HIV Pol inhibits HIV budding and mediates the severe budding defect of Gag-Pol

The prevailing hypothesis of HIV budding posits that the viral Gag protein drives budding, and that the Gag p6 peptide plays an essential role by recruiting host-cell budding factors to sites of HIV assembly. HIV also expresses a second Gag protein, p160 Gag-Pol, which lacks p6 and fails to bud from cells, consistent with the prevailing hypothesis of HIV budding. However, we show here that the severe budding defect of Gag-Pol is not caused by the absence of p6, but rather, by the presence of Pol. Specifically, we show that (i) the budding defect of Gag-Pol is unaffected by loss of HIV protease activity and is therefore an intrinsic property of the Gag-Pol polyprotein, (ii) the N-terminal 433 amino acids of Gag and Gag-Pol are sufficient to drive virus budding even though they lack p6, (iii) the severe budding defect of Gag-Pol is caused by a dominant, cis-acting inhibitor of budding in the HIV Pol domain, and (iv) Gag-Pol inhibits Gag and virus budding in trans, even at normal levels of Gag and Gag-Pol expression. These and other data support an alternative hypothesis of HIV budding as a process that is mediated by the normal, non-viral pathway of exosome/microvesicle biogenesis.     

Cleavage of Human Immunodeficiency Virus Type 1 Proteinase from the N-Terminally Adjacent p6* Protein Is Essential for Efficient Gag Polyprotein Processing and Viral Infectivity

Maturation of infectious human immunodeficiency virus (HIV) particles requires proteolytic cleavage of the structural polyproteins by the viral proteinase (PR), which is itself encoded as part of the Gag-Pol polyprotein. Expression of truncated PR-containing sequences in heterologous systems has mostly led to the autocatalytic release of an 11-kDa species of PR which is capable of processing all known cleavage sites on the viral precursor proteins. Relatively little is known about cleavages within the nascent virus particle, on the other hand, and controversial results concerning the active PR species inside the virion and the relative activities of extended PR species have been reported. Here, we report that HIV type 1 (HIV-1) particles of four different strains obtained from different cell lines contain an 11-kDa PR, with no extended PR proteins detectable. Furthermore, mutation of the N-terminal PR cleavage site leading to production of an N-terminally extended 17-kDa PR species caused a severe defect in Gag polyprotein processing and a complete loss of viral infectivity. We conclude that N-terminal release of PR from the HIV-1 polyprotein is essential for viral replication and suggest that extended versions of PR may have a transient function in the proteolytic cascade.     

Loss of Viral Fitness Associated with Multiple Gag and Gag-Pol Processing Defects in Human Immunodeficiency Virus Type 1 Variants Selected for Resistance to Protease Inhibitors In Vivo

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.     

Mutations in Multiple Domains of Gag Drive the Emergence of In Vitro Resistance to the Phosphonate-Containing HIV-1 Protease Inhibitor GS-8374

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.     

Free energy calculations on dimer stability of the HIV protease using molecular dynamics and a continuum solvent model

Dimerization of HIV-I protease (HIV PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disrupting dimerization of the enzyme can inhibit its activity. We have calculated the relative binding free energies between different dimers of the HIV protease using molecular dynamics and a continuum model, which we call MM/PBSA. We examined the dominant negative inhibition of the HIV PR by a mutated form of the protease and found relative dimerization free energies of homo- and hetero-dimerization consistent with experimental data. We also developed a rapid screening method, which was called the virtual mutagenesis method to consider other mutations which might stabilize non-wild-type heterodimers. Using this approach, we considered the mutations near the dimer interface which might cause dominant negative inhibition of the HIV PR. The rapid method we developed can be used in studying any ligand-protein and protein-protein interaction, in order to identify mutations that can enhance the binding affinities of the complex.     

Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations.     

Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain

Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed. Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2). A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly. Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype. This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target.     

Placement of leucine zipper motifs at the carboxyl terminus of HIV-1 protease significantly reduces virion production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.     

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.     

Loss of protease dimerization inhibition activity of darunavir is associated with the acquisition of resistance to darunavir by HIV-1

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.     

A p6Pol-protease fusion protein is present in mature particles of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites.     

Yeast two-hybrid assay for examining human immunodeficiency virus protease heterodimer formation with dominant-negative inhibitors and multidrug-resistant variants

The yeast two-hybrid assay was used to study the dimerization of engineered and naturally occurring variants of human immunodeficiency virus (HIV) protease (PR) monomers. Defective monomers that were previously shown to exhibit a dominant-negative (D-N) effect in cultured mammalian cells were tested for their ability to interact in the two-hybrid assay. Similarly, monomers with dimer-interface substitutions and monomers harboring in vivo selected mutations that confer multidrug resistance (mdr) in an AIDS patient were tested for interaction in yeast. Dimer formation between wt monomers with catalytic aspartates was not detected in yeast, whereas the dimerization of PR monomers harboring the acid active site substitution D25N was readily demonstrated. The use of inactive monomers harboring the D25N substitution as a genetic background for studying additional HIV PR mutations allowed for the probing of interactions between monomers with mdr-associated mutations with those based on the HIV-1 HXB2R sequence. The HTLVIII/HIV-1 HXB2R clone has been the basis for a large number of HIV-related plasmids, primers, antibodies, and other specific reagents throughout the HIV research community. The results of our assay suggest that HXB2R-based D-N PR inhibitors associate with variant monomers based on the recently obtained nucleotide sequence from an AIDS patient with a multidrug-resistant virus. These results further encourage the use of D-N PR inhibitors as antiviral agents which may complement existing small-molecule combination therapies.     

Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA.

Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles. To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus. A point mutation in the protease (PR) active site completely prevents Pr76gag processing. The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it. A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA. This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging. These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.     

Competitive inhibition of human immunodeficiency virus type-1 protease by the Gag-Pol transframe protein.

The human immunodeficiency virus type-1 (HIV-1) transframe protein p6* is located between the structural and enzymatic domains of the Gag-Pol polyprotein, flanked by the nucleocapsid (NC) and the protease (PR) domain at its amino and carboxyl termini, respectively. Here, we report that recombinant highly purified HIV-1 p6* specifically inhibits mature HIV-1 PR activity. Kinetic analyses and cross-linking experiments revealed a competitive mechanism for PR inhibition by p6*. We further demonstrate that the four carboxyl-terminal residues of p6* are essential but not sufficient for p6*-mediated inhibition of PR activity. Based on these results, we suggest a role of the transframe protein p6* in regulating HIV-1 PR activity during viral replication.