Quantitative Fluorescence Resonance Energy Transfer Microscopy Analysis of the Human Immunodeficiency Virus Type 1 Gag-Gag Interaction: Relative Contributions of the CA and NC Domains and Membrane Binding

The human immunodeficiency virus type 1 structural polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.The human immunodeficiency virus type 1 (HIV-1) structural precursor polyprotein Pr55^Gag is necessary and sufficient for the assembly of virus-like particles (VLPs). Gag is composed of four major structural domains, matrix (MA), capsid (CA), nucleocapsid (NC), and p6, as well as two spacer peptides, SP1 and SP2 (3, 30, 94). Following particle assembly and release, cleavage by HIV-1 protease separates these domains. However, these domains must work together in the context of the full-length Gag polyprotein to drive particle assembly.Previous studies have mapped two major functional domains involved in the early steps of assembly: first, Gag associates with cellular membranes via basic residues and N-terminal myristoylation of the MA domain (10, 17, 20, 35, 39, 87, 91, 106); second, the Gag-Gag interaction domains that span the CA C-terminal domain (CA-CTD) and NC domain promote Gag multimerization (3, 11, 14, 16, 18, 23, 27, 29, 30, 33, 36, 46, 64, 88, 94, 102, 103). Structural and genetic studies have identified two residues (W184 and M185) within a dimerization interface in the CA-CTD that are critical to CA-CA interactions (33, 51, 74, 96). Analytical ultracentrifugation of heterodimers formed between wild-type (WT) Gag and Gag mutants with changes at these residues suggests that the dimerization interface consists of two reciprocal interactions, one of which can be disrupted to form a “half-interface” (22).In addition to the CA-CTD, NC contributes to assembly via 15 basic residues (8, 9, 11, 14, 18, 23, 25, 28, 34, 40, 43, 54, 57, 58, 74, 79, 88, 97, 104, 105), although some researchers have suggested that NC instead contributes to the stability of mature virions after assembly (75, 98, 99). It is thought that the contribution of NC to assembly is due to its ability to bind RNA, since the addition of RNA promotes the formation of particles in vitro (14-16, 37, 46), and RNase treatment disrupts Gag-Gag interactions (11) and immature viral cores (67). However, RNA is not necessary per se, since dimerization motifs can substitute for NC (1, 4, 19, 49, 105). This suggests a model in which RNA serves a structural role, such as a scaffold, to promote Gag-Gag interactions through NC. Based on in vitro studies, it has been suggested that this RNA scaffolding interaction facilitates the low-order Gag multimerization mediated by CA-CTD dimerization (4, 37, 49, 62, 63, 85). Despite a wealth of biochemical data, the relative contributions of the CA-CTD and NC to Gag multimerization leading to assembly are yet to be determined in cells.Mutations in Gag interaction domains alter membrane binding in addition to affecting Gag multimerization. In particular, mutations or truncations of CA reduce membrane binding (21, 74, 82), and others previously reported that mutations or truncations of NC affect membrane binding (13, 78, 89, 107). These findings are consistent with a myristoyl switch model of membrane binding in which Gag can switch between high- and low-membrane-affinity states (38, 71, 76, 83, 86, 87, 92, 95, 107). Many have proposed, and some have provided direct evidence (95), that Gag multimerization mediated by CA or NC interactions promotes the exposure of the myristoyl moiety to facilitate membrane associations.Gag membrane binding and multimerization appear to be interrelated steps of virus assembly, since membrane binding also facilitates Gag multimerization. Unlike betaretroviruses that fully assemble prior to membrane targeting and envelopment (type B/D), lentiviruses, such as HIV, assemble only on cellular membranes at normal Gag expression levels (type C), although non-membrane-bound Gag complexes exist (45, 58, 60, 61, 65). Consistent with this finding, mutations that reduce Gag membrane associations cause a defect in Gag multimerization (59, 74). Therefore, in addition to their primary effects on Gag-Gag interactions, mutations in Gag interaction domains cause a defect in membrane binding, which, in turn, causes a secondary multimerization defect. To determine the relative contributions of the CA-CTD and the NC domain to Gag-Gag interactions at the plasma membrane, it is essential to eliminate secondary effects due to a modulation of membrane binding.Except for studies using a His-tag-mediated membrane binding system (5, 46), biochemical studies of C-type Gag multimerization typically lack membranes. Therefore, these studies do not fully represent particle assembly, which occurs on biological membranes in cells. Furthermore, many biochemical and structural approaches are limited to isolated domains or