitis virus (MHV), and the human alphacoronavirus HCoV-229E. In humans, the circulating coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 generally cause mild upper respiratory illness and collectively are associated with 10–30% of common cold cases (
      • Paules C.I.
      • Marston H.D.
      • Jama A.F.
      Coronavirus infections—more than just the common cold.
      ). However, within the past two decades, three highly pathogenic coronaviruses have emerged into the human population as the result of spillover events from wildlife that can cause severe respiratory illness: severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2011, and most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019. These outbreaks, together with estimates suggesting that hundreds to thousands of additional coronaviruses may reside in bats alone (
      • He B.
      • Zhang Y.
      • Xu L.
      • Yang W.
      • Yang F.
      • Feng Y.
      • Xia L.
      • Zhou J.
      • Zhen W.
      • Feng Y.
      • Guo H.
      • Zhang H.
      • Tu C.
      Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China.
      ), highlight the potential for future coronavirus zoonotic transmission.
      In this article, we provide an overview of the coronavirus life cycle with an eye toward its notable molecular features and potential targets for therapeutic interventions (Fig. 1). Much of the information presented is derived from studies of the betacoronaviruses MHV, SARS-CoV, and MERS-CoV, with a rapidly expanding number of reports on SARS-CoV-2. The first portion of the review focuses on the molecular basis of coronavirus entry and its replication cycle. We highlight several notable properties, such as the sophisticated viral gene expression and replication strategies that enable maintenance of a remarkably large, single-stranded, positive-sense (+) RNA genome and the extensive remodeling of cellular membranes to form specialized viral replication and assembly compartments. The second portion explores the mechanisms by which these viruses manipulate the host cell environment during infection including diverse alterations to host gene expression and immune response pathways. This article is intended as a more in-depth companion piece to our online “Coronavirus 101” lecture (https://youtu.be/8_bOhZd6ieM).
      Figure thumbnail gr1
      Figure 1Coronaviruses engage with a host cell-surface receptor and deposit their RNA genomes into the host cytoplasm through endocytosis or directmembrane fusion (1). The positive-sense RNA genome is translated by the host translation machinery (2) to make polyproteins that are cotranslationally cleaved by proteases encoded in the polyprotein to generate components of RdRp complex (3). The RdRp complex uses the genome as a template to generate negative-sense subgenome and genome-length RNAs (4), which are in turn used as templates for synthesis of positive-sense full-length progeny genomes and subgenomic mRNAs (5). Transcription and replication occur in convoluted membranes (CM) adjacent to DMVs that are both derived from rough endoplasmic reticulum(see for more details). The subgenomic mRNAs are translated into structural and accessory proteins (6). The positive sense genomic RNA is bound by nucleocapsid and buds into the ERGIC, which is decorated with structural proteins S, E, and M translated from positive-sense subgenomic RNAs (steps 6 and 7). The enveloped virion is then exported from the cell by exocytosis (steps 8 and 9).

      Part I: The viral life cycle

      Viral entry

      Coronavirus particles consist of a ∼30-kb strand of positive-sense RNA that forms the genome; this genome is coated with nucleocapsid (N) protein and enclosed in a lipid bilayer containing three membrane proteins: spike (S), membrane (M), and envelope (E) (
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      The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.
      ). For all studied coronaviruses, the M protein is critical for incorporating essential viral components into new virions during morphogenesis, and N protein associates with the viral genome and M to direct genome packaging into new viral particles. The E protein forms an ion channel in the viral membrane and participates in viral assembly. The S protein is required for viral entry, as it binds to the target cell and initiates fusion with the host cell membrane (reviewed in Ref.
      • Li F.
      Structure, function, and evolution of coronavirus spike proteins.
      ). S is homotrimeric, with each subunit consisting of two domains, S1 and S2. S1 contains the receptor-binding domain (RBD) and engages with the host receptor, whereas S2 mediates subsequent membrane fusion to enable the virus to enter the host cytoplasm. Activation of the S protein fusion activity requires prior proteolytic cleavage at two sites. The first cleavage site is at the S1/S2 boundary, leading to structural changes in the S2 domain that place it in a prefusion conformation. This cleavage event also separates S2 from S1, although the two domains remain noncovalently associated. The second cleavage site is at S2′, which drives fusion of the viral and cellular membranes to enable release of the N-coated RNA genome into the cytoplasm.
      Whereas coronaviruses use the above general strategy to enter target cells, the receptors and proteases used as well as subcellular sites of S cleavage differ depending on the virus (reviewed in Ref.
      • Millet J.K.
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      ). The S proteins of both SARS-CoV and SARS-CoV-2 use host ACE2 as their receptor (
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      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
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      ) (Fig. 2). ACE2 is a cell-surface peptidase that hydrolyzes angiotensin II and is expressed in most organs, with particularly high expression in the epithelia of lung and small intestine (
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      ). After ACE2 receptor binding, SARS-CoV and SARS-CoV-2 S proteins are subsequently cleaved and activated by the host cell-surface protease TMPRSS2 at the S1/S2 and S2′ sites, leading to membrane fusion (
      • Hoffmann M.
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      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
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      ). Some coronavirus S proteins are precleaved at the S1/S2 site by the cellular protease furin during their biosynthesis in the producer cell, as has been shown for both MHV and MERS-CoV (
      • Millet J.K.
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      ,
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      Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion.
      ), priming them for entry upon receptor binding on the target cell. MERS-CoV S protein uses DPP4 as its receptor (
      • Qian Z.
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      ,
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      Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4.
      ), and multiple cellular proteases, including TMPRSS2, endosomal cathepsins, and furin, have been implicated in the subsequent cleavage at the S2′ site (
      • Qian Z.
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      ,
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      Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2.
      ). The MHV S protein uses host CEACAM1a as its receptor and is subsequently cleaved at S2′ by lysosomal proteases (
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      ,
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      Crystal structure of murine sCEACAM1a[1,4]: a coronavirus receptor in the CEA family.
      ).
      Figure thumbnail gr2
      Figure 2Mechanism of SARS-CoV-2 viral entry. The SARS-CoV-2 S protein engages with the host ACE2 receptor and is subsequently cleaved at S1/S2 and S2′ sites by TMPRSS2 protease. This leads to activation of the S2 domain and drives fusion of the viral and host membranes. See section on ‘viral entry’ for details.
      The extent to which specific coronaviruses fuse at the plasma membrane versus during endocytosis remains incompletely resolved. In the cases of SARS-CoV, MERS-CoV, and MHV, the involvement of endosomal and lysosomal proteases in cleavage of their S proteins suggests that entry can occur during endocytosis. MHV enters predominantly through clathrin-mediated endocytosis and fusion with lysosomal membranes, as lysosomal proteases activate the S protein (
      • Burkard C.
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      • Bosch B.J.
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      Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
      ,
      • Eifart P.
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      • de Haan C.A.M.
      • Rottier P.J.M.
      • Korte T.
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      ). For SARS-CoV and MERS-CoV, both the endocytic and direct membrane fusion pathways may be used for entry. Studies in which components of endocytosis and endosomal proteases have been blocked demonstrate that SARS-CoV and MERS-CoV can exploit the endocytic pathway to enter target cells (
      • Simmons G.
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      ,
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      ,
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      Glycopeptide antibiotics potently inhibit cathepsin L in the late endosome/lysosome and block the entry of Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus (SARS-CoV).
      ). For these viruses, it is likely that the producer and target cell type influence which pathway they use for viral entry. For instance, when MERS-CoV S is precleaved in the producer cell, it gets activated by cell-surface proteases and enters the target cell by direct membrane fusion (
      • Park J.-E.
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      • Barlan A.
      • Fehr A.R.
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      • McCray P.B.
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      Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism.
      ). In contrast, when MERS-CoV S is uncleaved in the producer cell, it enters the target cell through endocytosis and is instead activated by endosomal cathepsins. MERS-CoV with S that has not been precleaved during morphogenesis is incapable of infecting target cell types that have low expression of cathepsins. There are reports demonstrating that inhibition of endosomal cathepsins reduces the efficiency of SARS-CoV-2 entry, suggesting that this virus also exploits endocytosis as another route of entry in addition to direct membrane fusion (
      • Hoffmann M.
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      • Schroeder S.
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      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Shang J.
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      Cell entry mechanisms of SARS-CoV-2.
      ,
      • Wang M.
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      ).
      There has already been considerable research on the SARS-CoV-2 S protein, given the crucial role it plays during viral entry (reviewed in Ref.
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      The proximal origin of SARS-CoV-2.
      ). Comparing the SARS-CoV-2 S protein sequence with that of closely related SARS-CoV–like viruses revealed that almost all the residues important for ACE2 engagement are not conserved in SARS-CoV-2 (
      • Wan Y.
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      ), although the SARS-CoV-2 S RBD has a 10–20-fold higher binding affinity to ACE2 than SARS-CoV S RBD (
      • Wrapp D.
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      Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.
      ). The mechanistic basis for the enhanced binding affinity is not entirely clear, as ACE2 engagement is structurally similar between SARS-CoV S and SARS-CoV-2 S (
      • Lan J.
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      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.
      ). However, there is a unique salt-bridge interaction present between SARS-CoV-2 S and ACE2, and this may contribute to the enhanced binding affinity. Furthermore, the S1/S2 site in SARS-CoV-2 S contains an insertion of polybasic residues (
      • Walls A.C.
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      The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade.
      ). The stretch of polybasic residues contains a furin recognition motif, and recent data suggest that furin can cleave at the S1/S2 site on SARS-CoV-2 S, but not SARS-CoV S, in producer cells (
      • Shang J.
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      Cell entry mechanisms of SARS-CoV-2.
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      A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells.
      ). This precleavage event is analogous to the processing of MERS-CoV S and MHV S, both of which also contain a furin cleavage site at S1/S2. A precleavage event at the S1/S2 site implies that SARS-CoV-2 S may only require cleavage at the S2′ site on the target cell surface, which would potentiate the membrane fusion process. Notably, acquisition of polybasic cleavage sites occurs during experimental selection for increased transmissibility and expanded tropism in other viruses, suggesting that it may have played a role in the bat-to-human spillover of SARS-CoV-2 (
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      Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens.
      ). Further investigation into the properties of S protein from SARS-CoV-2 and other closely related viruses may provide insight into the origin of SARS-CoV-2 as well as the mechanism behind its high transmissibility.
      Numerous therapeutic strategies are being explored to inhibit SARS-CoV-2 entry, including blocking ACE2 engagement, inactivating host proteases, and inhibiting S2-mediated membrane fusion. Neutralizing antibodies against SARS-CoV S display moderate efficacy in blocking SARS-CoV-2 infection due to significant differences in the epitope region (
      • Hoffmann M.
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      • Schiergens T.S.
      • Herrler G.
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      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ,
      • Walls A.C.
      • Park Y.-J.
      • Tortorici M.A.
      • Wall A.
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      Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein.
      ,
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      Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV.
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      The trinity of COVID-19: immunity, inflammation and intervention.
      ). A recent study isolated neutralizing antibodies capable of blocking the interaction between S and ACE2 from convalescent SARS-CoV-2 patients and demonstrated that they effectively reduce viral load in a mouse model, garnering optimism about the possible use of neutralizing antibodies for treatment (
      • Wu Y.
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      A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.
      ). Other strategies include development of lipopeptides that block S2-mediated membrane fusion (
      • Xia S.
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      Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion.
      ) and use of a clinically tested TMPRSS2 inhibitor (
      • Hoffmann M.
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      • Schroeder S.
      • Krüger N.
      • Herrler T.
      • Erichsen S.
      • Schiergens T.S.
      • Herrler G.
      • Wu N.-H.
      • Nitsche A.
      • Müller M.A.
      • Drosten C.
      • Pöhlmann S.
      SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.
      ). Not surprisingly, generating protective immunity against the S protein has been the major focus of SARS-CoV-2 vaccine efforts. S protein–directed vaccine platforms under development include production of recombinant S protein, use of nonpathogenic viral vectors to direct expression of S, and nucleic acid–based vaccines in which sequence encoding the S protein is delivered as an mRNA or on a DNA backbone (
      • Corey B.L.
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      • Fauci A.S.
      • Collins F.S.
      A strategic approach to COVID-19 vaccine R&D.
      ). The viral vector and nucleic acid vaccine strategies rely on host ribosomes to translate the S sequence into protein, which would then be subsequently processed and presented to the immune system.

