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.     

Molecular characterization of feline immunodeficiency virus budding

Infection of domestic cats with feline immunodeficiency virus (FIV) is an important model system for studying human immunodeficiency virus type 1 (HIV-1) infection due to numerous similarities in pathogenesis induced by these two lentiviruses. However, many molecular aspects of FIV replication remain poorly understood. It is well established that retroviruses use short peptide motifs in Gag, known as late domains, to usurp cellular endosomal sorting machinery and promote virus release from infected cells. For example, the Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif of HIV-1 Gag interacts directly with Tsg101, a component of the endosomal sorting complex required for transport I (ESCRT-I). A Tyr-Pro-Asp-Leu (YPDL) motif in equine infectious anemia virus (EIAV), and a related sequence in HIV-1, bind the endosomal sorting factor Alix. In this study we sought to identify and characterize FIV late domain(s) and elucidate cellular machinery involved in FIV release. We determined that mutagenesis of a PSAP motif in FIV Gag, small interfering RNA-mediated knockdown of Tsg101 expression, and overexpression of a P(T/S)AP-binding fragment of Tsg101 (TSG-5′) each inhibited FIV release. We also observed direct binding of FIV Gag peptides to Tsg101. In contrast, mutagenesis of a potential Alix-binding motif in FIV Gag did not affect FIV release. Similarly, expression of the HIV-1/EIAV Gag-binding domain of Alix (Alix-V) did not disrupt FIV budding, and FIV Gag peptides showed no affinity for Alix-V. Our data demonstrate that FIV relies predominantly on a Tsg101-binding PSAP motif in the C terminus of Gag to promote virus release in HeLa cells, and this budding mechanism is highly conserved in feline cells.     

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.     

The HIV-1 p6/EIAV p9 docking site in Alix is autoinhibited as revealed by a conformation-sensitive anti-Alix monoclonal antibody

Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], a component of the endosomal sorting machinery, contains a three-dimensional docking site for HIV-1 p6(Gag) or EIAV (equine infectious anaemia virus) p9(Gag), and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. In the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6(Gag)/p9(Gag) and we used this antibody to probe the accessibility of the docking site in Alix. Our results show that the docking site is not available in cytosolic or recombinant Alix under native conditions and becomes available upon addition of the detergent Nonidet P40 or SDS. In HEK (human embryonic kidney)-293 cell lysates, an active p6(Gag)/p9(Gag) docking site is specifically available in Alix from the membrane fraction. The findings of the present study demonstrate that formation or exposure of the p6(Gag)/p9(Gag) docking site in Alix is a regulated event and that Alix association with the membrane may play a positive role in this process.     

Assembly and replication of HIV-1 in T cells with low levels of phosphatidylinositol-(4,5)-bisphosphate

HIV-1 Gag assembles into virus particles predominantly at the plasma membrane (PM). Previously, we observed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] is essential for Gag binding to the plasma membrane and virus release in HeLa cells. In the current study, we found that PI(4,5)P(2) also facilitates Gag binding to the PM and efficient virus release in T cells. Notably, serial passage of HIV-1 in an A3.01 clone that expresses polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), yielded an adapted mutant with a Leu-to-Arg change at matrix residue 74 (74LR). Virus replication in T cells expressing 5ptaseIV was accelerated by the 74LR mutation relative to replication of wild type HIV-1 (WT). This accelerated replication of the 74LR mutant was not due to improved virus release. In control T cells, the 74LR mutant releases virus less efficiently than does the WT, whereas in cells expressing 5ptaseIV, the WT and the 74LR mutant are similarly inefficient in virus release. Unexpectedly, we found that the 74LR mutation increased virus infectivity and compensated for the inefficient virus release. Altogether, these results indicate that PI(4,5)P(2) is essential for Gag-membrane binding, targeting of Gag to the PM, and efficient virus release in T cells, which in turn likely promotes efficient virus spread in T cell cultures. In T cells with low PI(4,5)P(2) levels, however, the reduced virus particle production can be compensated for by a mutation that enhances virus infectivity.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

Cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding defect

The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.     

