Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.     

Pokeweed antiviral protein increases HIV-1 particle infectivity by activating the cellular mitogen activated protein kinase pathway

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase that exhibits antiviral activity against several viruses. The enzyme removes purine bases from the messenger RNAs of the retroviruses Human immunodeficiency virus-1 and Human T-cell leukemia virus-1. This depurination reduces viral protein synthesis by stalling elongating ribosomes at nucleotides with a missing base. Here, we transiently expressed PAP in cells with a proviral clone of HIV-1 to examine the effect of the protein on virus production and quality. PAP reduced virus production by approximately 450-fold, as measured by p24 ELISA of media containing virions, which correlated with a substantial decline in virus protein synthesis in cells. However, particles released from PAP-expressing cells were approximately 7-fold more infectious, as determined by single-cycle infection of 1G5 cells and productive infection of MT2 cells. This increase in infectivity was not likely due to changes in the processing of HIV-1 polyproteins, RNA packaging efficiency or maturation of virus. Rather, expression of PAP activated the ERK1/2 MAPK pathway to a limited extent, resulting in increased phosphorylation of viral p17 matrix protein. The increase in infectivity of HIV-1 particles produced from PAP-expressing cells was compensated by the reduction in virus number; that is, virus production decreased upon de novo infection of cells over time. However, our findings emphasize the importance of investigating the influence of heterologous protein expression upon host cells when assessing their potential for antiviral applications.     

Conserved Cysteines of the Human Immunodeficiency Virus Type 1 Protease Are Involved in Regulation of Polyprotein Processing and Viral Maturation of Immature Virions

We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H₂O₂ and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.     

Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.     

Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in?vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.     

Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease

The processing of precursor proteins (Gag and Gag-pol) by the viral protease is absolutely required in order to generate infectious particles. This prompted us to consider novel strategies that target viral maturation. Towards this end, we have engineered an HIV-1 virion associated protein, Vpr, to contain protease cleavage signal sequences from Gag and Gag-pol precursor proteins. We previously reported that virus particles derived from HIV-1 proviral DNA, encoding chimeric Vpr, showed a lack of infectivity, depending on the fusion partner. As an extension of that work, the potential of chimeric Vpr as a substrate for HIV-1 protease was tested utilizing an epitope-based assay. Chimeric Vpr molecules were modified such that the Flag epitope is removed following cleavage, thus allowing us to determine the efficiency of protease cleavage. Following incubation with the protease, the resultant products were analyzed by radioimmunoprecipitation using antibodies directed against the Flag epitope. Densitometric analysis of the autoradiograms showed processing to be both rapid and specific. Further, the analysis of virus particles containing chimeric Vpr by immunoblot showed reactivities to antibodies against the Flag epitope similar to the data observed in vitro. These results suggest that the pseudosubstrate approach may provide another avenue for developing antiviral agents.     

The cellular protein lyric interacts with HIV-1 Gag

Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein driving assembly and release of virions from infected cells. Gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. In a series of affinity purification experiments, we identified the cellular protein Lyric to be an HIV-1 Gag-interacting protein. Lyric was previously described to be an HIV-inducible gene and is involved in various signaling pathways. Gag interacts with endogenous Lyric via its matrix (MA) and nucleocapsid (NC) domains. This interaction requires Gag multimerization and Lyric amino acids 101 to 289. Endogenous Lyric is incorporated into HIV-1 virions and is cleaved by the viral protease. Gag-Lyric interaction was also observed for murine leukemia virus and equine infectious anemia virus, suggesting that it represents a conserved feature among retroviruses. Expression of the Gag binding domain of Lyric increased Gag expression levels and viral infectivity, whereas expression of a Lyric mutant lacking the Gag binding site resulted in lower Gag expression and decreased viral infectivity. The results of the current study identify Lyric to be a cellular interaction partner of HIV-1 Gag and hint at a potential role in regulating infectivity. Further experiments are needed to elucidate the precise role of this interaction.     

Chimeric human immunodeficiency virus type 1 (HIV-1) virions containing HIV-2 or simian immunodeficiency virus Nef are resistant to cyclosporine treatment

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function.     

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines.

The enzymatic activity of the human immunodeficiency type 1 (HIV-1) protease (PR) is crucial to render HIV-1 virions mature and infectious. Hence, genetic intervention strategies based on trans-dominant (td) variants of the HIV-1 PR might be an alternative to current pharmacological and gene therapy regimens for AIDS. CD4-positive human CEM-SS T-cell lines were generated which constitutively expressed HIV-1 td PR variants. HIV-1 infection experiments demonstrated severely reduced HIV-1 replication in these td PR CEM-SS cell lines compared with control T cells expressing wild-type PR. Furthermore, replication of an HIV-1 isolate bearing a PR inhibitor-resistant PR was blocked, showing that genetic intervention strategies based on td PRs can be effective against HIV-1 isolates containing PR inhibitor-resistant mutants.     