      Genome organization, polyprotein synthesis, and proteolysis

      Coronaviruses have one of the largest known genomes among RNA viruses, ranging from 27 to 32 kb in length, more than double the length of the average RNA virus genome, and encode for ∼22-29 proteins (
      • Kim D.
      • Lee J.-Y.
      • Yang J.-S.
      • Kim J.W.
      • Kim V.N.
      • Chang H.
      The architecture of SARS-CoV-2 transcriptome.
      ,
      • Gorbalenya A.E.
      • Enjuanes L.
      • Ziebuhr J.
      • Snijder E.J.
      Nidovirales: evolving the largest RNA virus genome.
      ). Given the constraints of eukaryotic translation, which generally allow one protein to be translated per mRNA with ribosome scanning beginning near the 5′ end, it is worth pausing to consider how this number of viral proteins can be synthesized from the genome with a single ribosome entry site. Coronaviruses achieve this feat through the use of large, multiprotein fusions (termed polyproteins, described below) that are subsequently processed into individual proteins (
      • Brian D.A.
      • Baric R.S.
      Coronavirus genome structure and replication.
      ), as well as through synthesis of sub-genome-length mRNAs using an unusual transcription mechanism (discussed in the subsequent section).
      All of the viral nonstructural proteins (nsps) are encoded in two open reading frames (ORF1a and -b) that encompass roughly the first two-thirds of the viral genome (Fig. 3). ORF1a/b is translated from the 5′-capped RNA genome by cap-dependent translation to produce a shorter polyprotein (the ∼440–500-kDa pp1a, which includes nsps 1-11) or a longer polyprotein (the ∼740–810-kDa pp1ab, which includes nsp1 to -16), depending on whether the stop codon at the end of ORF1a is recognized or bypassed. Bypassing the ORF1a stop codon occurs through a −1 ribosomal frameshift in the overlapping region between ORF1a and -1b just upstream of the stop codon, enabling production of the larger pp1ab polyprotein. Frameshifting occurs with ∼20–50% efficiency (
      • Irigoyen N.
      • Firth A.E.
      • Jones J.D.
      • Chung B.Y.W.
      • Siddell S.G.
      • Brierley I.
      High-resolution analysis of coronavirus gene expression by RNA sequencing and ribosome profiling.
      ) and is triggered by the presence of a slippery sequence, UUUAAAC, followed by an RNA pseudoknot structure (
      • Bredenbeek P.J.
      • Pachuk C.J.
      • Noten A.F.
      • Charité J.
      • Luytjes W.
      • Weiss S.R.
      • Spaan W.J.
      The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.
      ), the disruption of which affects frameshifting efficiency (
      • Plant E.P.
      • Sims A.C.
      • Baric R.S.
      • Dinman J.D.
      • Taylor D.R.
      Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.
      ). Whereas nsp1 to -11 from ORF1a are involved in a broad range of functions from blocking the initial immune response to functioning as cofactors for replication and transcription proteins, the core components of the replication and transcription machinery, such as the RNA-dependent RNA polymerase (RdRp), helicase, and other RNA-modifying enzymes, are present in the ORF1b portion of pp1ab. This frameshifting-based translational control strategy helps the virus maintain a stoichiometry of pp1a and pp1ab proteins that is optimal for infectivity and replication (
      • Plant E.P.
      • Rakauskaite R.
      • Taylor D.R.
      • Dinman J.D.
      Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.
      ,
      • Plant E.P.
      • Dinman J.D.
      The role of programmed-1 ribosomal frameshifting in coronavirus propagation.
      ). Due to this requirement of precise ratios of pp1a and pp1ab, frameshifting has been explored as a novel drug target (
      • Park S.-J.
      • Kim Y.-G.
      • Park H.-J.
      Identification of RNA pseudoknot-binding ligand that inhibits the −1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening.
      ,
      • Ahn D.-G.
      • Lee W.
      • Choi J.-K.
      • Kim S.-J.
      • Plant E.P.
      • Almazán F.
      • Taylor D.R.
      • Enjuanes L.
      • Oh J.-W.
      Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication.
      ) similar to such efforts in HIV (
      • Brakier-Gingras L.
      • Charbonneau J.
      • Butcher S.E.
      Targeting frameshifting in the human immunodeficiency virus.
      ). These drugs typically prevent frameshifting by binding to RNA structures that are required for frameshifting (
      • Park S.-J.
      • Kim Y.-G.
      • Park H.-J.
      Identification of RNA pseudoknot-binding ligand that inhibits the −1 ribosomal frameshifting of SARS-coronavirus by structure-based virtual screening.
      ,
      • Ahn D.-G.
      • Lee W.
      • Choi J.-K.
      • Kim S.-J.
      • Plant E.P.
      • Almazán F.
      • Taylor D.R.
      • Enjuanes L.
      • Oh J.-W.
      Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication.
      ).
      Figure thumbnail gr3
      Figure 3Genome organization of SARS-CoV. The RNA genome encodes two categories of proteins: nsps and structural and accessory proteins. The nonstructural proteins are encoded in ORF1a and ORF1b. Cap-dependent translation begins at ORF1a and produces pp1a, encompassing nsp1–11, or pp1ab, a longer polypeptide that includes nsp12–16. The production of either polypeptide depends on whether the stop codon at ORF1a is recognized by the ribosome or is bypassed through a change in the reading frame by the ribosome frameshifting site. The structural and accessory proteins are synthesized by translation of their respective subgenomic mRNAs (see ). The proteins have been color-coded by functional categories for SARS-CoV (see ).
      To liberate the individual nsps, pp1a and pp1ab are proteolytically processed in cis and in trans by two viral proteases encoded by nsp3 and nsp5. Nsp3 contains one or two papain-like proteases (PLpro1 and PLpro2), and nsp5 contains a chymotrypsin-like cysteine protease (3CLpro) (reviewed in Ref.
      • Ziebuhr J.
      • Snijder E.J.
      • Gorbalenya A.E.
      Virus-encoded proteinases and proteolytic processing in the Nidovirales.
      ). The 3CLpro catalyzes the proteolytic cleavage of all nsps downstream of nsp4 and is thus referred to as the main protease. Inhibitors of 3CLpro and PLpro have long been considered as potential drug targets, as their cleavage recognition sequences are distinct from other human proteases and they are essential to viral replication (
      • Zhang L.
      • Lin D.
      • Sun X.
      • Curth U.
      • Drosten C.
      • Sauerhering L.
      • Becker S.
      • Rox K.
      • Hilgenfeld R.
      Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.
      ,
      • Kumar V.
      • Shin J.S.
      • Shie J.-J.
      • Ku K.B.
      • Kim C.
      • Go Y.Y.
      • Huang K.-F.
      • Kim M.
      • Liang P.-H.
      Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CLPro inhibitors.
      ,
      • Anand K.
      • Ziebuhr J.
      • Wadhwani P.
      • Mesters J.R.
      • Hilgenfeld R.
      Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.
      ). Although PLpro is responsible for fewer cleavage events in pp1a, it additionally functions as a deubiquitinase and deISGylating (removal of conjugated interferon-stimulated gene 15 from cellular proteins) enzyme (
      • Clementz M.A.
      • Chen Z.
      • Banach B.S.
      • Wang Y.
      • Sun L.
      • Ratia K.
      • Baez-Santos Y.M.
      • Wang J.
      • Takayama J.
      • Ghosh A.K.
      • Li K.
      • Mesecar A.D.
      • Baker S.C.
      Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases.
      ,
      • Mielech A.M.
      • Kilianski A.
      • Baez-Santos Y.M.
      • Mesecar A.D.
      • Baker S.C.
      MERS-CoV papain-like protease has deISGylating and deubiquitinating activities.
      ), activities that contribute to evasion of the initial antiviral response (
      • Mielech A.M.
      • Kilianski A.
      • Baez-Santos Y.M.
      • Mesecar A.D.
      • Baker S.C.
      MERS-CoV papain-like protease has deISGylating and deubiquitinating activities.
      ). It is therefore possible that targeting PLpro would inhibit viral replication as well as prevent dysregulation of cellular signaling pathways that could lead to cell death in surrounding cells (
      • Báez-Santos Y.M.
      • St John S.E.
      • Mesecar A.D.
      The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.
      ).

