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Amino Acid Substitutions in Gag Protein at Non-cleavage Sites Are Indispensable for the Development of a High Multitude of HIV-1 Resistance against Protease Inhibitors*

Open AccessPublished:December 10, 2001DOI:https://doi.org/10.1074/jbc.M108005200
      Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.
      Combination antiretroviral therapy using reverse transcriptase inhibitors and protease inhibitors (PIs)
      PIs
      protease inhibitors
      HIV-1
      human immunodeficiency virus type 1
      APV
      amprenavir
      CPE
      cytopathic effect
      PBMC
      peripheral blood mononuclear cell
      PBS
      phosphate-buffered saline
      CHRA
      competitive HIV-1 replication assay
      RT
      reverse transcriptase
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      CypA
      cyclophilin A
      MA
      matrix protein
      1PIs
      protease inhibitors
      HIV-1
      human immunodeficiency virus type 1
      APV
      amprenavir
      CPE
      cytopathic effect
      PBMC
      peripheral blood mononuclear cell
      PBS
      phosphate-buffered saline
      CHRA
      competitive HIV-1 replication assay
      RT
      reverse transcriptase
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      CypA
      cyclophilin A
      MA
      matrix protein
      produces substantial suppression of viral replication in HIV-1-infected patients. However, the emergence of drug-resistant HIV-1 variants in such patients has limited the efficacy of combination chemotherapy. HIV-1 variants resistant to any of the currently available antiretroviral therapeutics have emerged both in vitro and in vivo (
      • Mitsuya H.
      • Erickson J.
      ). In particular, HIV-1 resistant to one PI is often cross-resistant to another PI, presenting formidable challenges in the therapy of HIV-1 infection. Indeed, HIV-1 protease has been shown to tolerate extensive sequence variations, remaining functional even with as many as 15 amino acid substitutions accumulated in a molecule composed of 99 amino acids (
      • Yoshimura K.
      • Kato R.
      • Yusa K.
      • Kavlick M.F.
      • Maroun V.
      • Nguyen A.
      • Mimoto T.
      • Ueno T.
      • Shintani M.
      • Falloon J.
      • Masur H.
      • Hayashi H.
      • Erickson J.
      • Mitsuya H.
      ,
      • Erickson J.W.
      • Gulnik S.V.
      • Markowitz M.
      ).
      Amino acid substitutions in HIV-1 Gag precursor p7-p1 and p1-p6 cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy including PIs (
      • Doyon L.
      • Croteau G.
      • Thibeault D.
      • Poulin F.
      • Pilote L.
      • Lamarre D.
      ,
      • Zhang Y.M.
      • Imamichi H.
      • Imamichi T.
      • Lane H.C.
      • Falloon J.
      • Vasudevachari M.B.
      • Salzman N.P.
      ). Those substitutions are believed to compensate for the enzymatic impairment of protease per se resulting from the acquisition of PI resistance-conferring amino acid substitutions within the protease-encoding region of the HIV-1 genome. However, a number of highly PI-resistant HIV-1 variants lack such cleavage site amino acid substitutions. Furthermore, recombinant HIV-1 clones to which PI resistance-conferring substitutions in the protease-encoding region were introduced are known to often fail to propagate in vitro (
      • Rose R.E.
      • Gong Y.F.
      • Greytok J.A.
      • Bechtold C.M.
      • Terry B.J.
      • Robinson B.S.
      • Alam M.
      • Colonno R.J.
      • Lin P.F.
      ). Therefore, we thought that as yet unidentified amino acid substitutions in the Gag-Pol polyprotein, the substrate for the enzyme, compensate for the altered enzymatic function caused by the acquisition of amino acid substitutions in the viral protease. To this end, we generated PI-resistant HIV-1 variants by exposing HIV-1 to four different PIs including amprenavir (APV) (
      • Yoshimura K.
      • Kato R.
      • Kavlick M.F.
      • Nguyen A.
      • Maroun V.
      • Maeda K.
      • Hussain K.A.
      • Ghosh A.K.
      • Erickson J.
      • Mitsuya H.
      ), JE-2147 (
      • Yoshimura K.
      • Kato R.
      • Yusa K.
      • Kavlick M.F.
      • Maroun V.
      • Nguyen A.
      • Mimoto T.
      • Ueno T.
      • Shintani M.
      • Falloon J.
      • Masur H.
      • Hayashi H.
      • Erickson J.
      • Mitsuya H.
      ), KNI-272 (
      • Kageyama S.
      • Mimoto T.
      • Murakawa Y.
      • Nomizu M.
      • Ford H., Jr.
      • Shirasaka T.
      • Gulnik S.
      • Erickson J.
      • Takada K.
      • Hayashi H.
      • Broder S.
      • Kiso Y.
      • Mitsuya H.