truncated Gag constructs. Thus, some of these studies are perhaps more relevant to the behavior of protease-cleaved Gag in mature virions. With few exceptions (47, 74), cell-based studies of Gag multimerization have typically been limited to measuring how well mutant Gag is incorporated into VLPs when coexpressed or not with WT Gag. Since VLP production is a complex multistep process, effects of mutations on other steps in the process can confound this indirect measure. For example, NC contributes to VLP production by both promoting multimerization and interacting with the host factor ALIX to promote VLP release (26, 80). To directly assay Gag multimerization in cells, several groups (24, 45, 52, 56) developed microscopy assays based on fluorescence resonance energy transfer (FRET). These assays measure the transfer of energy between donor and acceptor fluorescent molecules that are brought within ∼5 nm by the association of the proteins to which they are attached (41, 48, 90). However, these microscopy-based Gag FRET assays have not been used to fully elucidate several fundamental aspects of HIV-1 Gag multimerization at the plasma membrane of cells, such as the relative contributions of the CA-CTD and NC and the effect of membrane binding on Gag-Gag interactions. In this study, we used a FRET stoichiometry method based on calibrated spectral analysis of fluorescence microscopy images (41). This algorithm determines the fractions of both donor and acceptor fluorescent protein-tagged Gag molecules participating in FRET. For cells expressing Gag molecules tagged with donor (cyan fluorescent protein [CFP]) and acceptor (yellow fluorescent protein [YFP]) molecules, this method measures the apparent FRET efficiency, which is proportional to the mole fraction of Gag constructs in complex. By measuring apparent FRET efficiencies, quantitative estimates of the mole fractions of interacting proteins can be obtained.Using this FRET-based assay, we aim to answer two questions: (i) what are the relative contributions of CA-CTD and NC domains to Gag multimerization when secondary effects via membrane binding are held constant, and (ii) what is the effect of modulating membrane binding on the ability of Gag mutants to interact with WT Gag?Our data demonstrate that the CA-CTD dimerization interface is essential for Gag multimerization at the plasma membrane, as fully disrupting the CA-CTD interaction abolishes FRET, whereas a modest level of FRET is still detected in the absence of NC. We also present evidence that the CA-CTD dimerization interface consists of two reciprocal interactions, allowing the formation of a half-interface that can still contribute to Gag multimerization. Notably, when Gag derivatives with an intact CA-CTD were coexpressed with WT Gag, either membrane binding ability or NC was required for the Gag mutants to interact with WT Gag, suggesting functional compensation between these factors.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Late Domain-Independent Rescue of a Release-Deficient Moloney Murine Leukemia Virus by the Ubiquitin Ligase Itch

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell''s budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.Retroviruses require access to the host budding machinery to exit the cell (5, 13, 40). To this end, retroviral Gag polyproteins use short sequences called late (L) domains to promote virus release by recruiting members of the host vacuolar protein sorting (vps) machinery. In the cell, vps proteins are involved in membrane dynamics that facilitate the separation of daughter cells at the completion of cytokinesis (9, 39) and the budding of vesicles into endosomal compartments or multivesicular bodies (MVB) (2, 23), a process topologically similar to virus budding (57). Class E vps proteins are organized into three heteromeric endosomal complexes (called endosomal sorting complexes) required for transport, namely, ESCRT-I, -II, and -III (2). In the current model for budding, sequential recruitment of ESCRT components on the cytoplasmic face of the membrane facilitates vesicle invagination into MVB compartments and viral egress from the cell (2). The disassembly of ESCRT-III components is catalyzed by the activity of VPS4 AAA-type ATPase, which in turn is presumed to trigger membrane fission events (3, 50). Any disruption in this sequence, such as mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase, adversely affects virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (19, 21, 34, 49, 57).Currently, three types of L domain motifs have been identified: PT/SAP, LYPX_nL, and PPPY. All retroviral Gag molecules contain at least one of these motifs, as multiple L domains are believed to synergistically function to ensure efficient viral release. Moloney murine leukemia virus (MoMLV) Gag carries all three L domain motifs, PSAP, LYPAL, and PPPY, which