      Replication and gene expression

      A subset of nsps generated by proteolytic cleavage of the polyproteins come together to form the replication and transcription complexes (RTCs) that copy and transcribe the genome. RTCs reside in convoluted membrane structures (discussed in detail below) derived from rough endoplasmic reticulum (ER) and are anchored in place by viral transmembrane proteins nsp3, nsp4, and nsp6 (
      • Knoops K.
      • Kikkert M.
      • Worm S.H.E.V.D.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Koster A.J.
      • Mommaas A.M.
      • Snijder E.J.
      SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.
      ,
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ,
      • Hagemeijer M.C.
      • Ulasli M.
      • Vonk A.M.
      • Reggiori F.
      • Rottier P.J.M.
      • de Haan C.A.M.
      Mobility and interactions of coronavirus nonstructural protein 4.
      ,
      • Hagemeijer M.C.
      • Monastyrska I.
      • Griffith J.
      • van der Sluijs P.
      • Voortman J.
      • van Bergen En Henegouwen P.M.
      • Vonk A.M.
      • Rottier P.J.M.
      • Reggiori F.
      • de Haan C.A.M.
      Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.
      ). Similar to other positive-strand RNA viruses, replication of coronaviruses involves synthesis of the complementary full-length negative-strand RNA, which serves as a template for generation of positive-strand progeny genomes (
      • Sawicki S.G.
      • Sawicki D.L.
      Coronavirus transcription: a perspective.
      ). The negative-strand templates get turned over via unknown mechanisms (
      • Wang T.
      • Sawicki S.G.
      Mouse hepatitis virus minus-strand templates are unstable and turnover during viral replication.
      ), and the positive-strand genomes are packaged into virions. Several cis-acting RNA elements in the 5′ and 3′ end of the genome are important for replication and transcription (reviewed in Refs.
      • Sola I.
      • Mateos-Gómez P.A.
      • Almazán F.
      • Zúñiga S.
      • Enjuanes L.
      RNA-RNA and RNA-protein interactions in coronavirus replication and transcription.
      and
      • Madhugiri R.
      • Fricke M.
      • Marz M.
      • Ziebuhr J.
      Coronavirus cis-acting RNA elements.
      ). These include conserved stem loop structures within ∼500 nucleotides of the 5′ end of the genome, structural elements in the 3′ UTR that are partially conserved across the different coronaviruses, and the 3′ poly(A) tail. Negative-strand synthesis is facilitated by the N protein interacting with both the poly(A) tail and the 5′ end of the genome to bring these termini in proximity (
      • Lo C.Y.
      • Tsai T.L.
      • Lin C.N.
      • Lin C.H.
      • Wu H.Y.
      Interaction of coronavirus nucleocapsid protein with the 5′- and 3′- ends of the coronavirus genome is involved in genome circularization and negative-strand RNA synthesis.
      ).
      In addition to genomic replication, the RTCs also carry out synthesis of subgenomic (sg RNA) mRNAs, which encode for the ORFs located in the 3′-proximal one-third of the genome. All sg mRNAs are co-terminal and contain a common 5′ leader sequence that is derived from the 5′ end of the viral genome (
      • Lai M.M.
      • Patton C.D.
      • Stohlman S.A.
      Further characterization of mRNA's of mouse hepatitis virus: presence of common 5-”end nucleotides.
      ). Placement of the common leader sequence at the 5′ end of all sg mRNAs involves an unusual and complex mechanism of discontinuous transcription (Fig. 4) (reviewed in Ref.
      • Sola I.
      • Almazán F.
      • Zúñiga S.
      • Enjuanes L.
      Continuous and discontinuous RNA synthesis in coronaviruses.
      ). During negative-strand synthesis, the RdRp complex terminates or pauses at specific sites along the genome called transcription regulatory sequences (TRSs). The TRSs are present downstream of the common leader sequence at the 5′ end of the genome (TRS-L) and 5′ of every viral ORF along the body of the viral genome (TRS-B) except ORF1a and -1b. Complementarity between sequences in TRS-B on the newly synthesized negative sense RNA and TRS-L allows for the transcription complex to switch templates—effectively jumping from a given TRS-B to the TRS-L at the 5′ end of the genome. Transcription then continues, copying the leader sequence to complete the negative-strand sg RNA (
      • Sola I.
      • Moreno J.L.
      • Zúñiga S.
      • Alonso S.
      • Enjuanes L.
      Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis.
      ,
      • Zúñiga S.
      • Sola I.
      • Alonso S.
      • Enjuanes L.
      Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis.
      ). The negative-strand sg RNAs subsequently serve as templates to generate large numbers of sg mRNAs; the positive-strand RNAs far outnumber the negative-strand RNAs (
      • Sawicki S.G.
      • Sawicki D.L.
      • Siddell S.G.
      A contemporary view of coronavirus transcription.
      ). Secondary structure analysis of the TRS-L region has shown that the context of the sequence and associated structures are important for ensuring that only the TRS-L, and not other TRS-B sequences, acts as the template for strand switching by the RdRp (
      • Di H.
      • McIntyre A.A.
      • Brinton M.A.
      New insights about the regulation of Nidovirus subgenomic mRNA synthesis.
      ). The purpose of the 5′ leader sequence in all sg mRNAs, other than to potentially prime sg mRNA synthesis, is not completely understood. One study with SARS-CoV suggested that the 5′ leader sequence could be important for protection against cleavage by viral nsp1 (
      • Huang C.
      • Lokugamage K.G.
      • Rozovics J.M.
      • Narayanan K.
      • Semler B.L.
      • Makino S.
      SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.
      ), although the mechanism by which protection is rendered is unclear. The efficiency with which the template switch occurs is an important determinant of the levels of the different sg mRNAs and the ratio of sg mRNAs to genome-length RNA, as failed template switching leads to read-through at TRSs and increases the probability of producing genome-length RNA (reviewed in Ref.
      • Di H.
      • McIntyre A.A.
      • Brinton M.A.
      New insights about the regulation of Nidovirus subgenomic mRNA synthesis.
      ). Most of what is known about this regulation is from studies on arteriviruses, which belong to the same order (Nidovirales) as coronaviruses and synthesize sg mRNAs by a similar mechanism. The levels of several sg mRNAs are correlated with the stability (ΔG) of the duplex between TRS-L and TRS-B (
      • Sola I.
      • Moreno J.L.
      • Zúñiga S.
      • Alonso S.
      • Enjuanes L.
      Role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mRNA synthesis.
      ), and hence duplex stability was thought to be an important regulator of this process. However, a recent sequencing study with an arterivirus showed that some TRS-B sequences with 100% similarity to TRS-L core sequences were not used as switching points for the transcription complex, suggesting that whereas duplex stability is necessary, it is not sufficient to dictate template switching (
      • Di H.
      • Madden J.C.
      • Morantz E.K.
      • Tang H.-Y.
      • Graham R.L.
      • Baric R.S.
      • Brinton M.A.
      Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus.
      ). Regulation of the levels of some sg mRNAs, such as the N protein sg mRNA in coronaviruses, was shown to be mediated by short- and long-range RNA-RNA interactions (
      • Moreno J.L.
      • Zúñiga S.
      • Enjuanes L.
      • Sola I.
      Identification of a coronavirus transcription enhancer.
      ,
      • Mateos-Gómez P.A.
      • Morales L.
      • Zúñiga S.
      • Enjuanes L.
      • Sola I.
      Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during transcription.
      ).
      Figure thumbnail gr4
      Figure 4Discontinuous transcription. The RdRp complex initiates transcription at the 39 end of the positive-sense genome (1). Upon copying the TRS-B sequences present at specific sites along the genome body (2), the RdRp complex may “jump” to the TRS-L sequence (3) owing to complementarity between the TRS-B sequence on the nascent sg RNA and TRS-L sequence on the genome. Transcription is resumed on the new template, and the leader sequence (shown in red) is copied to complete the negative-strand sg RNA. The RdRp complex does not always switch templates at TRS-B sequences, resulting in the synthesis of genome-length negative-strand RNA. The negative-strand RNAs serve as templates for the synthesis of genome-length positive-strand RNAs or sg mRNAs.
      Several proteins have also been implicated in regulating the levels of sg mRNAs and the switch between full-length negative-strand synthesis and sg RNA synthesis, although a clear picture of features that favor transcription or replication has not emerged. For example, the viral N protein (
      • Schelle B.
      • Karl N.
      • Ludewig B.
      • Siddell S.G.
      • Thiel V.
      Selective replication of coronavirus genomes that express nucleocapsid protein.
      ) and the cellular kinase GSK-3 and helicase DDX1 (
      • Wu C.-H.
      • Chen P.-J.
      • Yeh S.-H.
      Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription.
      ) have been shown to be important for producing full-length negative-strand genomic RNA and long sg RNAs, suggesting a role in read-through of TRSs. However, the N protein also has helicase-like activity (
      • Grossoehme N.E.
      • Li L.
      • Keane S.C.
      • Liu P.
      • Dann C.E.
      • Leibowitz J.L.
      • Giedroc D.P.
      Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes.
      ), promotes template switching, and appears dispensable for replication but required for efficient sg mRNA transcription (
      • Zúñiga S.
      • Cruz J.L.G.
      • Sola I.
      • Mateos-Gómez P.A.
      • Palacio L.
      • Enjuanes L.
      Coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription.
      ). It is also possible that the transcription complex that carries out negative-strand synthesis is distinct from the version that carries out positive-strand synthesis (
      • Sawicki S.G.
      • Sawicki D.L.
      Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.
      ).