      ), and UIC-94003 (
      • Yoshimura K.
      • Kato R.
      • Kavlick M.F.
      • Nguyen A.
      • Maroun V.
      • Maeda K.
      • Hussain K.A.
      • Ghosh A.K.
      • Erickson J.
      • Mitsuya H.
      ), identified up to 23 amino acid substitutions in Gag and protease, generated a variety of infectious HIV-1 clones containing such amino acid substitutions, and characterized their replication profiles. We conclude that Gag amino acid substitutions such as H219Q and R409K, located outside the cleavage sites, contribute to the development of HIV-1 resistance to PIs and are essential for the replication of HIV-1 variants in the presence of PIs.

      DISCUSSION

      The role and impact of amino acid substitutions in thegag gene of HIV-1 genome which emerge during therapy with protease inhibitors in patients with AIDS have been poorly understood. This is mainly due to the findings that most of the amino acid substitutions detected in the gag gene are not seen in common among clinical HIV-1 strains isolated from patients and even among HIV-1 clones generated from HIV-1 of a single patient. More problematic is that the functions and tertiary structures of HIV-1 Gag proteins remain largely to be determined. In the present study, we used a single HIV-1 clone obtained from newly transfected COS-7 cells, HIV-1NL4-3, as a starting HIV-1 strain and propagated it in the presence of increasing concentrations of four different PIs, APV, JE-2147, KNI-272, and UIC-94003 over 27–62 passages; and we studied 14 mutations identified in Gag (Table I), among which 9 mutations were seen in more than two PI-exposed HIV-1 preparations. We focused on six major Gag amino acid substitutions, L75R, H219Q, V390D/V390A, R409K, L449F, and E468K in the present study (Table I).
      When HIV-1 was passaged under APV pressure, L75R and H219Q mutations in the Gag protein emerged prior to the emergence of protease mutations, and we found that such Gag mutations were indispensable for the efficient replication of APV resistant HIV-1 (HIV-1AR). We thought that Gag polyprotein containing L75R or H219Q would be more sensitive to the cleavage by mutant protease because such a protease has impaired enzymatic activity due to the accumulation of amino acid substitutions within itself. Indeed, HIV-1AR whose mutantgag gene was substituted with a wild type gaggene (but with the mutant protease being intact) (HIV-1ARL75/H219/V390) failed to replicate (Fig. 2 A), in agreement with prior studies (
      • Rose R.E.
      • Gong Y.F.
      • Greytok J.A.
      • Bechtold C.M.
      • Terry B.J.
      • Robinson B.S.
      • Alam M.
      • Colonno R.J.
      • Lin P.F.
      ,
      • Yoshimura K.
      • Kato R.
      • Kavlick M.F.
      • Nguyen A.
      • Maroun V.
      • Maeda K.
      • Hussain K.A.
      • Ghosh A.K.
      • Erickson J.
      • Mitsuya H.
      ). However, Western blot analysis showed that the polyprotein cleavage pattern was comparable between the replication-incompetent clonal HIV-1 with wild type Gag (e.g.HIV-1ARL75/H219/V390) and the HIV-1 that recovered replication competence with amino acid substitutions in Gag (e.g. HIV-1ARL75R/H219Q/V390). It is possible that the difference in the Gag polyprotein processing is functionally significant but is not readily detected with Western blot analysis (
      • Kageyama S.
      • Mimoto T.
      • Murakawa Y.
      • Nomizu M.
      • Ford H., Jr.
      • Shirasaka T.
      • Gulnik S.
      • Erickson J.
      • Takada K.
      • Hayashi H.
      • Broder S.
      • Kiso Y.
      • Mitsuya H.
      ). It is also possible that such amino acid substitutions in Gag facilitate certain functions of Gag proteins such as assembly, packaging, and budding functions, and the recovery of polyprotein cleavage is not required.
      There are several lines of evidence suggesting that trimerized matrix proteins (MAs) serve as a fundamental building block for the formation of the MA shell within the mature HIV-1 (
      • Turner B.G.
      • Summers M.F.
      ). The x-ray structural analysis of HIV-1 MA suggests that residues Pro-66 and Gly-71, which are highly conserved, function as “hinges” that allow a structural reorientation of MA at the trimer interface and that Ser-72 and Leu-75 have a tight interaction, and when the distance between these amino acids changes, the conformational interconversion of Gag occurs (
      • Massiah M.A.
      • Worthylake D.
      • Christensen A.M.
      • Sundquist W.I.
      • Hill C.P.
      • Summers M.F.
      ). Yu et al. (
      • Yu X.
      • Yuan X.
      • Matsuda Z.
      • Lee T.H.
      • Essex M.
