The Impact Of HIV-1 CRF07_BC P6^GagMutations On Virus Release,Maturation,Infectivity And Replication

Background and ObjectiveCRF07 BC has become one of the most commonly transmitted HIV-1 subtypes across the country.Previous studies have reported that CRF07 BC-infected patients showed significantly lower viral loads,slower immunological progression,and delayed virus growth kinetics compared to the subjects infected with the CRF01 AE and subtype B.Subsequent sequence analysis of CRF07_BC p6Gag indicated a specific seven-amino-acid(7aa)(30PIDKELY36)deletion(p6?7)and/or a PTAPPE(6aa)duplication.These observations suggest that mutant CRF07_BC virus may have different replication capacity,which in turn could impact disease progression.In this study,we examined the effect of these p6 mutations on virus release,infectivity and replication to unveil the molecular mechanism of slow AIDS progression among CRF07_BC infected patients.MethodsPatient-derived mutations in p6Gag were introduced into the HIV-1 proviral clone pNL4-3 for the construction of three mutant plasmids:PTAP motif(PTAPPE)duplication,7aa(30PIDKELY36)deletion,and the double mutation of PTAPPE duplication and 7aa deletion.Wild type(WT)pNL4-3 and p6 mutant plasmids were used to investigate virus release,maturation,infectivity,and replication capacity.We next studied the molecular mechanism of virus defects caused by p6?7.To characterize the role of the 7aa in p6Gag or in the overlapping open reading frame,p6Pol,we used Gag-and GagPol-expressing clones encoding WT or mutant p6 domain to investigate virus release and maturation.It has previously reported that the deletion of 36th residual Tyrosine(Y36)in p6 caused severe defect in virus particle production.To further define the role of Tyrosine(Y36)in the defect caused by the deletion of the 7aa(PEDKELY),three mutant p6 HIV-1 proviral clones,K-?Y36,K-?630-35 and K-?730-36,together with WT KFS were used to investigate virus release efficiency and Gag processing.HIV-1 p6-YPXnL motif binds to host protein Alix promoting virus budding.The residual Tyrosine(Y36)in 7aa(PEDKELY)that overlaps with YPXnL motif may affect the binding to Alix.To verify this hypothesis,virus release of WT KFS,K-?Y36,K-?630-35,and K-?730-36 were measured in the context of exogenous Alix V(residues 364-716)expression.We further analyzed the effect of p6?7 mutation on HIV-1 reverse transcription.The products of HIV-1 DNA synthesis initiation(R-U5),minus-strand transfer(U3-U5)and plus-strand transfer(R-5'UTR and Gag)were quantified using qPCR.ResultsThe phenotypic data suggested that:(1)the PTAPPE duplication did not significantly impact virus release,infectivity,and replication capacity,but slightly inhibited Gag processing.(2)The mutations P?7 and ?7 decreased virus release to 62%and 76%,respectively,as compared to the WT.The P?7 and ?7 mutants were 36%and 52%of Gag processing,respectively,and were 69%and 56%of infectivity of WT levels,respectively.Replication kinetics showed that P?7 and ?7 mutant viruses replicated 2-7 folds less efficiency than that of WT in MT-4,Jurkat T-cells,and PBMCs.Taken together,the seven-amino-acid(PIDKELY)deletion in p6 causes defects in virus release,maturation,infectivity and replication.It was observed that the deletion occurring in p6111 resulted in slightly reduced virus production and Gag processing of 88%and 69%,respectively,as compared to the WT.In contrast,the deletion of 7aa in p6Gag region decreased virus release efficiency and Gag processing by?2-3 times.These data demonstrate that seven-amino-acid deletion in p6Gag but not p6Pol is responsible for the defects in virus release and maturation.Single-amino-acid analysis suggested that K-?630-35 slightly reduced virus release,but did not significant impact Gag processing.The mutations K-?Y36 and K-?730-36 reduced virus release to 28%and 57%of WT level,respectively.Gag processing of K-?Y36 and K-?730-36 mutants showed 46%and 53%efficiencies,respectively,relative to WT level.These data suggest that the phenotypic defects of?7 mutant virus may be mainly caused by Tyrosine(Y36)deletion.Analysis of virus-Alix protein interaction demonstrated that WT virus release was severely inhibited by exogenous Alix V expression,but not by an Alix V variant(Alix V/F676D)which abrogates Alix-YPXnL binding.In contrast,virus release of K-?Y36,K-?630-35 or K-?730-36 was unaffected by either Alix V or Alix/F676D overexpression.These results imply that Tyrosine(Y36)may play a critical role in p6-Alix binding.Virus release defect may be in part due to the abrogation of the binding ability of p6 to Alix.We further analyzed the effect of p6A7 mutation on HIV-1 reverse transcription.The GagA7 and GagPolA7 mutants were less efficiency in the progression of minus-and plus-strand synthesis,in particular,two GagA7 mutants exhibited about?30-40%reduction in minus-strand DNA production.These data indicated that the deletion of the seven-amino-acids in p6 may reduce DNA production during HIV-1 reverse transcription.ConclusionWe showed that the deletion of seven-amino-acid(30PIDDKELY36)in p6 caused defects in virus production,maturation,infectivity and replication.Further mechanism studies suggested that 7aa deletion in p6Gagbut not p6poI played a major role in virus release and Gag processing.Tyrosine(Y36)may be the critical residue within the 7aa sequence,and its deletion decreased virus particle production,which probably due to the abrogation of p6-Alix binding.Moreover,7aa deletion in p6 also reduced DNA synthesis in HIV-1 reverse transcription.These phenotypes synergistically contribute to the reduced replication capacity of CRF07_BC and may explain the significantly low viral load as well as the delayed virus growth kinetics observed in infected patients.