      Composition of the replication/transcription complex

      Coronavirus replication, discontinuous transcription, and RNA processing are orchestrated by a remarkably sophisticated replicase complex (Fig. 5). Unlike other RNA viruses, where replication is primarily dependent on the RdRp and a small number of cofactors, coronaviruses appear to use a multiprotein complex, including the RdRp (nsp12), processivity factors (nsp7-8), a helicase (nsp13), single-strand binding protein (nsp9), a proofreading exonuclease (nsp14), other cofactors (e.g. nsp10), and capping enzymes (e.g. nsp16). This is more reminiscent of replisomes from DNA-based organisms and is potentially a consequence of their unusually large genomes (
      • Smith E.C.
      • Sexton N.R.
      • Denison M.R.
      Thinking outside the triangle: replication fidelity of the largest RNA viruses.
      ).
      Figure thumbnail gr5
      Figure 5Model of putative coronavirus replisome. Shown is a model of how the different proteins in the coronavirus replisome come together on the viral negative strand during synthesis of the positive-strand RNA. The core replicase is predicted to consist of the RdRp (nsp12), processivity factors (nsp7-8), and ExoN complex (nsp14, nsp10). The helicase is shown to be unwinding the dsRNA ahead of the replisome, and the SSB (nsp9) is shown as a dimer protecting single-stranded regions of the RNA. Additionally, the 2′-O-MTase (nsp16), which is predicted to be involved in RNA capping, is also indicated. The model is based on known structures and interactions between the proteins (see Refs.
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      ,
      • Jia Z.
      • Yan L.
      • Ren Z.
      • Wu L.
      • Wang J.
      • Guo J.
      • Zheng L.
      • Ming Z.
      • Zhang L.
      • Lou Z.
      • Rao Z.
      Delicate structural coordination of the severe acute respiratory syndrome coronavirus Nsp13 upon ATP hydrolysis.
      , and
      • Brunn, von A.
      • Teepe C.
      • Simpson J.C.
      • Pepperkok R.
      • Friedel C.C.
      • Zimmer R.
      • Roberts R.
      • Baric R.
      • Haas J.
      Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.
      • Pan J.
      • Peng X.
      • Gao Y.
      • Li Z.
      • Lu X.
      • Chen Y.
      • Ishaq M.
      • Liu D.
      • DeDiego M.L.
      • Enjuanes L.
      • Guo D.
      Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.
      • Imbert I.
      • Snijder E.J.
      • Dimitrova M.
      • Guillemot J.-C.
      • Lécine P.
      • Canard B.
      The SARS-coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein.
      and references within) (
      • Snijder E.J.
      • Decroly E.
      • Ziebuhr J.
      The nonstructural proteins directing coronavirus RNA synthesis and processing.
      ).
      In vitro studies showed that whereas the SARS-CoV RdRp nsp12 has some minimal activity on its own, its activity and processivity are greatly stimulated in the presence of nsp7-nsp8 cofactors (
      • Subissi L.
      • Posthuma C.C.
      • Collet A.
      • Zevenhoven-Dobbe J.C.
      • Gorbalenya A.E.
      • Decroly E.
      • Snijder E.J.
      • Canard B.
      • Imbert I.
      One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
      ). Cryo-EM structures of the SARS-CoV and SARS-CoV-2 nsp12-nsp7-nsp8 tripartite complex revealed that nsp8 binds nsp12 as both a heterodimer (nsp7-nsp8) and by itself to stabilize the regions of nsp12 involved in RNA binding (
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      ,
      • Gao Y.
      • Yan L.
      • Huang Y.
      • Liu F.
      • Zhao Y.
      • Cao L.
      • Wang T.
      • Sun Q.
      • Ming Z.
      • Zhang L.
      • Ge J.
      • Zheng L.
      • Zhang Y.
      • Wang H.
      • Zhu Y.
      • et al.
      Structure of the RNA-dependent RNA polymerase from COVID-19 virus.
      ). Whether the RdRp is capable of de novo initiation or requires a primer-template substrate remains heavily debated (
      • Velthuis, Te A.J.W.
      • Arnold J.J.
      • Cameron C.E.
      • van den Worm S.H.E.
      • Snijder E.J.
      The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent.
      ,
      • Ahn D.-G.
      • Choi J.-K.
      • Taylor D.R.
      • Oh J.-W.
      Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates.
      ). Coronavirus RdRps also have a conserved N-terminal domain that has nucleotidylation activity (NiRAN domain), which is essential for coronavirus replication (
      • Lehmann K.C.
      • Gulyaeva A.
      • Zevenhoven-Dobbe J.C.
      • Janssen G.M.C.
      • Ruben M.
      • Overkleeft H.S.
      • van Veelen P.A.
      • Samborskiy D.V.
      • Kravchenko A.A.
      • Leontovich A.M.
      • Sidorov I.A.
      • Snijder E.J.
      • Posthuma C.C.
      • Gorbalenya A.E.
      Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.
      ). Structural homology analysis of the NiRAN domain suggests that it shares significant homology with the nucleotide-binding site of protein kinases (
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      ), although how it might mediate nucleotidyltransferase or the function of this domain is not known.
      In addition to its role as a processivity factor for the RdRp, nsp8 was first thought to function as a primase during replication (
      • Imbert I.
      • Guillemot J.-C.
      • Bourhis J.-M.
      • Bussetta C.
      • Coutard B.
      • Egloff M.-P.
      • Ferron F.
      • Gorbalenya A.E.
      • Canard B.
      A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus.
      ,
      • Velthuis, Te A.J.W.
      • van den Worm S.H.E.
      • Snijder E.J.
      The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
      ). However, whereas nsp8 has polyadenylation activity that is stimulated by the presence of a polyU stretch on the template strand, it is unable to incorporate other nucleotides on heteropolymeric templates (
      • Tvarogová J.
      • Madhugiri R.
      • Bylapudi G.
      • Ferguson L.J.
      • Karl N.
      • Ziebuhr J.
      Identification and characterization of a human coronavirus 229E nonstructural protein 8-associated RNA 3′-terminal adenylyltransferase activity.
      ), suggesting that it might not be a primase. Additionally, the cryo-EM structure with nsp7 and nsp12 does not suggest a mechanism for nucleotide incorporation by nsp8. It has been proposed that the presence of polyU sequences at the 5′ end of the negative-strand viral RNA could promote polyadenylation of the viral positive-strand RNAs by nsp8, but this remains to be experimentally validated. The poly(A) tail length also varies during infection (
      • Wu H.Y.
      • Ke T.-Y.
      • Liao W.-Y.
      • Chang N.-Y.
      Regulation of coronaviral poly(A) tail length during infection.
      ), and it would be interesting to explore whether nsp8 has a role in this process.
      One of the interacting partners of nsp8 in the RTC is nsp9, a single-strand (ss) nucleic acid–binding protein (
      • Egloff M.-P.
      • Ferron F.
      • Campanacci V.
      • Longhi S.
      • Rancurel C.
      • Dutartre H.
      • Snijder E.J.
      • Gorbalenya A.E.
      • Cambillau C.
      • Canard B.
      The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world.
      ,
      • Sutton G.
      • Fry E.
      • Carter L.
      • Sainsbury S.
      • Walter T.
      • Nettleship J.
      • Berrow N.
      • Owens R.
      • Gilbert R.
      • Davidson A.
      • Siddell S.
      • Poon L.L.M.
      • Diprose J.
      • Alderton D.
      • Walsh M.
      • et al.
      The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.
      ) with no obvious sequence specificity or function. It binds ssDNA and ssRNA with equal affinity, although ssRNA is the presumed substrate during infection. Structural studies have shown that it dimerizes, and this is important for viral replication but dispensable for RNA binding (
      • Miknis Z.J.
      • Donaldson E.F.
      • Umland T.C.
      • Rimmer R.A.
      • Baric R.S.
      • Schultz L.W.
      Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth.
      ). It is possible that nsp9 binds to single-stranded regions of the viral genome and protects them from nucleases, akin to the role played by ssDNA-binding proteins in DNA replication systems. Indeed, other ss nucleic acid–binding proteins are also known to play roles in recombination and homologous base pairing (
      • Marceau A.H.
      Functions of single-strand DNA-binding proteins in DNA replication, recombination, and repair.
      ), processes that occur during discontinuous negative-strand synthesis in coronaviruses.
      Another key component of the RTC is nsp13, a superfamily 1 (SF1) 5′ → 3′ helicase (
      • Adedeji A.O.
      • Marchand B.
      • Velthuis, Te A.J.W.
      • Snijder E.J.
      • Weiss S.
      • Eoff R.L.
      • Singh K.
      • Sarafianos S.G.
      Mechanism of nucleic acid unwinding by SARS-CoV helicase.
      ) that interacts with nsp12 (
      • Jia Z.
      • Yan L.
      • Ren Z.
      • Wu L.
      • Wang J.
      • Guo J.
      • Zheng L.
      • Ming Z.
      • Zhang L.
      • Lou Z.
      • Rao Z.
      Delicate structural coordination of the severe acute respiratory syndrome coronavirus Nsp13 upon ATP hydrolysis.
      ) and several other components of the RTC. The functional role of helicases in replication of RNA viruses is largely unknown, although they are one of the most conserved proteins encoded by coronaviruses (reviewed in Ref.
      • Lehmann K.C.
      • Snijder E.J.
      • Posthuma C.C.
      • Gorbalenya A.E.
      What we know but do not understand about nidovirus helicases.
      ). Helicases use the energy from nucleotide hydrolysis to translocate on nucleic acids. In addition to its (d)NTPase activity, nsp13 also has a 5′-triphosphophatase activity, suggesting a role for it in RNA capping (
      • Ivanov K.A.
      • Thiel V.
      • Dobbe J.C.
      • van der Meer Y.
      • Snijder E.J.
      • Ziebuhr J.
      Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase.
      ). The helicase domain of MERS and SARS-CoV nsp13 shows remarkable similarity to the cellular Upf1 helicase, a protein involved in the nonsense-mediated decay pathway. Based on this observation, it has been proposed that nsp13 could also play a role in quality control of RNAs (
      • Hao W.
      • Wojdyla J.A.
      • Zhao R.
      • Han R.
      • Das R.
      • Zlatev I.
      • Manoharan M.
      • Wang M.
      • Cui S.
      Crystal structure of Middle East respiratory syndrome coronavirus helicase.
      ).
      One of the central outstanding questions about the role of helicases in RNA viruses is whether they function similarly to replicative helicases or if they are involved in unwinding local structures and removing obstacles for the polymerase. Replicative helicases typically work together with the polymerase to unwind the double-stranded nucleic acid ahead of the polymerase. The 5′ → 3′ directionality of the helicase is reminiscent of prokaryotic replisomes, where the helicase and polymerase translocate on different strands and the helicase helps in unwinding the duplex ahead of the polymerase. Thus, during the synthesis of full-length progeny genomes using the negative-strand RNA as a template, nsp13 could be bound to the positive-strand RNA and assist the RdRp as it copies the negative strand (Fig. 5). Cooperativity between the replicative helicase and polymerase is a conserved feature of DNA replisomes. The RdRp stimulates the activity of the helicase (
      • Jia Z.
      • Yan L.
      • Ren Z.
      • Wu L.
      • Wang J.
      • Guo J.
      • Zheng L.
      • Ming Z.
      • Zhang L.
      • Lou Z.
      • Rao Z.
      Delicate structural coordination of the severe acute respiratory syndrome coronavirus Nsp13 upon ATP hydrolysis.
      ), but whether the helicase has a reciprocal effect on RdRp activity, similar to DNA replisomes, would be interesting to test. A non-mutually exclusive possibility is that the helicase facilitates RdRp template switching during discontinuous transcription by releasing subgenomic RNAs at TRS sites during negative-strand synthesis, similar to a role played by some other SF1 helicases in recombination (
      • Raney K.D.
      • Byrd A.K.
      • Aarattuthodiyil S.
      Structure and mechanisms of SF1 DNA helicases.
      ).