bind the vps protein Tsg101, the ESCRT-associated protein Alix (46), and members of the Nedd4-ubiquitin ligase family (33), respectively. In HIV-1, the PTAP motif in the p6 region of Gag binds Tsg101 (16, 56), which functions in viral budding (16, 35) as a member of ESCRT-I (16, 36, 57). The LYPX_nL motif is also located in p6 and is the binding site for Alix (49, 57), a protein that also interacts with the nucleocapsid domain of HIV-1 Gag (14, 43) and links Gag to components of ESCRT-III (14). Similarly, the human T-cell leukemia virus (HTLV-I) Gag carries PPPY and PTAP L domains, which both contribute to efficient HTLV-1 release (6, 7, 21). The PPPY L domain motif, which is found in numerous retroviral Gag polyproteins (6, 7, 19, 21, 27, 28, 61, 62), plays a critical role in MoMLV release, as mutations disrupting its sequence lead to significant decreases in virus budding and release (33, 62). PSAP and LYPAL, the additional L domain motifs, are believed to serve little to no role in the release of MoMLV Gag virus-like particles (45, 46).The role of Nedd4-like ubiquitin ligases in budding events was initially established by data obtained with the yeast Nedd4-like ligase Rsp5, an enzyme that ubiquitinates surface proteins, thus signaling their incorporation into the MVB pathway (26). From retroviral budding studies, multiple findings support the notion that Nedd4-like ubiquitin ligases link PPPY-containing Gag proteins to the host ESCRT machinery. For example, mutations in the PPPY motif or expression of dominant-negative versions of Nedd4-like ligases resulted in budding defects similar to those seen upon interference with the function of ESCRT-III members (7, 21, 27, 28, 33, 62). Overexpression of Nedd4-like ligases WWP1 and Itch corrected the budding defects of a MoMLV PPPY mutant that retained residual binding to both ligases (33). Also, when transplanted to a heterologous retroviral Gag, the PPPY L domain creates a requirement for Nedd4-like ubiqutin ligase activity to facilitate viral release that is dependent on the presence of a functional ESCRT pathway (63). Collectively, these observations support the notion that Nedd4-like ubiquitin ligases link retroviral Gag polyproteins to components of the ESCRT pathway necessary for budding.Both endosomal and viral budding require the ubiquitin conjugation properties of Nedd4-like ligases, indicating that ubiquitin transfer to a key protein(s) is necessary to promote budding. A role for Gag ubiquitination in viral budding has been suggested (8, 20, 22, 48). In fact, ubiquitin attachment to equine infectious anemia virus (EIAV) Gag can substitute for the lack of L domains and rescue viral budding (25), suggesting that ubiquitin molecules conjugated to Gag can signal the recruitment of the host ESCRT machinery. For feline immunodeficiency virus, efficient budding seems to require L domain-dependent ubiquitination of Gag proteins (8) that is independent of the L domain ability to directly recruit Nedd4-like ubiquitin ligases (i.e., by means of the PT/SAP L domain motif) (8). Similarly, ubiquitination of HTLV-1 Gag was also shown to play a significant role in viral release (22). Conversely, data arguing in favor of a role for the ubiquitination of transacting factors, but not Gag, in the facilitation of viral budding have also been reported (10, 63). Thus Gag polyproteins recruit, in a PPPY-dependent or -independent manner, enzymatically active Nedd4-like ubiquitin ligases that conjugate ubiquitin molecules to Gag or to Gag-binding host factors. Such interactions, whether direct or indirect, are believed to link the viral protein to the host ESCRT pathway and facilitate release.In addition to the well-characterized cellular proteins that bind primary L domain motifs, retroviral Gag can recruit other host factors, either via secondary L domains or independently of L domains (10, 24, 29, 55, 59). These cellular factors are believed to promote virus production by facilitating Gag protein trafficking to the plasma membrane and/or providing additional L domain-independent links to the host vps pathway. Examples of these parallel pathways are illustrated in the rescue of a budding-defective HIV-1 lacking the PTAP domain by overexpression of Alix (15, 54) and in the remarkably potent rescue of HIV-1 lacking all known L domains by the overexpression of Nedd4.2s, a Nedd4.2 isoform that belongs to the Nedd4-like ubiquitin ligase family (10, 55). In this study, we sought to identify host cell factors that rescue budding defects of the MoMLV mutant lacking the PPPY motif (MoMLV AAAY mutant). Our studies provide evidence that Itch overexpression rescued budding and infectivity defects of the MoMLV AAAY mutant virus, indicating that Gag can recruit the ubiquitin ligase Itch in an L domain-independent manner to facilitate MoMLV release via a mechanism that involves Gag ubiquitination.     

Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.The encapsidation of the RNA genome is essential for retrovirus replication. Because the viral genomic RNA (gRNA) constitutes only a small fraction of the total cellular mRNA, a specific Gag-RNA interaction is thought to be required for viral genome packaging (2). The determinants of virus-specific gRNA incorporation include the cis-acting element at the 5′end of the viral gRNA, known as the packaging signal (ψ), and the nucleocapsid (NC) domain of the Gag polyprotein (3, 14, 62). In Rous sarcoma virus (RSV), the NC domain contains basic residues that are required for the recognition of and binding to ψ, as well as two Cys-His motifs that maintain the overall conformation of NC and are essential for RNA packaging (30, 31).Packaging of gRNA into progeny virions requires that the unspliced viral mRNA be exported from the nucleus. However, cellular proofreading mechanisms ensure that unspliced or intron-containing mRNAs are retained in the nucleus until splicing occurs. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) overcome this export block of unspliced genomes by encoding the Rev protein, which interacts with a cis-acting sequence in the viral RNA (the Rev-responsive element [RRE]) to facilitate cytoplasmic accumulation of intron-containing viral mRNA (16, 35). The export of the Rev-viral RNA complex is mediated through the interaction of a leucine-rich nuclear export signal (NES) in Rev with the CRM1 nuclear export factor (17, 18, 37, 41). Simple retroviruses do not encode Rev-like regulatory proteins, so other strategies for the export of unspliced viral RNAs are needed. For Mason-Pfizer monkey virus, a cis-acting constitutive transport element induces nuclear export of the unspliced viral RNA in a process mediated by the cellular mRNA nuclear export factor TAP (5, 25, 46, 63). In RSV, an RNA element composed of either of the two direct repeats flanking the src gene mediates the cytoplasmic accumulation of unspliced viral RNA by using host export proteins TAP and Dpb5 (29, 42, 44).The findings of recent studies suggest that specific RNA export pathways direct viral gRNA to sites of virion assembly (56); for example, HIV-1 gRNA export out of the nucleus by the Rev-RRE-CRM1 complex is required for the proper subcellular localization of Gag and efficient virus particle production (26, 57). In the case of RSV, little is known about the trafficking of the viral RNA destined for virion encapsidation or the effects of the gRNA nuclear export pathway on Gag trafficking and virus particle production. However, we do know that RSV Gag enters the nucleus during infection, owing to nuclear localization signals (NLSs) in the matrix (MA) and NC domains. The nuclear localization of Gag is transient, and export is mediated by a CRM1-dependent NES in the p10 region (6, 52, 53). Thus, it is feasible that Gag may facilitate the nuclear export of the gRNA, either directly or indirectly, to promote particle assembly (53).In support of this idea, Gag mutants engineered to be more efficiently directed to the plasma membrane than wild-type Gag by the addition of the Src membrane-binding domain (in Myr1E virus) or by the insertion of extra basic residues (in SuperM virus) are not concentrated in nuclei when cells are treated with the CRM1 inhibitor leptomycin B (LMB) (8, 20, 53). Moreover, Myr1E and SuperM virus particles incorporate reduced levels of viral gRNA compared to the levels incorporated by wild-type particles. Thus, there is a correlation between the nuclear transit of Gag and gRNA packaging, although the Myr1E and SuperM viruses may be deficient in gRNA encapsidation because they are transported to the plasma membrane too rapidly (8). To test the hypothesis that the loss of Gag nuclear trafficking is responsible for the gRNA packaging defect, we used a gain-of-function genetic approach whereby a heterologous NLS was inserted into Myr1E Gag, yielding mutant virus Myr1E.NLS. Our results revealed that restoring the nuclear trafficking of Myr1E Gag also restored the incorporation of gRNA into mutant virus particles.     

Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability,Localization, and Subsequent Budding of Virus-Like Particles

The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the ₉₆LPLGVA₁₀₁ sequence of eVP40 and the corresponding ₈₄LPLGIM₈₉ sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the ₉₆LPLGVA₁₀₁ motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the ₉₆LPLGVA₁₀₁ motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.Ebola and Marburg viruses are members of the family Filoviridae. Filoviruses are filamentous, negative-sense, single-stranded RNA viruses that cause lethal hemorrhagic fevers in both humans and nonhuman primates (5). Filoviruses encode seven viral proteins including: NP (major nucleoprotein), VP35 (phosphoprotein), VP40 (matrix protein), GP (glycoprotein), VP30 (minor nucleoprotein), VP24 (secondary matrix protein), and L (RNA-dependent RNA polymerase) (2, 5, 10, 12, 45). Numerous studies have shown that expression of Ebola virus VP40 (eVP40) alone in mammalian cells leads to the production of virus-like particles (VLPs) with filamentous morphology which is indistinguishable from infectious Ebola virus particles (12, 17, 18, 25, 26, 27, 30, 31, 34, 49). Like many enveloped viruses such as rhabdovirus (11) and arenaviruses (44), Ebola virus encodes late-assembly or L domains, which are sequences required for the membrane fission event that separates viral and cellular membranes to release nascent virion particles (1, 5, 7, 10, 12, 18, 25, 27, 34). Thus far, four classes of L domains have been identified which were defined by their conserved amino acid core sequences: the Pro-Thr/Ser-Ala-Pro (PT/SAP) motif (25, 27), the Pro-Pro-x-Tyr (PPxY) motif (11, 12, 18, 19, 41, 53), the Tyr-x-x-Leu (YxxL) motif (3, 15, 27, 37), and the Phe-Pro-Ile-Val (FPIV) motif (39). Both PTAP and the PPxY motifs are essential for efficient particle release for eVP40 (25, 27, 48, 49), whereas mVP40 contains only a PPxY motif. L domains are believed to act as docking sites for the recruitment of cellular proteins involved in endocytic trafficking and multivesicular body biogenesis to facilitate virus-cell separation (8, 13, 14, 16, 28, 29, 33, 36, 43, 50, 51).In addition to L domains, oligomerization, and plasma-membrane localization of VP40 are two functions of the protein that are critical for efficient budding of VLPs and virions. Specific sequences involved in self-assembly and membrane localization have yet to be defined precisely. However, recent reports have attempted to identify regions of VP40 that are important for its overall function in assembly and budding. For example, the amino acid region ₂₁₂KLR₂₁₄ located at the C-terminal region was found to be important for efficient release of eVP40 VLPs, with Leu₂₁₃ being the most critical (30). Mutation of the ₂₁₂KLR₂₁₄ region resulted in altered patterns of cellular localization and oligomerization of eVP40 compared to those of the wild-type genotype (30). In addition, the proline at position 53 was also implicated as being essential for eVP40 VLP release and plasma-membrane localization (54).In a more recent study, a YPLGVG motif within the M protein of Nipah virus (NiV) was shown to be important for stability, membrane binding, and budding of NiV VLPs (35). Whether this NiV M motif represents a new class of L domain remains to be determined. However, it is clear that this YPLGVG motif of NiV M is important for budding, perhaps involving a novel mechanism (35). Our rationale for investigating the corresponding, conserved motifs present within the Ebola and Marburg virus VP40 proteins was based primarily on these findings with NiV. In addition, Ebola virus VP40 motif maps close to the hinge region separating the N- and C-terminal domains of VP40 (4). Thus, the ₉₆LPLGVA₁₀₁ motif of eVP40 is predicted to be important for the overall stability and function of VP40 during egress. Findings presented here indicate that disruption of these filovirus VP40 motifs results in a severe defect in VLP budding, due in part to impairment in overall VP40 structure, stability and/or intracellular localization.     