Human immunodeficiency virus type 1 Gag engages the Bro1 domain of ALIX/AIP1 through the nucleocapsid

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.     

Potent rescue of human immunodeficiency virus type 1 late domain mutants by ALIX/AIP1 depends on its CHMP4 binding site

The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.     

Budding of retroviruses utilizing divergent L domains requires nucleocapsid

We recently reported that human immunodeficiency virus type 1 (HIV-1) carrying PTAP and LYPX(n)L L domains ceased budding when the nucleocapsid (NC) domain was mutated, suggesting a role for NC in HIV-1 release. Here we investigated whether NC involvement in virus release is a property specific to HIV-1 or a general requirement of retroviruses. Specifically, we examined a possible role for NC in the budding of retroviruses relying on divergent L domains and structurally homologous NC domains that harbor diverse protein sequences. We found that NC is critical for the release of viruses utilizing the PTAP motif whether it functions within its native Gag in simian immunodeficiency virus cpzGAB2 (SIVcpzGAB2) or SIVsmmE543 or when it is transplanted into the heterologous Gag protein of equine infectious anemia virus (EIAV). In both cases, virus release was severely diminished even though NC mutant Gag proteins retained the ability to assemble spherical particles. Moreover, budding-defective NC mutants, which displayed particles tethered to the plasma membrane, were triggered to release virus when access to the cell endocytic sorting complex required for transport pathway was restored (i.e., in trans expression of Nedd4.2s). We also examined the role of NC in the budding of EIAV, a retrovirus relying exclusively on the (L)YPX(n)L-type L domain. We found that EIAV late budding defects were rescued by overexpression of the isolated Alix Bro1 domain (Bro1). Bro1-mediated rescue of EIAV release required the wild-type NC. EIAV NC mutants lost interactions with Bro1 and failed to produce viruses despite retaining the ability to self-assemble. Together, our studies establish a role for NC in the budding of retroviruses harboring divergent L domains and evolutionarily diverse NC sequences, suggesting the utilization of a common conserved mechanism and/or cellular factor rather than a specific motif.     

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.     

Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body

The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6(Gag) and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.     

The Phe105 loop of Alix Bro1 domain plays a key role in HIV-1 release

Alix and cellular paralogs HD-PTP and Brox contain N-terminal Bro1 domains that bind ESCRT-III CHMP4. In contrast to HD-PTP and Brox, expression of the Bro1 domain of Alix alleviates HIV-1 release defects that result from interrupted access to ESCRT. In an attempt to elucidate this functional discrepancy, we solved the crystal structures of the Bro1 domains of HD-PTP and Brox. They revealed typical "boomerang" folds they share with the Bro1 Alix domain. However, they each contain unique structural features that may be relevant to their specific function(s). In particular, phenylalanine residue in position 105 (Phe105) of Alix belongs to a long loop that is unique to its Bro1 domain. Concurrently, mutation of Phe105 and surrounding residues at the tip of the loop compromise the function of Alix in HIV-1 budding without affecting its interactions with Gag or CHMP4. These studies identify a new functional determinant in the Bro1 domain of Alix.     

p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease.

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

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.     

Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-[3',3'-dimethylsuccinyl]-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.     

Structural basis for viral late-domain binding to Alix

The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of human immunodeficiency virus (HIV)-1. The Gag p6 protein of HIV-1 contains a LYPx(n)LxxL motif that is required for Alix-mediated budding and binds a region of Alix spanning residues 360-702. The structure of this fragment of Alix has the shape of the letter 'V' and is termed the V domain. The V domain has a topologically complex arrangement of 11 alpha-helices, with connecting loops that cross three times between the two arms of the V. The conserved residue Phe676 is at the center of a large hydrophobic pocket and is crucial for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.     

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.     

Defects in human immunodeficiency virus budding and endosomal sorting induced by TSG101 overexpression

Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5') suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5' on the inhibition of HIV-1 release. We observed that a mutation in TSG-5' that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5' to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3') potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3' overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5' suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3' globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5', target HIV-1 without disrupting endosomal sorting.