      Mechanisms underlying high-fidelity replication

      RNA viruses typically have high mutation rates due to lack of RdRp proofreading activity, which promotes viral genetic diversity and increases their adaptive potential. However, the potential for accumulation of deleterious mutations leading to collapse of the viral population through error catastrophe caps the size of most RNA virus genomes to ∼15 kb (reviewed in Ref.
      • Smith E.C.
      • Sexton N.R.
      • Denison M.R.
      Thinking outside the triangle: replication fidelity of the largest RNA viruses.
      ). The ∼30-kb coronavirus genome far exceeds this threshold, indicating that they must have specialized mechanisms to counteract this mutational burden. In this regard, they are one of the few RNA viruses apart from toroviruses and roniviruses (which are also exceptionally large) that have an exonuclease activity and associated high-fidelity replication (
      • Smith E.C.
      • Denison M.R.
      Coronaviruses as DNA wannabes: a new model for the regulation of RNA virus replication fidelity.
      ). The discovery of this exonuclease (nsp14-ExoN) in the coronavirus genome (
      • Minskaia E.
      • Hertzig T.
      • Gorbalenya A.E.
      • Campanacci V.
      • Cambillau C.
      • Canard B.
      • Ziebuhr J.
      Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis.
      ) showed for the first time the potential for proofreading activity in RNA viruses and explained how coronaviruses maintain their genome integrity. Indeed, the mutation rates of coronaviruses are an order of magnitude lower (10−6 to 10−7) than that of most RNA viruses, and mutating the SARS-CoV or MHV ExoN gene causes the error frequency to jump to that observed in many other RNA viruses (10−3 to 10−5) (
      • Eckerle L.D.
      • Lu X.
      • Sperry S.M.
      • Choi L.
      • Denison M.R.
      High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants.
      ,
      • Eckerle L.D.
      • Becker M.M.
      • Halpin R.A.
      • Li K.
      • Venter E.
      • Lu X.
      • Scherbakova S.
      • Graham R.L.
      • Baric R.S.
      • Stockwell T.B.
      • Spiro D.J.
      • Denison M.R.
      Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.
      ,
      • Smith E.C.
      • Blanc H.
      • Surdel M.C.
      • Vignuzzi M.
      • Denison M.R.
      Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.
      ).
      Active-site mutants that abolish the exonuclease activity of ExoN are lethal for HCoV-229E and transmissible gastroenteritis virus (TGEV) and cause impaired growth for MHV and SARS-CoV (
      • Minskaia E.
      • Hertzig T.
      • Gorbalenya A.E.
      • Campanacci V.
      • Cambillau C.
      • Canard B.
      • Ziebuhr J.
      Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis.
      ), suggesting that ExoN is important but may not be essential under all conditions. Why MHV and SARS-CoV but not HCoV-229E and TGEV can tolerate ExoN mutants is unclear, although it is possible that ExoN is essential only in alphacoronaviruses (HCoV and TGEV) and not in betacoronaviruses (MHV and SARS-CoV). It is also possible that the active-site mutation in SARS and MHV did not fully deactivate the enzyme or that other proteins in the replicase can compensate for the absence of an active ExoN. For example, nsp10 stimulates the catalytic activity of nsp14-ExoN to remove a mismatched nucleotide at the 3′ end of the RNA by >35-fold (
      • Bouvet M.
      • Imbert I.
      • Subissi L.
      • Gluais L.
      • Canard B.
      • Decroly E.
      RNA 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex.
      ), and the high replication fidelity depends on the nsp10-nsp14 interaction (
      • Smith E.C.
      • Case J.B.
      • Blanc H.
      • Isakov O.
      • Shomron N.
      • Vignuzzi M.
      • Denison M.R.
      Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity.
      ). ExoN (nsp14) also interacts with the nsp12-nsp8-nsp7 tripartite complex (
      • Subissi L.
      • Posthuma C.C.
      • Collet A.
      • Zevenhoven-Dobbe J.C.
      • Gorbalenya A.E.
      • Decroly E.
      • Snijder E.J.
      • Canard B.
      • Imbert I.
      One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
      ), providing biochemical evidence for its role in proofreading during transcription/replication. Nsp10 also interacts with nsp16 (a potential RNA-modifying enzyme), and it has been proposed that all of these proteins could come together to form a larger complex during replication similar to DNA replisome complexes. In vitro biochemical studies comparing the activity of ExoN from MHV and SARS-CoV and HCoV-229E together with the accessory proteins could shed mechanistic light on these phenotypic differences between the ExoN mutants.
      Replication fidelity is inherently tied to viral fitness and, in most cases, changes to replication fidelity decrease fitness (reviewed in Ref.
      • Smith E.C.
      • Sexton N.R.
      • Denison M.R.
      Thinking outside the triangle: replication fidelity of the largest RNA viruses.
      ). This suggests that mutants with altered replication fidelity (such as the ExoN mutant) have potential therapeutic value as live attenuated vaccines (
      • Graham R.L.
      • Becker M.M.
      • Eckerle L.D.
      • Bolles M.
      • Denison M.R.
      • Baric R.S.
      A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease.
      ). Indeed, the SARS-CoV ExoN mutant had decreased pathogenesis and did not revert to virulence even after persistent infection in vivo (
      • Graham R.L.
      • Becker M.M.
      • Eckerle L.D.
      • Bolles M.
      • Denison M.R.
      • Baric R.S.
      A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease.
      ). The ExoN mutation did not revert to WT even over 250 viral passages, although it accumulated a variety of mutations that partially compensated for the replication defect and decreased the population sensitivity to mutagens (
      • Graepel K.W.
      • Lu X.
      • Case J.B.
      • Sexton N.R.
      • Smith E.C.
      • Denison M.R.
      Proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations.
      ). Several components of the replicase complex, including nsp8, nsp9, nsp12, and nsp13, had mutations in the coding region, underscoring the complexity and interdependence of the RTC and how that helps the virus circumvent the consequences of decreased fidelity. A better understanding of the mechanism of replication fidelity will also allow for the exploration of mutants that increase replication fidelity and thereby reduce diversity and potentially fitness of the population, as has been shown for polioviruses (
      • Vignuzzi M.
      • Wendt E.
      • Andino R.
      Engineering attenuated virus vaccines by controlling replication fidelity.
      ).
      Recombination, which is generally high in RNA viruses and is linked to their virulence and pathogenicity (
      • Xiao Y.
      • Rouzine I.M.
      • Bianco S.
      • Acevedo A.
      • Goldstein E.F.
      • Farkov M.
      • Brodsky L.
      • Andino R.
      RNA recombination enhances adaptability and is required for virus spread and virulence.
      ), may also influence coronavirus diversity. In coronaviruses, recombination occurs as an inherent part of the replication cycle during the synthesis of sg RNAs and is tied to the ability of the RdRp to switch templates from the TRS-B sequence to the TRS-L sequence to copy the leader sequence from the 5′ end of the genome. Such recombination events can also occur between co-infecting coronaviruses with different genotypes (reviewed in Ref.
      • Su S.
      • Wong G.
      • Shi W.
      • Liu J.
      • Lai A.C.K.
      • Zhou J.
      • Liu W.
      • Bi Y.
      • Gao G.F.
      Epidemiology, genetic recombination, and pathogenesis of coronaviruses.
      ). Recombination can lead to defective copies of RNA that can no longer be replicated (
      • Nagy P.D.
      • Simon A.E.
      New insights into the mechanisms of RNA recombination.
      ) or recombinants with new properties, such as the ability to replicate in a new host (
      • Su S.
      • Wong G.
      • Shi W.
      • Liu J.
      • Lai A.C.K.
      • Zhou J.
      • Liu W.
      • Bi Y.
      • Gao G.F.
      Epidemiology, genetic recombination, and pathogenesis of coronaviruses.
      ), leading to new outbreaks. Mutational reversion and recombination-driven processes can pose significant challenges to the use of live attenuated vaccines (
      • Vignuzzi M.
      • Wendt E.
      • Andino R.
      Engineering attenuated virus vaccines by controlling replication fidelity.
      ), emphasizing the need to engineer recombination-resistant strains (
      • Graham R.L.
      • Deming D.J.
      • Deming M.E.
      • Yount B.L.
      • Baric R.S.
      Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform.
      ). A recent study suggests the involvement of nsp14-ExoN in mediating recombination frequency and junction site selection in several coronaviruses (
      • Gribble J.
      • Pruijssers A.J.
      • Agostini M.L.
      • Anderson-Daniels J.
      • Chappell J.D.
      • Lu X.
      • Stevens L.J.
      • Routh A.L.
      • Denison M.R.
      The coronavirus proofreading exoribonuclease mediates extensive viral recombination.
      ), opening up an exciting avenue of exploration for nsp14 in vaccine development.

      Viral RNA processing

      Capping the 5′ end of the viral mRNA is important for viral mRNA stability, translation initiation, and escape from the cellular innate immune system (
      • Hyde J.L.
      • Diamond M.S.
      Innate immune restriction and antagonism of viral RNA lacking 2′-O methylation.
      ). Capping typically occurs co-transcriptionally in the nucleus, so RNA viruses that replicate in the cytoplasm encode their own enzymes or incorporate other strategies, such as cap snatching (as in bunyaviruses) (
      • Cheng E.
      • Mir M.A.
      Signatures of host mRNA 5′ terminus for efficient hantavirus cap snatching.
      ), to protect the 5′ end of their RNAs. The coronavirus capping mechanism is not completely understood, although it appears to follow the canonical capping pathway. Capping begins with hydrolysis of the γ-phosphate of the 5′ end nucleotide; although not yet directly shown, this is thought to be mediated by the nucleotide triphosphatase activity of nsp13-helicase (
      • Ivanov K.A.
      • Ziebuhr J.
      Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5′-triphosphatase activities.
      ). This is followed by the addition of a guanosine monophosphate to the diphosphate RNA by a guanylyl transferase that has remained elusive in coronaviruses, although the NiRAN domain of nsp12 could be involved in this process (
      • Lehmann K.C.
      • Gulyaeva A.
      • Zevenhoven-Dobbe J.C.
      • Janssen G.M.C.
      • Ruben M.
      • Overkleeft H.S.
      • van Veelen P.A.
      • Samborskiy D.V.
      • Kravchenko A.A.
      • Leontovich A.M.
      • Sidorov I.A.
      • Snijder E.J.
      • Posthuma C.C.
      • Gorbalenya A.E.
      Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses.
      ). The guanosine is then methylated at the N7 position, likely by N7-methyltransferase (MTase) activity that resides in the C-terminal part of ExoN (nsp14) (
      • Chen Y.
      • Cai H.
      • Pan J.
      • Xiang N.
      • Tien P.
      • Ahola T.
      • Guo D.
      Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase.
      ). Finally, nsp16 is thought to methylate the first and second nucleotides at the 2′-O position (
      • Bouvet M.
      • Debarnot C.
      • Imbert I.
      • Selisko B.
      • Snijder E.J.
      • Canard B.
      • Decroly E.
      In vitro reconstitution of SARS-coronavirus mRNA cap methylation.
      ). This activity requires interaction with nsp10, which appears to improve substrate and RNA binding by nsp16 (
      • Decroly E.
      • Debarnot C.
      • Ferron F.
      • Bouvet M.
      • Coutard B.
      • Imbert I.
      • Gluais L.
      • Papageorgiou N.
      • Sharff A.
      • Bricogne G.
      • Ortiz-Lombardia M.
      • Lescar J.
      • Canard B.
      Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-O-methyltransferase nsp10/nsp16 complex.
      ). The 2′-O-methylation is important for evasion of the type-I interferon (IFN) response (which is discussed below) (
      • Züst R.
      • Cervantes-Barragan L.
      • Habjan M.
      • Maier R.
      • Neuman B.W.
      • Ziebuhr J.
      • Szretter K.J.
      • Baker S.C.
      • Barchet W.
      • Diamond M.S.
      • Siddell S.G.
      • Ludewig B.
      • Thiel V.
      Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.
      ). Of the enzymes involved in capping, the N7-MTase of nsp14 is an attractive antiviral target, as this domain exhibits a noncanonical MTase fold different from cellular MTases (
      • Chen Y.
      • Cai H.
      • Pan J.
      • Xiang N.
      • Tien P.
      • Ahola T.
      • Guo D.
      Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase.
      ).
      The 3′ end of coronavirus mRNAs are polyadenylated. The length of the polyadenylated tail regulates translation efficiency of the mRNAs (
      • Wu H.Y.
      • Ke T.-Y.
      • Liao W.-Y.
      • Chang N.-Y.
      Regulation of coronaviral poly(A) tail length during infection.
      ) and is essential for negative-strand synthesis (
      • Lin Y.J.
      • Liao C.L.
      • Lai M.M.
      Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription.
      ). Whereas polyadenylation-related elements, such as a AGUAAA hexamer and the poly(A) tail, work in concert to ensure polyadenylation of the genome (
      • Peng Y.-H.
      • Lin C.-H.
      • Lin C.N.
      • Lo C.Y.
      • Tsai T.L.
      • Wu H.Y.
      Characterization of the role of hexamer AGUAAA and poly(A) tail in coronavirus polyadenylation.
      ), the precise mechanism by which this occurs is not known. It is also unclear whether the RdRp carries out the polyadenylation or if cellular poly(A) polymerases are recruited for this process.
      Given that translation of coronavirus mRNAs relies on host cap–dependent translation machinery, a number of cellular cap-binding complex factors are candidates for therapeutic targeting (
      • Müller C.
      • Schulte F.W.
      • Lange-Grünweller K.
      • Obermann W.
      • Madhugiri R.
      • Pleschka S.
      • Ziebuhr J.
      • Hartmann R.K.
      • Grünweller A.
      Broad-spectrum antiviral activity of the eIF4A inhibitor silvestrol against corona- and picornaviruses.
      ,
      • Cencic R.
      • Desforges M.
      • Hall D.R.
      • Kozakov D.
      • Du Y.
      • Min J.
      • Dingledine R.
      • Fu H.
      • Vajda S.
      • Talbot P.J.
      • Pelletier J.
      Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication.
      ). Systematic mapping of the interaction between SARS-CoV-2 proteins and the host proteome has revealed interactions between viral proteins and host translation machinery, and an inhibitor of cap-dependent translation initiation reduced viral infectivity in cell culture (
      • Gordon D.E.
      • Jang G.M.
      • Bouhaddou M.
      • Xu J.
      • Obernier K.
      • White K.M.
      • O'Meara M.J.
      • Rezelj V.V.
      • Guo J.Z.
      • Swaney D.L.
      • Tummino T.A.
      • Hüttenhain R.
      • Kaake R.M.
      • Richards A.L.
      • Tutuncuoglu B.
      • et al.
      A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.
      ). These data point to the possible effectiveness of a host-directed antiviral therapeutic strategy in treating COVID-19.

