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.     

Basic residues in the nucleocapsid domain of Gag are critical for late events of HIV-1 budding

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind the cellular proteins Tsg101 and Alix, respectively. These interactions are thought to recruit members of the host fission machinery (ESCRT) to facilitate HIV-1 release. Here we report a new role for the p6-adjacent nucleocapsid (NC) domain in HIV-1 release. The mutation of basic residues in NC caused a pronounced decrease in virus release from 293T cells, although NC mutant Gag proteins retained the ability to interact with cellular membranes and RNAs. Remarkably, electron microscopy analyses of these mutants revealed arrested budding particles at the plasma membrane, analogous to those seen following the disruption of the PTAP motif. This result indicated that the basic residues in NC are important for virus budding. When analyzed in physiologically more relevant T-cell lines (Jurkat and CEM), NC mutant viruses remained tethered to the plasma membrane or to each other by a membranous stalk, suggesting membrane fission impairment. Remarkably, NC mutant release defects were alleviated by the coexpression of a Gag protein carrying a wild-type (WT) NC domain but devoid of all L domain motifs and by providing alternative access to the ESCRT pathway, through the in trans expression of the ubiquitin ligase Nedd4.2s. Since NC mutant Gag proteins retained the interaction with Tsg101, we concluded that NC mutant budding arrests might have resulted from the inability of Gag to recruit or utilize members of the host ESCRT machinery that act downstream of Tsg101. Together, these data support a model in which NC plays a critical role in HIV-1 budding.     

Human immunodeficiency virus type 1 Gag contains a dileucine-like motif that regulates association with multivesicular bodies

Multivesicular bodies (MVBs) are cholesterol-enriched organelles formed by the endocytic pathway. The topology of vesicle formation in MVBs is identical to that of retroviral budding from the plasma membrane, and budding of human immunodeficiency virus type 1 (HIV-1) into MVBs in macrophages has recently been visualized. The Gag proteins from HIV-1, as well as many other retroviruses, contain short motifs that mediate interactions with MVBs and other endocytic components, suggesting that Gag proteins directly interface with the endocytic pathway. Here, we show that HIV-1 Gag contains an internalization signal that promotes endocytosis of a chimeric transmembrane fusion protein. Mutation of this motif within Gag strongly inhibits virus-like particle production. Moreover, wild-type Gag, but not the internalization-defective mutation, can be induced to accumulate within CD63-positive MVBs by treatment of cells with U18666A, a drug that redistributes cholesterol from the plasma membrane to MVBs. We propose that HIV-1 Gag contains a signal that promotes interaction with the cellular endocytic machinery and that the site of particle production is regulated by the subcellular distribution of cholesterol.     

Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.     

The PTAP sequence within the p6 domain of human immunodeficiency virus type 1 Gag regulates its ubiquitination and MHC class I antigen presentation

Endogenous peptides presented by MHC class I (MHC-I) molecules are mostly derived from de novo synthesized, erroneous proteins, so-called defective ribosomal products (DRiPs), which are rapidly degraded via the ubiquitin-proteasome pathway. We have previously shown that the HIV-1 Gag protein represents a bona fide substrate for the DRiP pathway and that the amount of Gag-DRiPs can be enhanced by the introduction of an N-end rule degradation signal, leading to increased MHC-I presentation and immunogenicity of Gag. Based on these findings, we sought to identify a naturally occurring sequence motif within Gag that regulates its entry into the DRiP pathway. As the PTAP late assembly domain motif in the C-terminal p6 domain of Gag has been shown to negatively regulate the ubiquitination of Gag, we analyzed the correlation between ubiquitination and MHC-I presentation of PTAP-deficient Gag. Intriguingly, mutation of PTAP not only reduces the release of virus-like particles, but also increases ubiquitination of Gag and, consistently, enhances MHC-I presentation of a Gag-derived epitope. Although the half-life of the PTAP mutant was only mildly reduced, the entry into the DRiP pathway was significantly increased, as demonstrated by short-term pulse-chase analyses under proteasome inhibition. Collectively, these results indicate that, besides driving virus release, the PTAP motif regulates the entry of Gag into the DRiP pathway and, thus, into the MHC-I pathway. Although there are no naturally occurring PTAP mutants of HIV-1, mutations of PTAP might enhance the immunogenicity of Gag and, thus, be considered for the improvement of vaccine development.     

Evidence of a role for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery in HIV-1 assembly and release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.     

Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.     

Role of the feline immunodeficiency virus L-domain in the presence or absence of Gag processing: involvement of ubiquitin and Nedd4-2s ligase in viral egress

RNA-enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L-domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non-primate lentivirus, is strictly dependent for its budding on a "PSAP"-type L-domain, mapping in the carboxy-terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L-domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4-2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L-domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non-primate lentivirus shares with HIV-1 a novel mechanism of connection to the cellular budding machinery.     

Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 gag polyprotein and results in increased levels of ubiquitinated gag

The final stages of budding and release of a retroviral particle from the cell require the late (L) domain of Gag. Recently, ubiquitin and ubiquitin ligases have been implicated in the late stages of retroviral budding. In a yeast two-hybrid screen of a T-cell cDNA library to identify cellular proteins that interact with human immunodeficiency virus type 2 (HIV-2) Gag polyprotein, we identified Tsg101, an inactive homologue of ubiquitin ligase E2. Tsg101 and HIV-2 Gag interact specifically in vitro and in vivo. The interaction requires the L domain PTAPP motif in the p6 domain of HIV-2 Gag and the N-terminal Ubc-conjugation homology domain of Tsg101. Tsg101 is incorporated into HIV-2 virions. Expression of the N-terminal Ubc-conjugation homology domain of Tsg101 inhibits the release of HIV-2 virus particles. Overexpression of Tsg101 results in an increase in the level of ubiquitination of HIV-2 Gag. Our results provide evidence for recruitment of the ubiquitination machinery of the cell during late stages of the viral life cycle, mediated by the viral Gag protein.     

Human Endogenous Retrovirus K Gag Coassembles with HIV-1 Gag and Reduces the Release Efficiency and Infectivity of HIV-1

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.     

Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag

The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P(2) interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P(2) dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P(2) is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P(2). Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P(2). Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P(2) and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.     

The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding

Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.     

Cellular motor protein KIF-4 associates with retroviral Gag

Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4-Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.     

BCA2/Rabring7 Targets HIV-1 Gag for Lysosomal Degradation in a Tetherin-Independent Manner

BCA2 (Rabring7, RNF115 or ZNF364) is a RING-finger E3 ubiquitin ligase that was identified as a co-factor in the restriction imposed by tetherin/BST2 on HIV-1. Contrary to the current model, in which BCA2 lacks antiviral activity in the absence of tetherin, we found that BCA2 possesses tetherin-independent antiviral activity. Here we show that the N-terminus of BCA2 physically interacts with the Matrix region of HIV-1 and other retroviral Gag proteins and promotes their ubiquitination, redistribution to endo-lysosomal compartments and, ultimately, lysosomal degradation. The targeted depletion of BCA2 in tetherin-expressing and tetherin-deficient cells results in a significant increase in virus release and replication, indicating that endogenous BCA2 possesses antiviral activity. Therefore, these results indicate that BCA2 functions as an antiviral factor that targets HIV-1 Gag for degradation, impairing virus assembly and release.     

Late assembly domain function can exhibit context dependence and involves ubiquitin residues implicated in endocytosis

Retroviral Gag polyproteins contain regions that promote the separation of virus particles from the plasma membrane and from each other. These Gag regions are often referred to as late assembly (L) domains. The L domain of human immunodeficiency virus type 1 (HIV-1) is in the C-terminal p6(gag) domain and harbors an essential P(T/S)APP motif, whereas the L domains of oncoretroviruses are in the N-terminal half of the Gag precursor and have a PPXY core motif. We recently observed that L domains induce the ubiquitination of a minimal HIV-1 Gag construct and that point mutations which abolish L domain activity prevent Gag ubiquitination. In that study, a peptide from the Ebola virus L domain with overlapping P(T/S)APP and PPXY motifs showed exceptional activity in promoting Gag ubiquitination and the release of virus-like particles. We now show that a substitution which disrupts the PPXY motif but leaves the P(T/S)APP motif intact abolishes L domain activity in the minimal Gag context, but not in the context of a near full-length HIV-1 Gag precursor. Our results reveal that the P(T/S)APP motif does not function autonomously and indicate that the HIV-1 nucleocapsid-p1 region, which is proximal to p6(gag), can cooperate with the conserved L domain core motif. We have also examined the effects of ubiquitin mutants on virus-like particle production, and the results indicate that residues required for the endocytosis function of ubiquitin are also involved in virus budding.     

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.     

Inhibition of HIV-1 maturation via drug association with the viral Gag protein in immature HIV-1 particles

The small molecule 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently inhibits human immunodeficiency virus, type 1 (HIV-1) replication by interfering with proteolytic cleavage of the viral Gag protein at a specific site. Here we have demonstrated that the antiviral mechanism involves the association of DSB with Gag at a 1:1 stoichiometry within immature HIV-1 particles. The binding was specific, as mutations in Gag that confer resistance to DSB inhibited the association, which could be competed by DSB but not by the inactive compound betulinic acid. The addition of DSB to purified immature viral cores inhibited the cleavage of Gag at the CA-SP1 junction in vitro, thus reproducing the effect of the drug when present during maturation of HIV-1 particles. Based on these findings, we propose a model in which a trimer of DSB associates with the CA-SP1 junction of adjacent subunits within the Gag polymer. The model may explain the ability of highly similar compounds to specifically target the seemingly unrelated steps of HIV-1 maturation and virus entry.     

Filamin A protein interacts with human immunodeficiency virus type 1 Gag protein and contributes to productive particle assembly

HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection.     

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Important role for the CA-NC spacer region in the assembly of bovine immunodeficiency virus Gag protein

Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.