Mumps Virus Matrix,Fusion, and Nucleocapsid Proteins Cooperate for Efficient Production of Virus-Like Particles

Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells. To define mumps virus (MuV) proteins important for this process, viral proteins were expressed either singly or in combination in mammalian cells to produce virus-like particles (VLPs). Only the MuV matrix (M) protein when expressed by itself was capable of inducing particle release, but the quantity of these M-alone particles was very small. Efficient production of mumps VLPs occurred only when the M protein was coexpressed together with other viral proteins, with maximum production achieved upon coexpression of the viral M, nucleocapsid (NP), and fusion (F) proteins together. Electron microscopy analysis confirmed that VLPs were morphologically similar to MuV virions. The two MuV glycoproteins were not equal contributors to particle formation. The F protein was a major contributor to VLP production, while the hemagglutinin-neuraminidase protein made a smaller contribution. Evidence for the involvement of class E protein machinery in VLP budding was obtained, with mumps VLP production inhibited upon expression of dominant-negative versions of the class E proteins Vps4A and Chmp4b. Disruption of the sequence 24-FPVI-27 within the MuV M protein led to poor VLP production, consistent with findings of earlier studies of a related sequence, FPIV, important for the budding of parainfluenza virus 5. Together, these results demonstrate that different MuV structural proteins cooperate together for efficient particle production and that particle budding likely involves host class E protein machinery.Mumps virus (MuV) is a paramyxovirus from the Rubulavirus genus. Prior to mass vaccination, mumps was a very common childhood illness, with characteristic symptoms including fever, fatigue, and inflammation of the salivary glands. Less frequently, MuV infection results in serious complications including aseptic meningitis and encephalitis (22). Significant outbreaks of mumps have occurred recently in the United Kingdom (6), Canada (40), and the United States (7, 14), highlighting the continued relevance of this disease even in countries where vaccination is widespread. Like other paramyxoviruses, MuV possesses a genome that consists of single-stranded negative-sense RNA, encapsidated by a nucleocapsid (NP) protein and associated with an RNA-dependent RNA polymerase complex composed of large protein and phosphoprotein subunits. This core is linked to the virion membrane by matrix (M) protein. The outer surface of the virion is covered with glycoprotein spikes consisting of the hemagglutinin-neuraminidase (HN) protein, which binds sialic acid to allow virion attachment to cells, and fusion (F) protein, which induces viral and cellular membranes to fuse together during virus entry. Additional components of MuV include the small hydrophobic protein, which prevents infected cells from undergoing apoptosis (67), and V protein, which prevents induction of interferon-induced antiviral responses (29, 30, 62). The late steps of the MuV life cycle that allow for assembly and budding of MuV virions remain for the most part unexplored.Enveloped virus particles are formed by budding from cellular membranes at specific locations at which viral proteins, and often host factors, have assembled together. For the negative-strand RNA viruses, coordination among the different viral components during virus assembly appears to be directed by the viral matrix proteins, which have the potential to interact with the cytoplasmic tails of the viral glycoproteins and with viral ribonucleoproteins (RNPs) in the cytoplasms of infected cells. M proteins likely assemble as layers beneath the plasma membranes of infected cells and induce other viral components to gather at these locations, from which virus budding occurs (reviewed in references 49 and 57).For many viruses, it has been possible to achieve assembly and budding of particles from cells that have been transfected to produce one or more viral proteins in the absence of virus infection. These particles often resemble virions morphologically and have been termed virus-like particles (VLPs). VLP production provides a useful means for determining the individual roles of different virus proteins in particle formation, and in some cases the VLPs themselves have shown promise as vaccines (45). For most negative-strand RNA viruses, VLP formation is critically dependent on the presence of the viral matrix proteins (49). Indeed, in the cases of Newcastle disease virus (NDV) (37) and Nipah virus (11, 38), M protein expression is sufficient for highly efficient VLP production, with no apparent need for assistance from any of the other viral structural components, such as the viral glycoproteins or NP proteins. In the case of NDV, incorporation of glycoproteins and NP proteins into the budding VLPs requires specific interactions involving the M protein, but these interactions do not appear to facilitate the budding process itself (37).Although expression of viral matrix protein is sufficient for robust VLP production in the above cases, it has long been thought that additional viral components are also important for efficient budding of many negative-strand RNA viruses. For example, an important role for viral glycoproteins in virus assembly has been established based on studies with recombinant viruses that contain glycoproteins lacking their cytoplasmic tails (4, 17, 26, 34, 35, 48, 52, 66) and analyses of assembly-defective subacute sclerosing panencephalitis measles virus strains (5, 47). In fact, recent evidence suggests that for influenza virus it is the viral glycoproteins (and not viral matrix protein) that are the main drivers of virus budding (9). For other negative-strand RNA viruses, expression of viral glycoproteins together with matrix proteins in some cases significantly enhances the efficiency of VLP release. Ebola VLPs (31), Sendai VLPs (55, 56), and parainfluenza virus 5 (PIV5)-like particles (51) are all produced more efficiently in the presence of viral glycoprotein expression. Ebola virus glycoprotein in some cell types functions during virus release to inhibit the action of tetherin, a cellular protein which functions to prevent the release of enveloped virus particles from infected cells (28). In addition to the viral glycoproteins, other viral components can also enhance the production of VLPs. Production of Ebola VLPs and PIV5-like particles can be further enhanced through expression of the corresponding NP proteins (31, 51), and Sendai VLP production is enhanced through expression of Sendai virus C protein (55). Hence, for these viruses, multiple proteins cooperate with one another to achieve maximum VLP production. The extent to which particle formation actually requires this cooperation differs, however. In the case of PIV5, it is absolutely essential; expression of the M protein alone does not lead to VLP production (51). On the other hand, cooperation among viral proteins is beneficial but not strictly required for the production of Sendai or Ebola VLPs, since expression of the matrix proteins of these viruses is sufficient for VLP production (20, 55, 56, 61).The late steps of negative-strand RNA virus budding may occur in a way that is analogous to the budding of retroviruses, which employ protein-protein interaction domains called late domains to manipulate host machinery and allow release of virus particles (reviewed in references 1 and 3). Cellular factors recruited by late domains in many cases are class E proteins that are part of the vacuolar protein sorting (Vps) pathway of the cell. Indeed, disruption of the Vps pathway through expression of dominant-negative (DN) versions of the Vps4 ATPase protein blocks the budding of many retroviruses (reviewed in reference 1), as well as the budding of Ebola virus (32), Lassa fever virus (63), and PIV5 (50). However, other negative-strand RNA viruses, such as influenza virus, bud particles in ways that are not substantially affected by disruption of the cellular Vps pathway (reviewed in reference 8).Here, experiments are described which define MuV proteins important for the assembly and budding of VLPs. Using proteins derived from the 88-1961 wild-type (wt) strain of MuV, optimal production of mumps VLPs is shown to occur upon coexpression of the MuV M, F, and NP proteins together in transiently transfected mammalian cells. Evidence is also provided that supports a role for cellular class E protein machinery in the budding of mumps VLPs.     

Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus

VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism.In recent years, virus-like particles (VLPs), which are formed upon recombinant expression of the viral matrix and/or surface glycoproteins, have been recognized as representing powerful tools for developing novel vaccines and investigating certain aspects of the viral replication cycle (24, 44, 59, 63). Matrix proteins from many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and arenaviruses, are able to induce VLPs (10, 14, 18, 28-30, 48, 49, 52). Increasing evidence also indicates that budding activity, and thus the release of VLPs, is often influenced by a complex interplay with components of the endosomal sorting complexes required for transport (ESCRTs), which mainly constitute the vacuolar protein sorting (VPS) pathway (16, 38, 42, 54). ESCRTs trigger the formation and budding of vesicles into the lumina of multivesicular bodies (MVBs), and the constituents of the ESCRTs are recycled by the activity of VPS4, an AAA-type ATPase. Expression of dominant-negative (DN) VPS4 mutants, which lack the ability to bind or hydrolyze ATP, blocks recycling of the ESCRTs and induces the formation of enlarged endosomes lacking internal vesicle accumulation (2, 3, 7). The inward budding of vesicles into the MVBs is topologically similar to the budding of viruses, since the vesicles bud away from the cytosol and into the lumen (reviewed in references 1, 20, and 26). Therefore, it is not entirely surprising that viruses use the cellular ESCRT machinery to organize the budding of viral progeny. Interactions between viral matrix proteins and ESCRTs occur through tetrapeptide motifs, known as late domains, which were first identified in retroviruses. Known late domains consist of the amino acid sequence P(T/S)AP, PPxY, or YxxL, where “x” represents any amino acid (19, 25, 62). The P(T/S)AP motif, for example, mediates interaction with tumor susceptibility gene 101 (Tsg101) (16, 36, 57); the PPxY motif mediates binding to WW domains of Nedd4-like ubiquitin ligases (9, 22); and the YxxL motif mediates interaction with AIP1/Alix (35, 47, 58). Recently, a novel late-domain motif, FPIV, has been identified in paramyxoviruses (46), and it is thought that additional late-domain motifs remain to be discovered (for a review, see reference 5).Inhibition of the VPS pathway has been shown to inhibit the budding of various viruses that are released with the help of ESCRTs. However, the budding of viruses and VLPs depends on the activity of ESCRTs to different degrees. Downregulation of Tsg101, a member of the ESCRT-I complex, inhibited the release of VLPs mediated by lymphocytic choriomeningitis virus Z protein and Marburg virus (MARV) VP40 (42, 54) but did not substantially inhibit the release of Gag-induced VLPs of Moloney murine leukemia virus and Rous sarcoma virus or that of matrix protein-induced VLPs of rabies virus (16, 27, 38). Expression of DN VPS4 inhibited the