      Replication/transcription complex proteins as drug targets

      Whereas the complexity of the coronavirus replisome may have enabled the virus to expand its genome, it also presents numerous targets for the development of antivirals (
      • Subissi L.
      • Imbert I.
      • Ferron F.
      • Collet A.
      • Coutard B.
      • Decroly E.
      • Canard B.
      SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.
      ). Most prominent is the RdRp, as it is essential for the virus and lacks homologs in the host. Nucleoside analogs, which are nucleotide triphosphate (NTP) mimics, are commonly used RdRp inhibitors (
      • Pruijssers A.J.
      • Denison M.R.
      Nucleoside analogues for the treatment of coronavirus infections.
      ). However, designing nucleoside analogs as inhibitors is particularly challenging for coronaviruses due to the presence of the exonuclease, which can exise incorporated analogs and thus provide resistance. An exception to this has been the adenosine analog remedesivir, which is currently in phase 3 clinical trials for treating coronavirus infections (
      • Agostini M.L.
      • Andres E.L.
      • Sims A.C.
      • Graham R.L.
      • Sheahan T.P.
      • Lu X.
      • Smith E.C.
      • Case J.B.
      • Feng J.Y.
      • Jordan R.
      • Ray A.S.
      • Cihlar T.
      • Siegel D.
      • Mackman R.L.
      • Clarke M.O.
      • et al.
      Coronavirus susceptibility to the antiviral remdesivir (GS-5734) is mediated by the viral polymerase and the proofreading exoribonuclease.
      ). A recent in vitro study with purified RdRp-nsp8 complex from several coronaviruses showed that remedesivir incorporation blocks chain elongation 3 nucleotides downstream of its incorporation site, which potentially protects it from ExoN cleavage (
      • Gordon C.J.
      • Tchesnokov E.P.
      • Feng J.Y.
      • Porter D.P.
      • Götte M.
      The antiviral compound remdesivir potently inhibits RNA-dependent RNA polymerase from Middle East respiratory syndrome coronavirus.
      ,
      • Gordon C.J.
      • Tchesnokov E.P.
      • Woolner E.
      • Perry J.K.
      • Feng J.Y.
      • Porter D.P.
      • Gotte M.
      Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency.
      ). Additionally, remedesivir is selectively incorporated by the RdRp over the natural substrate ATP. Better in vitro reconstitution systems incorporating the other components of the RTC (nsp7-, nsp13-, and nsp14-exonuclease) will further help to elucidate the mechanism of inhibition.
      It may also be of interest to develop nonnucleoside RdRp inhibitors, as have been developed for other RNA viruses, such as hepatitis C virus (
      • Cannalire R.
      • Tarantino D.
      • Astolfi A.
      • Barreca M.L.
      • Sabatini S.
      • Massari S.
      • Tabarrini O.
      • Milani M.
      • Querat G.
      • Mastrangelo E.
      • Manfroni G.
      • Cecchetti V.
      Functionalized 2,1-benzothiazine 2,2-dioxides as new inhibitors of Dengue NS5 RNA-dependent RNA polymerase.
      ). Nonnucleoside inhibitors typically function allosterically and hence are potentially immune to the resistance conferred by the exonuclease activity of ExoN. Combining compounds that inhibit ExoN together with nucleoside analogs to inhibit the RdRp or using small molecules that increase the mutation load of the virus by other mechanisms that are not sensitive to the exonuclease are other viable options (
      • Agostini M.L.
      • Pruijssers A.J.
      • Chappell J.D.
      • Gribble J.
      • Lu X.
      • Andres E.L.
      • Bluemling G.R.
      • Lockwood M.A.
      • Sheahan T.P.
      • Sims A.C.
      • Natchus M.G.
      • Saindane M.
      • Kolykhalov A.A.
      • Painter G.R.
      • Baric R.S.
      • et al.
      Small-molecule antiviral β-d-N4-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance.
      ). Finally, other components of the RTC, such as the helicase (
      • Adedeji A.O.
      • Singh K.
      • Calcaterra N.E.
      • DeDiego M.L.
      • Enjuanes L.
      • Weiss S.
      • Sarafianos S.G.
      Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.
      ), exonuclease (
      • Smith E.C.
      • Blanc H.
      • Surdel M.C.
      • Vignuzzi M.
      • Denison M.R.
      Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.
      ), and capping machinery (
      • Decroly E.
      • Debarnot C.
      • Ferron F.
      • Bouvet M.
      • Coutard B.
      • Imbert I.
      • Gluais L.
      • Papageorgiou N.
      • Sharff A.
      • Bricogne G.
      • Ortiz-Lombardia M.
      • Lescar J.
      • Canard B.
      Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-O-methyltransferase nsp10/nsp16 complex.
      ,
      • Ferron F.
      • Decroly E.
      • Selisko B.
      • Canard B.
      The viral RNA capping machinery as a target for antiviral drugs.
      ), have also been considered as potential druggable targets.

      Coronavirus replication occurs within heavily modified membranes

      A defining feature of many positive-strand RNA viruses, including CoVs, is their ability to hijack and reform intracellular membranes to create a cellular niche for the replication of their RNA genome. Ultrastructural characterization of mainly MHV– and SARS-CoV–infected cells has revealed the membranes that anchor RTCs in CoV-infected cells to be quite striking, consisting of double membrane vesicles (DMVs) among other intricate convoluted membrane structures that isolate CoV RNA from the rest of the cellular environment (Fig. 6) (
      • Gosert R.
      • Kanjanahaluethai A.
      • Egger D.
      • Bienz K.
      • Baker S.C.
      RNA replication of mouse hepatitis virus takes place at double-membrane vesicles.
      ,
      • Goldsmith C.S.
      • Tatti K.M.
      • Ksiazek T.G.
      • Rollin P.E.
      • Comer J.A.
      • Lee W.W.
      • Rota P.A.
      • Bankamp B.
      • Bellini W.J.
      • Zaki S.R.
      Ultrastructural characterization of SARS coronavirus.
      ,
      • Snijder E.J.
      • van der Meer Y.
      • Zevenhoven-Dobbe J.
      • Onderwater J.J.M.
      • van der Meulen J.
      • Koerten H.K.
      • Mommaas A.M.
      Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex.
      ,
      • Ulasli M.
      • Verheije M.H.
      • de Haan C.A.M.
      • Reggiori F.
      Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus.
      ). Conceptually, RTC formation leads to the concentration of viral replication machinery, spatially separating the sites of viral RNA replication from downstream virion assembly in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). Additionally, RTCs likely prevent detection of viral dsRNA replication products from innate immune sensors.
      Figure thumbnail gr6
      Figure 6Diagram of convoluted membranes/double membrane vesicles. Coronavirus infection leads to ER membrane modification as RTCs are formed. Nsp3 and nsp4 are co-translationally embedded in the ER membrane and interact via their luminal loops. This leads to “zippering” of ER membranes and induced curvature (1). These interactions yield a complex array of convoluted membranes (CM) and DMVs that are contiguous with the rough ER (2). The protein components of RTCs are mainly localized to the convoluted membranes. The DMVs contain dsRNA, thought to be sequestered replication intermediates. The DMV inner membrane has no ribosomes, connections to the cytoplasm or connections to the rest of the network. The mechanism of DMV formation and the exact site of CoV RNA replication within this membrane network are currently unknown. See section, ‘Coronavirus replication occurs within heavily modified membranes’ for references.
      The DMVs and convoluted membranes in CoV-infected cells form at the nuclear periphery and are derived from host ER membrane (
      • Knoops K.
      • Kikkert M.
      • Worm S.H.E.V.D.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Koster A.J.
      • Mommaas A.M.
      • Snijder E.J.
      SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.
      ). The majority of the membrane manipulation is carried out by three nonstructural proteins with integral transmembrane domains: nsp3, nsp4, and nsp6 (
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ). Although biochemical characterization of these proteins is hindered by their hydrophobic nature, protein-protein interaction studies performed in cells suggest that nsp3, nsp4, and nsp6 can oligomerize and form complexes through their luminal loops (
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ,
      • Hagemeijer M.C.
      • Ulasli M.
      • Vonk A.M.
      • Reggiori F.
      • Rottier P.J.M.
      • de Haan C.A.M.
      Mobility and interactions of coronavirus nonstructural protein 4.
      ,
      • Hagemeijer M.C.
      • Monastyrska I.
      • Griffith J.
      • van der Sluijs P.
      • Voortman J.
      • van Bergen En Henegouwen P.M.
      • Vonk A.M.
      • Rottier P.J.M.
      • Reggiori F.
      • de Haan C.A.M.
      Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.
      ). Expression of these RTC proteins individually in uninfected cells is sufficient to cause membrane proliferation and various perturbations of membrane morphology (
      • Hagemeijer M.C.
      • Monastyrska I.
      • Griffith J.
      • van der Sluijs P.
      • Voortman J.
      • van Bergen En Henegouwen P.M.
      • Vonk A.M.
      • Rottier P.J.M.
      • Reggiori F.
      • de Haan C.A.M.
      Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.
      ). Co-expression of nsp3 and nsp4 leads to their colocalization in perinuclear foci by fluorescence microscopy and the formation of membrane structures with increased curvature by EM (
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ). Because the specific interaction of nsp3 and nsp4 is required for these structures to form, it is hypothesized that nsp3 and nsp4 rearrange membranes and introduce curvature by a “zipper” mechanism, essentially bringing ER membranes together through nsp3/4 interactions (
      • Hagemeijer M.C.
      • Monastyrska I.
      • Griffith J.
      • van der Sluijs P.
      • Voortman J.
      • van Bergen En Henegouwen P.M.
      • Vonk A.M.
      • Rottier P.J.M.
      • Reggiori F.
      • de Haan C.A.M.
      Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.
      ) (Fig. 6). The nsp3/4 interaction also recruits other proteins, including nsp6, to anchor RTCs. Finally, triple transfection of nsp3, nsp4, and nsp6 together results in the formation of DMVs in uninfected cells (
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ). Due to the importance of membrane modification during viral replication, CoV transmembrane proteins may be attractive drug targets. In fact, a small molecule screen for antiviral activity yielded a compound that targets the transmembrane protein nsp6 and essentially blocks viral RNA replication and DMV formation (
      • Lundin A.
      • Dijkman R.
      • Bergström T.
      • Kann N.
      • Adamiak B.
      • Hannoun C.
      • Kindler E.
      • Jónsdóttir H.R.
      • Muth D.
      • Kint J.
      • Forlenza M.
      • Müller M.A.
      • Drosten C.
      • Thiel V.
      • Trybala E.
      Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.
      ).
      During CoV infection, the inner membrane of the DMV is sealed while the outer membrane of the DMVs forms a contiguous network with the convoluted membranes and modified ER membranes (Fig. 6). When this network is isolated from cells, it is capable of producing both genomic and subgenomic RNAs in vitro even in the presence of RNases and proteases, but not detergent, thus implicating the membrane network in shielding viral RNA replication (
      • van Hemert M.J.
      • van den Worm S.H.E.
      • Knoops K.
      • Mommaas A.M.
      • Gorbalenya A.E.
      • Snijder E.J.
      SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.
      ). The anchored RTC complexes consist of viral proteins nsp2–10, nsp12–16, and N protein, which have diverse enzymatic functions required for RNA replication as discussed above (
      • Ulasli M.
      • Verheije M.H.
      • de Haan C.A.M.
      • Reggiori F.
      Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus.
      ,
      • Denison M.R.
      • Spaan W.J.
      • van der Meer Y.
      • Gibson C.A.
      • Sims A.C.
      • Prentice E.
      • Lu X.T.
      The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis.
      ,
      • V'kovski P.
      • Gerber M.
      • Kelly J.
      • Pfaender S.
      • Ebert N.
      • Braga Lagache S.
      • Simillion C.
      • Portmann J.
      • Stalder H.
      • Gaschen V.
      • Bruggmann R.
      • Stoffel M.H.
      • Heller M.
      • Dijkman R.
      • Thiel V.
      Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling.
      ). The RTC microenvironment also includes numerous host proteins that participate in CoV biology, such as proteins involved in vesicular trafficking and translation initiation factors, the latter of which are suggestive of active translation near sites of viral RNA replication (
      • V'kovski P.
      • Gerber M.
      • Kelly J.
      • Pfaender S.
      • Ebert N.
      • Braga Lagache S.
      • Simillion C.
      • Portmann J.
      • Stalder H.
      • Gaschen V.
      • Bruggmann R.
      • Stoffel M.H.
      • Heller M.
      • Dijkman R.
      • Thiel V.
      Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling.
      ). The site of RNA replication inside this membrane network is currently unknown. Whereas viral RTC proteins labeled by immuno-EM primarily localize to convoluted membranes between DMVs, dsRNA (presumed to be of viral origin) labeled by the J2 antibody localizes inside the DMVs (Fig. 6). However, there is no experimental evidence demonstrating whether dsRNAs inside the DMV represent nascent viral transcripts, viral RNA replication byproducts, or even host dsRNAs. Recently, nascent viral RNA was visualized by metabolic labeling and quantitative EM autoradiography, revealing that viral transcription does in fact occur in association with the DMVs rather than convoluted membranes (
      • Snijder E.J.
      • Limpens R.W.A.L.
      • de Wilde A.H.
      • de Jong A.W.M.
      • Zevenhoven-Dobbe J.C.
      • Maier H.J.
      • Faas F.F.G.A.
      • Koster A.J.
      • Bárcena M.
      A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis.
      ). The spatial resolution of this technique, while clearly demonstrating viral transcription within the vicinity of the DMVs, was not sufficient to pinpoint the localization of nascent viral RNA within DMVs and/or in association with DMV membranes. Because no visible pores or openings in the inner membrane of the DMV have been detected with conventional EM techniques, viral RNA synthesis regardless of locale would rely on a yet unidentified transport mechanism capable of moving viral proteins and/or RNA in and out of the DMV inner membrane (
      • Knoops K.
      • Kikkert M.
      • Worm S.H.E.V.D.
      • Zevenhoven-Dobbe J.C.
      • van der Meer Y.
      • Koster A.J.
      • Mommaas A.M.
      • Snijder E.J.
      SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.
      ,
      • Snijder E.J.
      • Limpens R.W.A.L.
      • de Wilde A.H.
      • de Jong A.W.M.
      • Zevenhoven-Dobbe J.C.
      • Maier H.J.
      • Faas F.F.G.A.
      • Koster A.J.
      • Bárcena M.
      A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis.
      ).

