The hydrophobic pocket of cyclophilin is the binding site for the human immunodeficiency virus type 1 Gag polyprotein.

Completion of an early step in the human immunodeficiency virus type 1 (HIV-1) life cycle requires incorporation into virions of the cellular peptidyl-prolyl isomerase cyclophilin A (CyPA) by the Gag polyprotein. Elucidation of the biochemical role of CyPA would be aided by a detailed analysis of the genetic requirements for the formation of the Gag-CyPA complex; previous experiments have demonstrated the requirement for a critical proline and the immediately preceding glycine, located within the capsid domain of Gag, but nothing is known about the necessary CyPA residues. Cyclophilins possess a hydrophobic pocket where proline-containing peptide substrates and the immunosuppressive drug cyclosporine A bind. In this study, we engineered five CyPA mutations, each of which alters a residue that contributes to the hydrophobic pocket. Compared with the wild-type protein, all of the mutants drastically reduced CyPA binding to HIV-1 Gag and similarly inhibited CyPA incorporation into virions. In addition, we demonstrated that previously reported differences between the Gag-binding properties of CyPA and CyPB are due to adventitious association involving residues in the signal sequence of CyPB and that the core domain of CyPB interacts with Gag in a fashion which is indistinguishable from that of CyPA. These studies indicate that, as with other proline-containing peptides or cyclosporine A, HIV-1 Gag directly contacts residues in the hydrophobic pocket of CyPA.     

Cyclophilin binding to the human immunodeficiency virus type 1 Gag polyprotein is mimicked by an anti-cyclosporine antibody.

Human immunodeficiency virus type 1 is unique among retroviruses in that infectivity requires specific incorporation into virions of the cellular protein cyclophilin A through interactions with the Gag polyprotein. Here we show that monoclonal antibody B11 1.4, which recognizes a cyclophilin-binding epitope on cyclosporine, detects denatured or native human immunodeficiency virus type 1 capsid. B11 1.4 does not recognize the capsids of other retroviruses, and binding is inhibited by cyclosporine or by cyclophilin A.     

Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.     

Kinesin KIF4 regulates intracellular trafficking and stability of the human immunodeficiency virus type 1 Gag polyprotein

Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.     

Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A

Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.     

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.     

Structural consequences of cyclophilin A binding on maturational refolding in human immunodeficiency virus type 1 capsid protein

While several cellular proteins are incorporated in the human immunodeficiency virus type 1 virion, cyclophilin (CyP) A is the only one whose absence has been demonstrated to impair infectivity. Incorporation of the cytosolic protein results from interaction with a highly exposed Pro-rich loop in the N-terminal region of the capsid (CA) domain of the precursor polyprotein, Pr55(Gag). Even when prevented from interacting with CyP A, Pr55Gag still forms particles that proceed to mature into morphologically wild-type virions, suggesting that CyP A influences a postassembly event. The nature of this CyP A influence has yet to be elucidated. Here, we show that while CyP A binds both Gag and mature CA proteins, the two binding interactions are actually different. Tryptophan 121 (W121) in CyP A distinguished the two proteins: a phenylalanine substitution (W121F) impaired binding of mature CA protein but not of Gag. This indicates the occurrence of a maturation-dependent switch in the conformation of the Pro-rich loop. A structural consequence of Gag binding to CyP A was to block this maturational refolding, resulting in a 24-kDa CA protein retaining the immature Pro-rich loop conformation. Using trypsin as a structure probe, we demonstrate that the conformation of the C-terminal region in mature CA is also a product of maturational refolding. Binding to wild-type CyP A altered this conformation, as indicated by a reduction in the accessibility of Cys residue(s) in the region to chemical modification. Hence, the end result of binding to CyP A, whether the Pro-rich loop is in the context of Gag or mature CA protein, is a structurally modified mature CA protein. The postassembly role of CyP A may be mediated through these modified mature CA proteins.     

Ubiquitination of human immunodeficiency virus type 1 Gag is highly dependent on Gag membrane association

Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.     

Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus

Binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55(Gag), to membrane is an indispensable step in virus assembly. Previously, we reported that a matrix (MA) residue 6 substitution (6VR) imposed a virus assembly defect similar to that observed with myristylation-defective mutants, suggesting that the 6VR change impaired membrane binding. Intriguingly, the 6VR mutation had no effect on Gag myristylation. The defective phenotype imposed by 6VR was reversed by changes at other positions in MA, including residue 97. In this study, we use several biochemical methods to demonstrate that the residue 6 mutation, as well as additional substitutions in MA amino acids 7 and 8, reduce membrane binding without affecting N-terminal myristylation. This effect is observed in the context of Pr55(Gag), a truncated Gag containing only MA and CA, and in MA itself. The membrane binding defect imposed by the 6VR mutation is reversed by second-site changes in MA residues 20 and 97, both of which, when present alone, increase membrane binding to levels greater than those for the wild type. Both reduced and enhanced membrane binding imposed by the MA substitutions depend upon the presence of the N-terminal myristate. The results support the myristyl switch model recently proposed for the regulation of Gag membrane binding, according to which membrane binding is determined by the degree of exposure or sequestration of the N-terminal myristate moiety. Alternatively, insertion of the myristate into the lipid bilayer might be a prerequisite event for the function of other distinct MA-encoded membrane binding domains.     

Analysis of human immunodeficiency virus type 1 Gag ubiquitination

Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.     

Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity

The peptidyl-prolyl isomerase cyclophilin A (CypA) increases the kinetics by which human immunodeficiency virus type 1 (HIV-1) spreads in tissue culture. This was conclusively demonstrated by gene targeting in human CD4(+) T cells, but the role of CypA in HIV-1 replication remains unknown. Though CypA binds to mature HIV-1 capsid protein (CA), it is also incorporated into nascent HIV-1 virions via interaction with the CA domain of the Gag polyprotein. These findings raised the possibility that CypA might act at multiple steps of the retroviral life cycle. Disruption of the CA-CypA interaction, either by the competitive inhibitor cyclosporine (CsA) or by mutation of CA residue G89 or P90, suggested that producer cell CypA was required for full virion infectivity. However, recent studies indicate that CypA within the target cell regulates HIV-1 infectivity by modulating Ref1- or Lv1-mediated restriction. To examine the relative contribution to HIV-1 replication of producer cell CypA and target cell CypA, we exploited multiple tools that disrupt the HIV-1 CA-CypA interaction. These tools included the drugs CsA, MeIle(4)-CsA, and Sanglifehrin; CA mutants exhibiting decreased affinity for CypA or altered CypA dependence; HeLa cells with CypA knockdown by RNA interference; and Jurkat T cells homozygous for a deletion of the gene encoding CypA. Our results clearly demonstrate that target cell CypA, and not producer cell CypA, is important for HIV-1 CA-mediated function. Inhibition of HIV-1 infectivity resulting from virion production in the presence of CsA occurs independently of the CA-CypA interaction or even of CypA.     

Multiple isoforms of cyclophilin A associated with human immunodeficiency virus type 1

It is well-known that a peptidyl-prolyl cis-trans isomerase cyclophilin A (CyPA) is incorporated into Human immunodeficiency virus type 1 (HIV-1) particle. The proteome analysis of the purified HIV-1 strain LAV-1 (HIV-1(LAV-1)) reveals that three isoforms of CyPA with an isoelectric point (pI) of 6.00, 6.40, and 6.53 are inside the viral membrane and another isoform with a pI of 6.88 is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N-acetylated. The mechanisms that permit the redistribution of CyPA with a pI of 6.88 on the viral surface have not yet been clarified, but it penetrates the viral membrane after budding.     

In vitro assembly of human immunodeficiency virus type 1 Gag protein.

Retroviral Gag protein is sufficient to produce Gag virus-like particles when expressed in higher eukaryotic cells. Here we describe the in vitro assembly reaction of human immunodeficiency virus Gag protein, which consists of two sequential steps showing the optimal conditions for each reaction. Following expression and purification, Gag protein lacking only the C-terminal p6 domain was present as a monomer (50 kDa) by velocity sedimentation analysis. Initial assembly of the Gag protein to 60 S intermediates occurred by dialysis at 4 degrees C in low salt at neutral to alkaline pH. However, higher order of assembly required incubation at 37 degrees C and was facilitated by the addition of Mg(2+). Prolonged incubation under these conditions produced complete assembly (600 S), equivalent to Gag virus-like particles obtained from Gag-expressing cells. Neither form disassembled by treatment with nonionic detergent, suggesting that correct assembly might occur in vitro. Electron microscopic observation confirmed that the 600 S assembly products were spherical particles similar to authentic immature human immunodeficiency virus particles. The latter assembly stage but not the former was accelerated by the addition of RNA although not inhibited by RNaseA treatment. These results suggest that Gag protein alone assembles in vitro, but that additional RNA facilitates the assembly reaction.     

Analysis of human immunodeficiency virus type 1 Gag dimerization-induced assembly

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.     

Functional domains of the capsid protein of human immunodeficiency virus type 1.

A series of deletions was introduced into the CA domain of the human immunodeficiency virus type 1 Gag polyprotein to examine its role in virus particle and core formation. The mutations resulted in two phenotypes, indicating the existence of two functionally distinct regions within the CA domain. Deletions within a conserved stretch of 20 amino acids referred to as the major homology region (MHR) and deletions C terminal to this region blocked virus replication and significantly reduced the ability to form viral particles. Deletions N terminal to the MHR also prevented virus replication, but the mutants retained the ability to assemble and release viral particles with the same efficiency as the wild-type virus. The mutant particles contained circular rather than cone-shaped cores, and while they were of a density similar to that of wild-type particles, they were more heterogeneous in size. These results indicate that CA domain sequences N terminal to the MHR are essential for the morphogenesis of the mature cone-shaped core.     

Functional surfaces of the human immunodeficiency virus type 1 capsid protein

The human immunodeficiency virus type 1 initially assembles and buds as an immature particle that is organized by the viral Gag polyprotein. Gag is then proteolyzed to produce the smaller capsid protein CA, which forms the central conical capsid that surrounds the RNA genome in the mature, infectious virus. To define CA surfaces that function at different stages of the viral life cycle, a total of 48 different alanine-scanning surface mutations in CA were tested for their effects on Gag protein expression, processing, particle production and morphology, capsid assembly, and infectivity. The 27 detrimental mutations fall into three classes: 13 mutations significantly diminished or altered particle production, 9 mutations failed to assemble normal capsids, and 5 mutations supported normal viral assembly but were nevertheless reduced more than 20-fold in infectivity. The locations of the assembly-defective mutations implicate three different CA surfaces in immature particle assembly: one surface encompasses helices 4 to 6 in the CA N-terminal domain (NTD), a second surrounds the crystallographically defined CA dimer interface in the C-terminal domain (CTD), and a third surrounds the loop preceding helix 8 at the base of the CTD. Mature capsid formation required a distinct surface encompassing helices 1 to 3 in the NTD, in good agreement with a recent structural model for the viral capsid. Finally, the identification of replication-defective mutants with normal viral assembly phenotypes indicates that CA also performs important nonstructural functions at early stages of the viral life cycle.