Uncoupling human immunodeficiency virus type 1 Gag and Pol reading frames: role of the transframe protein p6* in viral replication.
ABSTRACT: Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6(gag) sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3' end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6(gag) reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.
Project description:Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within the pol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6(gag)-p6(pol) region of HIV-1, placed immediately upstream of pol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6(Gag)), or by changes in activation of the viral protease (p6(Pol)). Duplication of the proline-rich p6(Gag) PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naïve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6(Pol)), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.
Project description:The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.
Project description:The structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.
Project description:Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations.Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations.
Project description:A transframe region within HIV-1 Gag-Pol (referred to as p6* or p6pol), directly linked to the protease (PR) N-terminus, plays a pivotal role in modulating PR activation. To identify specific p6* residues involved in PR activation, we created a series of p6* mutants by making substitutions for conserved p6* residues. Our results indicate that some p6* mutants were defective in terms of virus infectivity, despite displaying a wild-type virus particle processing pattern. Mutations at p6* F8 reduced virus infectivity associated with insufficient virus processing, due in part to impaired PR maturation and RT packaging. Our data strongly suggest that conserved Phe (F) residues at position 8 of p6* are involved in the PR maturation process.
Project description:Human immunodeficiency virus type 1 (HIV-1) Gag is expressed as a polyprotein that is cleaved into six proteins by the viral protease in a maturation process that begins during assembly and budding. While processing of the N terminus of Gag is strictly required for virion maturation and infectivity, the necessity for the C-terminal cleavages of Gag is less well defined. To examine the importance of this process, we introduced a series of mutations into the C terminus of Gag that interrupted the cleavage sites that normally produce in the nucleocapsid (NC), spacer 2 (SP2), or p6(Gag) proteins. Protein analysis showed that all of the mutant constructs produced virions efficiently upon transfection of cells and appropriately processed Gag polyprotein at the nonmutated sites. Mutants that produced a p9(NC/SP2) protein exhibited only minor effects on HIV-1 infectivity and replication. In contrast, mutants that produced only the p8(SP2/p6) or p15(NC/SP2/p6) protein had severe defects in infectivity and replication. To identify the key defective step, we quantified reverse transcription and integration products isolated from infected cells by PCR. All mutants tested produced levels of reverse transcription products either similar to or only somewhat lower than that of wild type. In contrast, mutants that failed to cleave the SP2-p6(Gag) site produced drastically less provirus than the wild type. Together, our results show that processing of the SP2-p6(Gag) and not the NC-SP2 cleavage site is important for efficient viral DNA integration during infection in vitro. In turn, this finding suggests an important role for the p9(NC/SP2) species in some aspect of integration.
Project description:<h4>Background</h4>Polymorphisms at cleavage sites (CS) can influence Gag and Pol proteins processing by the viral protease (PR), restore viral fitness and influence the virological outcome of specific antiretroviral drugs. However, data of HIV-1 variant-associated CS variability is scarce.<h4>Methods</h4>In this descriptive research, we examine the effect of HIV-1 variants on CS conservation using all 9,028 gag and 3,906 pol HIV-1 sequences deposited in GenBank, focusing on the 110 residues (10 per site) involved at 11 CS: P17/P24, P24/P2, P2/P7, P7/P1, P1/P6 (gag) , NC/TFP, TFP/P6 (pol), P6 (pol) /PR, PR/RT(p51), RT(p51)/RT(p66) and RT(p66)/IN. CS consensus amino acid sequences across HIV-1 groups (M, O, N, P), group M 9 subtypes and 51 circulating recombinant forms (CRF) were inferred from our alignments and compared to the HIV-1 consensus-of-consensuses sequence provided by GenBank.<h4>Results</h4>In all HIV-1 variants, the most conserved CS were PR/RT(p51), RT(p51)/RT(p66), P24/P2 and RT(p66)/IN and the least P2/P7 and P6 (pol) /PR. Conservation was significantly lower in subtypes vs. recombinants in P2/P7 and TFP/P6 (pol) and higher in P17/P24. We found a significantly higher conservation rate among Group M vs. non-M Groups HIV-1. The late processing sites at Gag (P7/P1) and GagPol precursors (PR/RT(p51)) presented a significantly higher conservation vs. the first CS (P2/P7) in the 4 HIV-1 groups. Here we show 52 highly conserved residues across HIV-1 variants in 11 CS and the amino acid consensus sequence in each HIV-1 group and HIV-1 group M variant for each 11 CS.<h4>Conclusions</h4>This is the first study to describe the CS conservation level across all HIV-1 variants and 11 sites in one of the largest available sequence HIV-1 dataset. These results could help other researchers for the future design of both novel antiretroviral agents acting as maturation inhibitors as well as for vaccine targeting CS.
Project description:Naturally occurring polymorphisms in the protease of human immunodeficiency virus type 1 (HIV-1) subtype C would be expected to lead to adaptive (compensatory) changes in protease cleavage sites. To test this hypothesis, we examined the prevalences and patterns of cleavage site polymorphisms in the Gag, Gag-Pol, and Nef cleavage sites of C compared to those in non-C subtypes. Codon-based maximum-likelihood methods were used to assess the natural selection and evolutionary history of individual cleavage sites. Seven cleavage sites (p17/p24, p24/p2, NC/p1, NC/TFP, PR/RT, RT/p66, and p66/IN) were well conserved over time and in all HIV-1 subtypes. One site (p1/p6(gag)) exhibited moderate variation, and four sites (p2/NC, TFP/p6(pol), p6(pol)/PR, and Nef) were highly variable, both within and between subtypes. Three of the variable sites are known to be major determinants of polyprotein processing and virion production. P2/NC controls the rate and order of cleavage, p6(gag) is an important phosphoprotein required for virion release, and TFP/p6(pol), a novel cleavage site in the transframe domain, influences the specificity of Gag-Pol processing and the activation of protease. Overall, 58.3% of the 12 HIV-1 cleavage sites were significantly more diverse in C than in B viruses. When analyzed as a single concatenated fragment of 360 bp, 96.0% of group M cleavage site sequences fell into subtype-specific phylogenetic clusters, suggesting that they coevolved with the virus. Natural variation at C cleavage sites may play an important role, not only in regulation of the viral cycle but also in disease progression and response to therapy.
Project description:Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.
Project description:The structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol.In developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 x 106 TU/ml, unconcentrated).Here, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.