      Viral packaging and egress

      The assembly of an infectious CoV virion requires that its nucleocapsid, consisting of the viral RNA genome coated with N protein, and viral envelope coalesce into the same intracellular space. Viral glycoproteins that are incorporated into the envelope (M, E, and S proteins) are translated in the ER and retained at the site of budding in the ERGIC (Fig. 1). The ERGIC budding site is distinct from the site of viral genome synthesis in the RTC. The nucleocapsid core of the virion traffics from the RTC to ultimately bud into ERGIC membranes, which are decorated with M, E, and S protein and become the lipid envelope of the virion. The most abundant envelope component is the M protein, which plays a central role in viral egress. Outside of the context of infection, M protein expression alone is not sufficient to cause budding of virus-like particles, but co-expression with E (or N in the case of SARS-CoV) can result in virus-like particle formation in the absence of infection (
      • Bos E.C.
      • Luytjes W.
      • van der Meulen H.V.
      • Koerten H.K.
      • Spaan W.J.
      The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus.
      ,
      • Vennema H.
      • Godeke G.J.
      • Rossen J.W.
      • Voorhout W.F.
      • Horzinek M.C.
      • Opstelten D.J.
      • Rottier P.J.
      Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes.
      ,
      • Huang Y.
      • Yang Z.-Y.
      • Kong W.-P.
      • Nabel G.J.
      Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production.
      ). During infection, the M protein nucleates virion components within the ERGIC budding compartment, as M directly interacts with the virion proteins E, N, and S and the CoV genomic RNA (
      • Kuo L.
      • Masters P.S.
      Functional analysis of the murine coronavirus genomic RNA packaging signal.
      ,
      • Narayanan K.
      • Maeda A.
      • Maeda J.
      • Makino S.
      Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells.
      ,
      • Nguyen V.P.
      • Hogue B.G.
      Protein interactions during coronavirus assembly.
      ,
      • Opstelten D.J.
      • Raamsman M.J.
      • Wolfs K.
      • Horzinek M.C.
      • Rottier P.J.
      Envelope glycoprotein interactions in coronavirus assembly.
      ). The E protein, while not highly abundant in the envelope, is critical for viral envelope curvature and maturation and can form membrane ion channels, although the significance of this latter activity is not yet appreciated (
      • Wilson L.
      • Mckinlay C.
      • Gage P.
      • Ewart G.
      SARS coronavirus E protein forms cation-selective ion channels.
      ,
      • Venkatagopalan P.
      • Daskalova S.M.
      • Lopez L.A.
      • Dolezal K.A.
      • Hogue B.G.
      Coronavirus envelope (E) protein remains at the site of assembly.
      ). S protein assembly into virions is enhanced by C-terminal dilysine, dibasic, or tyrosine-based endoplasmic reticulum retention signals (
      • Lontok E.
      • Corse E.
      • Machamer C.E.
      Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site.
      ,
      • Schwegmann-Wessels C.
      • Al-Falah M.
      • Escors D.
      • Wang Z.
      • Zimmer G.
      • Deng H.
      • Enjuanes L.
      • Naim H.Y.
      • Herrler G.
      A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus.
      ,
      • McBride C.E.
      • Li J.
      • Machamer C.E.
      The cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and promotes interaction with membrane protein.
      ). Although the retention signals are quite divergent among CoVs, all serve to maintain S near the ERGIC-localized M protein, ensuring M-S interaction at the site of virion assembly. Following budding of the nucleocapsid core into the M-, E-, and S-containing ERGIC membranes, the newly enveloped virion then leaves the cell through the exocytic pathway.
      Although CoV replication produces an abundance of unique viral RNAs in the cell (positive-strand genomic RNAs, positive-strand sg mRNAs, and negative-strand RNAs), purified CoV virions house mainly full genome-length RNA (
      • Kuo L.
      • Masters P.S.
      Functional analysis of the murine coronavirus genomic RNA packaging signal.
      ,
      • Makino S.
      • Yokomori K.
      • Lai M.M.
      Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal.
      ,
      • Escors D.
      • Izeta A.
      • Capiscol C.
      • Enjuanes L.
      Transmissible gastroenteritis coronavirus packaging signal is located at the 5′ end of the virus genome.
      ). Conceptually, this specificity is thought to be driven by a packaging signal unique to the genome-length RNA. In MHV, a packaging signal has been mapped to ORF1b within the nsp15 gene (a region absent in sg RNAs) and is predicted to form a bulged stem-loop structure with repeating AGC/GUAAU motifs (
      • Fosmire J.A.
      • Hwang K.
      • Makino S.
      Identification and characterization of a coronavirus packaging signal.
      ,
      • Chen S.-C.
      • van den Born E.
      • van den Worm S.H.E.
      • Pleij C.W.A.
      • Snijder E.J.
      • Olsthoorn R.C.L.
      New structure model for the packaging signal in the genome of group IIa coronaviruses.
      ). This packaging signal specifically binds both the N and M proteins, but the order in which these interactions occur is not clear (reviewed in Ref.
      • Masters P.S.
      Coronavirus genomic RNA packaging.
      ). N protein must have broad RNA-binding activity, as it ultimately coats the length of the viral genome to form the nucleocapsid component of the virion and additionally forms complexes with sg RNAs (
      • Narayanan K.
      • Maeda A.
      • Maeda J.
      • Makino S.
      Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells.
      ,
      • Baric R.S.
      • Nelson G.W.
      • Fleming J.O.
      • Deans R.J.
      • Keck J.G.
      • Casteel N.
      • Stohlman S.A.
      Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription.
      ,
      • Cologna R.
      • Spagnolo J.F.
      • Hogue B.G.
      Identification of nucleocapsid binding sites within coronavirus-defective genomes.
      ). Thus, an additional role of M in recognizing the packaging signal and selecting full-length genomic RNA is an attractive model for genome packaging specificity, at least in the context of MHV infection (
      • Narayanan K.
      • Chen C.-J.
      • Maeda J.
      • Makino S.
      Nucleocapsid-independent specific viral RNA packaging via viral envelope protein and viral RNA signal.
      ). In contrast, the packaging signal identified in MHV is absent from other lineages of β-coronaviruses, including SARS-CoV and MERS (reviewed in Ref.
      • Masters P.S.
      Coronavirus genomic RNA packaging.
      ), leaving us with little understanding of how other CoVs selectively package genome-length RNAs.

      Part II: Viral manipulation of the host

      Viruses depend on host processes to complete their life cycle. In addition to employing cellular machines like the ribosome to translate their proteins and manipulating cellular membranes during RNA synthesis and viral morphogenesis, several coronavirus proteins modify the cellular environment in ways that may influence viral pathogenesis and replication in vivo. In this section, we discuss the roles of coronavirus proteins in altering the cellular signaling landscape as well as the ability of the virus to modulate host gene expression and its interactions with and counteraction of the host immune response.