      ) constructed a mutant HIV-1 with a wild type protease lacking 10 Gag amino acids (codons 68–77), which showed an impaired viral production, suggesting that that stretch of amino acids is essential for the replicative fitness of HIV-1. In the present study, the L75R substitution in Gag conferred replication advantages on HIV-1, particularly when combined with H219Q substitution. Taken together, one can speculate that the L75R substitution may alter the trimerization of MA and/or the interaction of MA with a lipid membrane of HIV-1 resulting in its “membrane anchoring” through altered electrostatic interactions. Such changes, with HIV-1AR background, may ultimately confer a replication advantage on the virus.
      The H219Q substitution in p24 was identified in all clones derived from four PI-resistant HIV-1 variants. In this regard, the amino acids 217–225 reportedly made a single exposed loop in the capsid protein that binds to the enzymatic active site of human CypA (
      • Gamble T.R.
      • Vajdos F.F.
      • Yoo S.
      • Worthylake D.K.
      • Houseweart M.
      • Sundquist W.I.
      • Hill C.P.
      ) whose incorporation into HIV-1 particles seems to be indispensable for the infectivity of HIV-1. In fact, HIV-1 replication is inhibited by cyclosporin A that binds to CypA and interferes with the virion association of CypA (
      • Braaten D.
      • Franke E.K.
      • Luban J.
      ,
      • Braaten D.
      • Aberham C.
      • Franke E.K.
      • Yin L.
      • Phares W.
      • Luban J.
      ). It has been noted that SIVmac capsid protein also has an exposed loop but has no affinity to CypA and is capable of replicating without CypA (
      • Bukovsky A.A.
      • Weimann A.
      • Accola M.A.
      • Gottlinger H.G.
      ). Moreover, the transfer of the HIV-1 loop to the corresponding position in SIVmac results in the efficient incorporation of CypA and confers HIV-1-like sensitivity to cyclosporin A (
      • Bukovsky A.A.
      • Weimann A.
      • Accola M.A.
      • Gottlinger H.G.
      ). Yoo et al. (
      • Yoo S.
      • Myszka D.G.
      • Yeh C.
      • McMurray M.
      • Hill C.P.
      • Sundquist W.I.
      ) generated a number of mutant p24 proteins, analyzed their binding affinity to CypA, and showed that the H219Q mutation in Gag of HIV-1NL4-3diminished CypA binding. It should be noted that H219Q represents a polymorphic amino acid substitution and is often seen particularly in clades F and G isolates (
      • Kuiken C.
      • Foley B.
      • Hahn B., P., M.
      • McCutchan F.
      • Mellors J.
      • Mullins J.
      • Wolinsky S.
      • Korber B.
      ). In the present study, we found that His-219 in HIV-1NL4-3 obtained from freshly transfected COS-7 cells spontaneously changed to glutamine over 10 passages in one of three independent experiments. In the two other experiments, A224V and V218M emerged when the H219Q substitution did not emerge, whereas no mutations occurred in common among clones in other regions of the entire gag gene (Table II). These data strongly suggest that the H219Q substitution brings about a conformational change in the loop formed by the stretch of the amino acids positioned 217–225 (corresponding to CypA binding loop).
      Based on these data, we postulated that the exposed loop of p24 might exert a negative effect(s) on the replication of HIV-1 either at the stages of assembly or disassembly and that the binding of CypA to the loop might induce conformational changes so that the negative effect(s) are canceled, thus restoring replicative fitness. Therefore, we compared the amounts of viral particle-associated CypA in H219Q-carrying and H219Q-lacking HIV-1 (HIV-1ARL75R/H219Q/V390 and HIV-1ARL75/H219/V390, respectively) using the Western blot assay, and we found that HIV-1ARL75R/H219Q/V390 contained less CypA (Fig. 6) but replicated faster than HIV-1ARL75/H219/V390 (Fig. 2, A andF). These data suggest that H219Q substitution changed the loop conformation so that the rate of replication became greater and presumably relatively independent of the binding of CypA. Indeed, in our preliminary structural characterization of HIV-1 matrix-capsid antigens, the N terminus of capsid antigen appears to take multiple conformations.
      C. Tang and M. F. Summers, unpublished data.
      It is possible that the cleavage of matrix and capsid antigens shifts the equilibrium toward a certain conformation (
      • Dietrich L.
      • Ehrlich L.S.
      • LaGrassa T.J.
      • Ebbets-Reed D.
      • Carter C.
      ), and CypA binding is required for the stabilization of that conformation. In this respect, the H219Q substitution per se might stabilize that conformation, and the requirement of CypA binding is reduced. It is worth noting that Western blot analysis has a limited utility in quantification and that enzyme-linked immunosorbent assay or its related method should enable us to quantitate more precisely the levels of virion-associated cyclophilin A than Western blot analysis. However, to date, no cyclophilin-specific monoclonal antibody has been generated or reported, and the establishment of a more quantitative method for cyclophilin A is awaited for further detailed investigation into the role of cyclophilin A in the replication of HIV-1.