Re-visiting the functional Relevance of the highly conserved Serine 40 Residue within HIV-1 p6<Superscript>Gag</Superscript>

Background

HIV-1 formation is driven by the viral structural polyprotein Gag, which assembles at the plasma membrane into a hexagonal lattice. The C-terminal p6^Gag domain harbors short peptide motifs, called late domains, which recruit the cellular endosomal sorting complex required for transport and promote HIV-1 abscission from the plasma membrane. Similar to late domain containing proteins of other viruses, HIV-1 p6 is phosphorylated at multiple residues, including a highly conserved serine at position 40. Previously published studies showed that an S40F exchange in p6^Gag severely affected virus infectivity, while we had reported that mutation of all phosphorylatable residues in p6^Gag had only minor effects.

Findings

We introduced mutations into p6^Gag without affecting the overlapping pol reading frame by using an HIV-1 derivative where gag and pol are genetically uncoupled. HIV-1 derivatives with a conservative S40N or a non-conservative S40F exchange were produced. The S40F substitution severely affected virus maturation and infectivity as reported before, while the S40N exchange caused no functional defects and the variant was fully infectious in T-cell lines and primary T-cells.

Conclusions

An HIV-1 variant carrying a conservative S40N exchange in p6^Gag is fully functional in tissue culture demonstrating that neither S40 nor its phosphorylation are required for HIV-1 release and maturation. The phenotype of the S40F mutation appears to be caused by the bulky hydrophobic residue introduced into a flexible region.

     

Optimized Infectivity of the Cell-Free Single-Cycle Human Immunodeficiency Viruses Type 1 (HIV-1) and Its Restriction by Host Cells

The infectivity of retroviruses such as HIV-1 in plasma or cultured media is less than 0.1% in general, the mechanisms of which are not yet fully understood. One possible explanation among others is the potential presence of large numbers of defective virions in a virus pool, which limits the apparent infectivity of HIV virions. To test this hypothesis, we have varied the culture conditions used to generate single-cycle HIV-1 virions. Among these culture variables, virion harvest time, media change after transfection, and envelope plasmid input can all improve HIV-1 infectivity by reducing the number of defective virions. A harvest time of 18–24 hours post transfection as opposed to 48 hours, and a media change six hours post transfection both improve viral infectivity. An optimal quantity of envelope plasmid input during transfection was also found. Collectively, these conditions increased the infectivity of HIV-1 virions by sevenfold compared to normally reported values in TZM-bl indicator cell lines. These conditions also increased the infectivity of HIV-1 in CD4⁺ T cells, suggesting that these conditions work by increasing the intrinsic infectivity of a virus pool. Nevertheless, these improvements on virion infectivity were marginal compared to the impact of host cells on HIV infection, which can decrease the apparent infectivity by 19-fold even for the most optimized viruses. These results suggest that the infectivity of HIV-1 virions can be optimized by reducing the number of defective virions; however, viral-cell interactions may pose a major barrier for HIV-1 infectivity.     

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.     

The Proteolytic Cleavage of Human Immunodeficiency Virus Type 1 Nef Does Not Correlate with Its Ability To Stimulate Virion Infectivity

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan⁵⁷ decreased the efficiency of Nef processing, while mutating leucine⁵⁸ had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a nef-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity.     

The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity

The relationship between virion protein maturation and genomic RNA dimerization of human immunodeficiency virus type 1 (HIV-1) remains incompletely understood. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. Within the virion maturation process, the RNA dimer stabilization begins during the primary cleavage (p2-NC) of Pr55 Gag. However, the primary cleavage alone is not sufficient, and the ensuing cleavages are required for the completion of dimerization. From our observations, the increase of cleavage products may not put a threshold on the transition from fragile to stable dimeric RNA. Most of the RNA dimerization process did not require viral core formation, and particle morphology dynamics during viral maturation did not completely synchronize with the transition of dimeric RNA status. Although the endogenous virion RT activity was fully acquired at the initial step of maturation, the following process was necessary for viral DNA production in infected cell, suggesting the maturation of viral RNA/protein plays critical role for viral infectivity other than RT process.     

Macrophages archive HIV-1 virions for dissemination in trans

Viruses have evolved various strategies in order to persist within the host. To date, most information on mechanisms of HIV-1 persistence has been derived from studies with lymphocytes, but there is little information regarding mechanisms that govern HIV-1 persistence in macrophages. It has previously been demonstrated that virus assembly in macrophages occurs in cytoplasmic vesicles, which exhibit the characteristics of multivesicular bodies or late endosomes. The infectious stability of virions that assemble intracellularly in macrophages has not been evaluated. We demonstrate that virions assembling intracellularly in primary macrophages retain infectivity for extended intervals. Infectious virus was recovered directly from cytoplasmic lysates of macrophages and could be transmitted from macrophages to peripheral blood lymphocytes in trans 6 weeks after ongoing viral replication was blocked. Cell-associated virus decayed significantly from 1 to 2 weeks post infection, but decreased minimally thereafter. The persistence of intracellular virions did not require the viral accessory proteins Vpu or Nef. The stable sequestration of infectious virions within cytoplasmic compartments of macrophages may represent an additional mechanism for viral persistence in HIV-1-infected individuals.     

HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice

Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.