      Accessory proteins and viral pathogenicity

      Coronavirus genomes contain a number of genes concentrated in the 3′ region of the genome that encode for accessory proteins that are largely dispensable for viral replication and growth in vitro (
      • de Haan C.A.M.
      • Masters P.S.
      • Shen X.
      • Weiss S.
      • Rottier P.J.M.
      The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host.
      ,
      • Haijema B.J.
      • Volders H.
      • Rottier P.J.M.
      Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis.
      ,
      • Yount B.
      • Roberts R.S.
      • Sims A.C.
      • Deming D.
      • Frieman M.B.
      • Sparks J.
      • Denison M.R.
      • Davis N.
      • Baric R.S.
      Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice.
      ,
      • Ontiveros E.
      • Kuo L.
      • Masters P.
      • Perlman S.
      Analysis of nonessential gene function in recombinant MHV-JHM. Gene 4 knockout recombinant virus.
      ,
      • Shen S.
      • Wen Z.L.
      • Liu D.X.
      Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication.
      ,
      • Hodgson T.
      • Britton P.
      • Cavanagh D.
      Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.
      ,
      • Casais R.
      • Davies M.
      • Cavanagh D.
      • Britton P.
      Gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication.
      ). The SARS-CoV genome encodes for eight accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b), which are the best-studied set of accessory proteins among β-coronaviruses (
      • Marra M.A.
      • Jones S.J.M.
      • Astell C.R.
      • Holt R.A.
      • Brooks-Wilson A.
      • Butterfield Y.S.N.
      • Khattra J.
      • Asano J.K.
      • Barber S.A.
      • Chan S.Y.
      • Cloutier A.
      • Coughlin S.M.
      • Freeman D.
      • Girn N.
      • Griffith O.L.
      • et al.
      The genome sequence of the SARS-associated coronavirus.
      ,
      • Rota P.A.
      • Oberste M.S.
      • Monroe S.S.
      • Nix W.A.
      • Campagnoli R.
      • Icenogle J.P.
      • Peñaranda S.
      • Bankamp B.
      • Maher K.
      • Chen M.-H.
      • Tong S.
      • Tamin A.
      • Lowe L.
      • Frace M.
      • DeRisi J.L.
      • et al.
      Characterization of a novel coronavirus associated with severe acute respiratory syndrome.
      ). Accessory proteins are specific to each CoV genus and exhibit little homology across the family; as such, this set of eight proteins are specific to human and animal isolates of SARS-CoV (
      • Lai M.M.
      • Cavanagh D.
      The molecular biology of coronaviruses.
      ). Additionally, no significant amino acid sequence similarity is shared between SARS-CoV accessory proteins and other known viral or cellular proteins, providing little insight to predict functional roles (
      • Liu D.X.
      • Fung T.S.
      • Chong K.K.-L.
      • Shukla A.
      • Hilgenfeld R.
      Accessory proteins of SARS-CoV and other coronaviruses.
      ). Despite being nonessential for viral replication in cultured cells, the accessory proteins presumably modulate virus-host interactions that are important during in vivo infection, including cell proliferation, programmed cell death, pro-inflammatory cytokine production, and IFN signaling (see Table 1) (
      • Liu D.X.
      • Fung T.S.
      • Chong K.K.-L.
      • Shukla A.
      • Hilgenfeld R.
      Accessory proteins of SARS-CoV and other coronaviruses.
      ,
      • Narayanan K.
      • Huang C.
      • Makino S.
      SARS coronavirus accessory proteins.
      ). Many SARS-CoV accessory proteins can also be incorporated into virions or virus-like particles during infection, potentially suggesting minor structural roles (
      • Narayanan K.
      • Huang C.
      • Makino S.
      SARS coronavirus accessory proteins.
      ).
      Table 1SARS-CoV proteins and their functions with SARS-CoV-2 variations
      NameFunctionsSARS-CoV-2 variationsReferences
      I. Structural proteins
      SSpike protein, host cell receptor binding for viral entry, JNK ↑, ERK ↑, CCL2 ↑27 aa substitutions (6 in RBD, 6 in subdomain, and 4 in known peptide antigen for SARS-CoV); no aa substitutions in CoV-2 polybasic cleavage site compared with CoV consensus sequence; cleavage site is absent in SARS-CoV
      • Chen I.-Y.
      • Chang S.C.
      • Wu H.Y.
      • Yu T.-C.
      • Wei W.-C.
      • Lin S.
      • Chien C.-L.
      • Chang M.-F.
      Upregulation of the chemokine (C-C motif) ligand 2 via a severe acute respiratory syndrome coronavirus spike-ACE2 signaling pathway.
      ,
      • Delmas B.
      • Laude H.
      Assembly of coronavirus spike protein into trimers and its role in epitope expression.
      ,
      • Beniac D.R.
      • Andonov A.
      • Grudeski E.
      • Booth T.F.
      Architecture of the SARS coronavirus prefusion spike.
      EEnvelope protein, viral assembly and release, p38 MAPK ↑
      • Venkatagopalan P.
      • Daskalova S.M.
      • Lopez L.A.
      • Dolezal K.A.
      • Hogue B.G.
      Coronavirus envelope (E) protein remains at the site of assembly.
      ,
      • Jiménez-Guardeño J.M.
      • Nieto-Torres J.L.
      • DeDiego M.L.
      • Regla-Nava J.A.
      • Fernandez-Delgado R.
      • Castaño-Rodriguez C.
      • Enjuanes L.
      The PDZ-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis.
      ,
      • Nieto-Torres J.L.
      • DeDiego M.L.
      • Álvarez E.
      • Jiménez-Guardeño J.M.
      • Regla-Nava J.A.
      • Llorente M.
      • Kremer L.
      • Shuo S.
      • Enjuanes L.
      Subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein.
      • DeDiego M.L.
      • Álvarez E.
      • Almazán F.
      • Rejas M.T.
      • Lamirande E.
      • Roberts A.
      • Shieh W.-J.
      • Zaki S.R.
      • Subbarao K.
      • Enjuanes L.
      A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro in vivo.
      • Kuo L.
      • Masters P.S.
      The small envelope protein E is not essential for murine coronavirus replication.
      MMembrane protein, virion shape, membrane curvature
      • Neuman B.W.
      • Kiss G.
      • Kunding A.H.
      • Bhella D.
      • Baksh M.F.
      • Connelly S.
      • Droese B.
      • Klaus J.P.
      • Makino S.
      • Sawicki S.G.
      • Siddell S.G.
      • Stamou D.G.
      • Wilson I.A.
      • Kuhn P.
      • Buchmeier M.J.
      A structural analysis of M protein in coronavirus assembly and morphology.
      ,
      • Nal B.
      • Chan C.
      • Kien F.
      • Siu L.
      • Tse J.
      • Chu K.
      • Kam J.
      • Staropoli I.
      • Crescenzo-Chaigne B.
      • Escriou N.
      • van der Werf S.
      • Yuen K.-Y.
      • Altmeyer R.
      Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.
      NNucleocapsid protein, binding to RNA genome, genome tethering to RTCs, type I IFN production and signaling inhibition, viral suppressor of RNA silencing, AP-1 ↑, JNK ↑, p38 MAPK ↑5 aa substitutions
      • He R.
      • Leeson A.
      • Andonov A.
      • Li Y.
      • Bastien N.
      • Cao J.
      • Osiowy C.
      • Dobie F.
      • Cutts T.
      • Ballantine M.
      • Li X.
      Activation of AP-1 signal transduction pathway by SARS coronavirus nucleocapsid protein.
      ,
      • Surjit M.
      • Liu B.
      • Jameel S.
      • Chow V.T.K.
      • Lal S.K.
      The SARS coronavirus nucleocapsid protein induces actin reorganization and apoptosis in COS-1 cells in the absence of growth factors.
      ,
      • Chang C.-K.
      • Sue S.-C.
      • Yu T.-H.
      • Hsieh C.-M.
      • Tsai C.-K.
      • Chiang Y.-C.
      • Lee S.-J.
      • Hsiao H.-H.
      • Wu W.-J.
      • Chang W.-L.
      • Lin C.-H.
      • Huang T.-H.
      Modular organization of SARS coronavirus nucleocapsid protein.
      • Cui L.
      • Wang H.
      • Ji Y.
      • Yang J.
      • Xu S.
      • Huang X.
      • Wang Z.
      • Qin L.
      • Tien P.
      • Zhou X.
      • Guo D.
      • Chen Y.
      The nucleocapsid protein of coronaviruses acts as a viral suppressor of RNA silencing in mammalian cells.
      • Hurst K.R.
      • Koetzner C.A.
      • Masters P.S.
      Identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein.
      II. Nonstructural proteins
      nsp1Cellular mRNA degradation and translation inhibition, type I IFN inhibition7 aa substitutions
      • Huang C.
      • Lokugamage K.G.
      • Rozovics J.M.
      • Narayanan K.
      • Semler B.L.
      • Makino S.
      SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.
      ,
      • Kamitani W.
      • Narayanan K.
      • Huang C.
      • Lokugamage K.
      • Ikegami T.
      • Ito N.
      • Kubo H.
      • Makino S.
      Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation.
      ,
      • Narayanan K.
      • Huang C.
      • Lokugamage K.
      • Kamitani W.
      • Ikegami T.
      • Tseng C.-T.K.
      • Makino S.
      Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells.
      ,
      • Wathelet M.G.
      • Orr M.
      • Frieman M.B.
      • Baric R.S.
      Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain.
      nsp2Unknown61 aa substitutions
      nsp3Papain-like protease, polypeptide cleaving, type I IFN production and signaling inhibition, IL-6 ↑102 aa substitutions
      • Lei J.
      • Kusov Y.
      • Hilgenfeld R.
      Nsp3 of coronaviruses: structures and functions of a large multi-domain protein.
      ,
      • Serrano P.
      • Johnson M.A.
      • Chatterjee A.
      • Neuman B.W.
      • Joseph J.S.
      • Buchmeier M.J.
      • Kuhn P.
      • Wüthrich K.
      Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3.
      nsp4DMV formation36 aa substitutions
      • Gadlage M.J.
      • Sparks J.S.
      • Beachboard D.C.
      • Cox R.G.
      • Doyle J.D.
      • Stobart C.C.
      • Denison M.R.
      Murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function.
      ,
      • Beachboard D.C.
      • Anderson-Daniels J.M.
      • Denison M.R.
      Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications.
      nsp53CLpro, polypeptides cleaving, type I IFN signaling inhibition5 aa substitutions
      • Stobart C.C.
      • Sexton N.R.
      • Munjal H.
      • Lu X.
      • Molland K.L.
      • Tomar S.
      • Mesecar A.D.
      • Denison M.R.
      Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.
      ,
      • Zhu X.
      • Wang D.
      • Zhou J.
      • Pan T.
      • Chen J.
      • Yang Y.
      • Lv M.
      • Ye X.
      • Peng G.
      • Fang L.
      • Xiao S.
      Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving STAT2.
      nsp6Restricting autophagosome expansion, DMV formation21 aa substitutions
      • Angelini M.M.
      • Akhlaghpour M.
      • Neuman B.W.
      • Buchmeier M.J.
      Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles.
      ,
      • Cottam E.M.
      • Whelband M.C.
      • Wileman T.
      Coronavirus NSP6 restricts autophagosome expansion.
      nsp7Processivity factor for RdRp, cofactor with nsp8 and nsp12
      • Subissi L.
      • Posthuma C.C.
      • Collet A.
      • Zevenhoven-Dobbe J.C.
      • Gorbalenya A.E.
      • Decroly E.
      • Snijder E.J.
      • Canard B.
      • Imbert I.
      One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      • Gao Y.
      • Yan L.
      • Huang Y.
      • Liu F.
      • Zhao Y.
      • Cao L.
      • Wang T.
      • Sun Q.
      • Ming Z.
      • Zhang L.
      • Ge J.
      • Zheng L.
      • Zhang Y.
      • Wang H.
      • Zhu Y.
      • et al.
      Structure of the RNA-dependent RNA polymerase from COVID-19 virus.
      nsp8Processivity factor for RdRp, cofactor with nsp7 and nsp124 aa substitutions
      • Subissi L.
      • Posthuma C.C.
      • Collet A.
      • Zevenhoven-Dobbe J.C.
      • Gorbalenya A.E.
      • Decroly E.
      • Snijder E.J.
      • Canard B.
      • Imbert I.
      One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.
      • Kirchdoerfer R.N.
      • Ward A.B.
      Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.
      • Gao Y.
      • Yan L.
      • Huang Y.
      • Liu F.
      • Zhao Y.
      • Cao L.
      • Wang T.
      • Sun Q.
      • Ming Z.
      • Zhang L.
      • Ge J.
      • Zheng L.
      • Zhang Y.
      • Wang H.
      • Zhu Y.
      • et al.
      Structure of the RNA-dependent RNA polymerase from COVID-19 virus.
      ,
      • Imbert I.
      • Guillemot J.-C.
      • Bourhis J.-M.
      • Bussetta C.
      • Coutard B.
      • Egloff M.-P.
      • Ferron F.
      • Gorbalenya A.E.
      • Canard B.
      A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus.
      ,
      • Velthuis, Te A.J.W.
      • van den Worm S.H.E.
      • Snijder E.J.
      The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
      nsp9Single-strand nucleic acid–binding protein1 aa substitution
      • Egloff M.-P.
      • Ferron F.
      • Campanacci V.
      • Longhi S.
      • Rancurel C.
      • Dutartre H.
      • Snijder E.J.
      • Gorbalenya A.E.
      • Cambillau C.
      • Canard B.
      The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world.
      ,
      • Sutton G.
      • Fry E.
      • Carter L.
      • Sainsbury S.
      • Walter T.
      • Nettleship J.
      • Berrow N.
      • Owens R.
      • Gilbert R.
      • Davidson A.
      • Siddell S.
      • Poon L.L.M.
      • Diprose J.
      • Alderton D.
      • Walsh M.
      • et al.
      The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.
      nsp10Catalytic activity of Nsp14-ExoN ↑, scaffold protein for nsp14 and nsp162 aa substitutions