Functional replacement of a retroviral late domain by ubiquitin fusion

Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/DeltaYPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/DeltaYPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.     

Identification of domains in gag important for prototypic foamy virus egress

Sequence motifs (L domains) have been described in viral structural proteins. Mutations in these lead to a defect at a late stage in virus assembly and budding. For several viruses, recruitment of an endosomal sorting complexes required for transport 1 subunit (Tsg101), a component of the class E vacuolar protein sorting (EVPS) machinery, is a prerequisite for virion budding. To effect this, Tsg101 interacts with the PT/SAP L domain. We have identified candidate L-domain motifs, PSAP, PPPI, and YEIL, in the prototypic foamy virus (PFV) Gag protein, based on their homology to known viral L domains. Mutation of the PSAP and PPPI motifs individually reduced PFV egress, and their combined mutation had an additive effect. When PSAP was mutated, residual infectious PFV release was unaffected by dominant negative Vps4 (an ATPase involved in the final stages of budding), and sensitivity to dominant negative Tsg101 was dramatically reduced, suggesting that the PSAP motif functions as a conventional class E VPS-dependent L domain. Consistent with this notion, yeast two-hybrid analysis showed a PSAP motif-dependent interaction between PFV Gag and Tsg101. Surprisingly, PFV release which is dependent on the PPPI motif was Vps4-independent and was partially inhibited by dominant negative Tsg101, suggesting that PPPI functions by an unconventional mechanism to facilitate PFV egress. Mutation of the YEIL sequence completely abolished particle formation and also reduced the rate of Gag processing by the viral protease, suggesting that the integrity of YEIL is required at an assembly step prior to budding and YEIL is not acting as an L domain.     

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.     

The C-terminal portion of the Hrs protein interacts with Tsg101 and interferes with human immunodeficiency virus type 1 Gag particle production

The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in "lump-like" structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.     

The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment

The endosomal sorting complex I required for transport (ESCRT-I) is composed of the three subunits Vps23/Tsg101, Vps28 and Vps37. ESCRT-I is recruited to cellular membranes during multivesicular endosome biogenesis and by enveloped viruses such as HIV-1 to mediate budding from the cell. Here, we describe the crystal structure of a conserved C-terminal domain from Sacharomyces cerevisiae Vps28 (Vps28-CTD) at 3.05 A resolution which folds independently into a four-helical bundle structure. Co-expression experiments of Vps28-CTD, Vps23 and Vps37 suggest that Vps28-CTD does not directly participate in ESCRT-I assembly and may thus act as an adaptor module for downstream interaction partners. We show through mutagenesis studies that Vps28-CTD employs its strictly conserved surface in the interaction with the ESCRT-III factor Vps20. Furthermore, we present evidence that Vps28-CTD is sufficient to rescue an equine infectious anaemia virus (EIAV) Gag late domain deletion. Vps28-CTD mutations abolishing Vps20 interaction in vitro also prevent the rescue of the EIAV Gag late domain mutant consistent with a potential direct Vps28-ESCRT-III Vps20 recruitment. Therefore, the physiological relevant EIAV Gag-Alix interaction can be functionally replaced by a Gag-Vps28-CTD fusion. Because both Alix and Vps28-CTD can directly recruit ESCRT-III proteins, ESCRT-III assembly coupled to Vps4 action may therefore constitute the minimal budding machinery for EIAV release.     

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.     

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.     

Tsg101 and Alix interact with murine leukemia virus Gag and cooperate with Nedd4 ubiquitin ligases during budding

Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses.     

The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.     

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.     

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.     

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.     

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.     

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.     

Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the mu2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an approximately 300-residue region of AIP1/Alix-(409-715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 mu2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant mu2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities.     

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.     

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.     

AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding

HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.     

ESCRT machinery potentiates HIV-1 utilization of the PI(4,5)P(2)-PLC-IP3R-Ca(2+) signaling cascade

Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P₇TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY₃₆PX_nL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca²⁺ release from intracellular stores, triggers Ca²⁺ cell influx and thereby functions as a major regulator of Ca²⁺ signaling. In the present study, we determined whether the L domain links Gag to Ca²⁺ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca²⁺ was elevated, events indicative of induction of Ca²⁺ signaling. The results suggest that L domain function, ESCRT machinery and Ca²⁺ signaling are linked events in Gag release.     

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.