      Following the emergence of protease gene mutations in HIV-1NL4-3 propagated in the presence of APV, two substitutions R409K and E468K emerged in the Gag protein. Both codons at 409 and 468 are known to be highly conserved (
      • Kuiken C.
      • Foley B.
      • Hahn B., P., M.
      • McCutchan F.
      • Mellors J.
      • Mullins J.
      • Wolinsky S.
      • Korber B.
      ). A newly generated HIV-1 clone carrying these two mutations in addition to the cleavage site mutation L449F (HIV-1ARR409K/L449F/E468K) predominated two HIV-1 clones lacking either of these two mutations in the presence of APV in the replication kinetics assay and the CHRA (Fig. 7 B and Fig. 8, C andD). In contrast, HIV-1 carrying these two mutations and wild type protease (HIV-1NLR409K/L449/E468K) was outgrown by HIV-1 lacking the two mutations (HIV-1NLR409/L449/E468) (Fig.8 E). Based on these data, we thought that these two mutations altered the conformation of the tertiary structure of the Gag precursor p7-p1-p6 and made the cleavage site more accessible to the mutated protease, the rate-limiting step for virus maturation (
      • Darke P.L.
      • Nutt R.F.
      • Brady S.F.
      • Garsky V.M.
      • Ciccarone T.M.
      • Leu C.T.
      • Lumma P.K.
      • Freidinger R.M.
      • Veber D.F.
      • Sigal I.S.
      ,
      • Tozser J.
      • Blaha I.
      • Copeland T.D.
      • Wondrak E.M.
      • Oroszlan S.
      ,
      • Wondrak E.M.
      • Louis J.M.
      • de Rocquigny H.
      • Chermann J.C.
      • Roques B.P.
      ). However, Western blot analysis failed to reveal the difference in Gag cleavage profiles. There is a possibility that the functions of Gag proteins are altered by L75R, H219Q, R409K, or E468K substitutions, resulting in enhanced viral replication. One of known Gag functions is the packaging of genomic RNA into HIV-1 particles (
      • Turner B.G.
      • Summers M.F.
      ). Therefore, we semi-quantified the amount of viral particle-associated genomic RNA in five different clonal HIV-1 preparations (HIV-1ARL75/H219/V390/R409K/L449F/E468K, HIV-1ARL75R/H219Q/V390/R409K/L449F/E468K, HIV-1JRL75/H219/V390/R409K/L449/E468, HIV-1JRL75/H219Q/V390/R409K/L449/E468, and HIV-1NLL75/H219/V390/R409/L449/E468) using Northern blot analysis (Fig. 5) and RT-PCR (Table III), but the amounts of RNA were all comparable.
      Most of the Gag mutations identified in this study have not been reported to be associated with the acquisition of HIV-1 resistance against PIs. For example, R409K occurred when HIV-1NL4-3was passaged in the presence of all four PIs, APV, JE-2147, KNI-272, and UIC-94003 (Table I). However, we failed to identify R409K in any of ∼30 clones generated from each of four heavily PI-treated HIV-1-infected patients whose HIV-1 was proved to harbor a number of mutations in the protease-encoding gene (
      • Yoshimura K.
      • Kato R.
      • Yusa K.
      • Kavlick M.F.
      • Maroun V.
      • Nguyen A.
      • Mimoto T.
      • Ueno T.
      • Shintani M.
      • Falloon J.
      • Masur H.
      • Hayashi H.
      • Erickson J.
      • Mitsuya H.
      ). It appears that the R409K substitution occurs specifically with the HIV-1NL4-3genetic background. In all the selection experiments in this study, we used a newly generated HIV-1NL4-3, a highly pure clonal HIV-1 preparation, which enabled us to identify such a mutation after drug selection procedure. In this context, it is likely that HIV-1 in patients represents the quasi-species carrying a myriad of genetic backgrounds and, in response to PI(s) including APV, HIV-1 develops a number of genetic background-specific Gag mutations, so that sequencing analyses of relatively few clones (i.e. ∼30 clones in our study) failed to identify common mutations.
      The present data, taken together, suggest that HIV-1 resistance to PIs is associated with primary and secondary mutations in the viral protease and is also associated with the cleavage site amino acid substitutions in Gag together with substitutions at non-cleavage sites. Such non-cleavage site Gag mutations should render the polyprotein cleavage sites more accessible to the protease, make polymerization of viral proteins more efficacious, and/or make assembly and disassembly more efficient. Alteration(s) of other unknown functions of Gag proteins may also contribute to the HIV-1 acquisition of resistance to PIs, but it appears that HIV-1 resistance to PIs is acquired with multiple mechanisms.

      Acknowledgments

      We thank Louis E. Henderson for helpful discussions.

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