release of VLPs induced by the Gag proteins of Rous sarcoma virus and Moloney murine leukemia virus (16, 38) as well as that of VLPs induced by Lassa virus Z protein (55) but had no effect on the budding of rabies virus and cytomegalovirus (13, 27). These data indicate that in spite of the presence of late-domain motifs, a block in the VPS pathway may not always be critical for the budding of VLPs. In addition, the lack of known late domains in many enveloped viruses raises the question of whether they use other entry points into the VPS pathway or whether they exploit entirely different mechanisms of budding (60). To date, knowledge of how viral matrix proteins engage cellular machineries, such as the VPS pathway, to induce viral budding at the plasma membrane is very limited (8).The matrix protein VP40 of MARV contains only one known late-domain motif, PPPY, and a recent study showed that mutation of this late domain inhibited the release of VP40-induced VLPs. In addition, depletion of Tsg101 reduced the release of VP40-induced VLPs, suggesting that ESCRT-I is involved in this process (54). Whether a functional VPS pathway is important for the release of MARV VP40-induced VLPs or MARV particles remains unknown.VLPs induced by many viral matrix proteins have a morphology similar to that of cellular vesicles, which makes it difficult to separate the spherical VLPs from released cellular vesicles (4, 17, 53). In contrast, VLPs induced by the filovirus matrix protein VP40 are elongated and similar in morphology to viral particles (30, 49). Nevertheless, we observed that the supernatants of cells expressing VP40 contained various populations of particles with different morphologies. This raised the questions of whether the different particles are released by the same mechanism, whether they are all induced by VP40, and whether they are dependent on the same cellular pathways.The aim of the present study was to analyze the populations of particles released from cells expressing the MARV matrix protein VP40 and to gain further insights into the interaction between MARV and the cellular machinery involved in the budding of VLPs and MARV particles.We found that cells expressing VP40 released vesicular and filamentous particles, which could be separated by gradient centrifugation. Fractions with mainly vesicular particles represented a mixture of vesicles containing exclusively cellular proteins and vesicles also containing VP40 and few short filamentous particles. Longer filamentous particles, whose morphology resembled that of MARV particles but which displayed a much higher variability in length (400 nm to 5 μm), were found in denser gradient fractions. Filamentous VP40-induced VLPs were able to sort out cellular proteins efficiently. Release of VP40-induced filamentous VLPs was supported by the late-domain motif present in VP40, and inhibition of the cellular ESCRT machinery reduced the amount of these VLPs in the supernatant. Interestingly, the release of VLPs induced by a mutant of VP40 lacking the late domain was also reduced by inhibition of the cellular ESCRT machinery. Expression of a DN mutant of VPS4 diminished the budding of infectious MARV particles by 50%, a finding consistent with the idea that the activity of the ESCRT machinery supports viral budding but is not essential.     

The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX_nL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host''s fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP⁻), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP⁻ budding defects is independent of cellular Tsg101, implying that Nedd4-1''s function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX_nL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP⁻. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX_nL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix''s facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX_nL/Alix budding pathway via a mechanism that involves Alix ubiquitination.Retroviral Gag polyproteins bear short conserved sequences that control virus budding and release. As such, these motifs have been dubbed late or L domains (49). Three types of L domains have thus far been characterized: PT/SAP, LYPX_nL, and PPPY motifs (5, 9, 32). They recruit host proteins known to function in the vacuolar protein sorting (vps) of cargo proteins and the generation of multivesicular bodies (MVB) compartments (2). It is currently accepted that budding of vesicles into MVB involves the sequential recruitment of endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) and the activity of the VPS4 AAA-ATPase (22). These sorting events are believed to be triggered by recognition of ubiquitin molecules conjugated to cargo proteins (20, 24, 41). For retrovirus budding, L domain motifs are the primary signals in Gag that elicit the recruitment of ESCRT components to facilitate viral budding. Consequently, mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase adversely affect virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (7, 10, 15, 16, 21, 28, 44).Two late domains have been identified within the p6 region of human immunodeficiency virus type 1 (HIV-1) Gag protein: the PTAP and LYPX_nL motifs. The PTAP motif binds the cellular protein Tsg101 (15, 39, 40, 47), whereas the LYPX_nL motif is the docking site for Alix (44). Tsg101 functions in HIV-1 budding (15) as a member of ESCRT-I (30, 48), a soluble complex required for the generation of MVB. This process is topologically similar to HIV-1 budding and requires the recruitment of ESCRT-III members called the charged-multivesicular body proteins (3, 29, 48) and the activity of the VPS4 AAA-ATPase (4, 48). In addition to binding the LYPX_nL motif, Alix also interacts with the nucleocapsid (NC) domain of HIV-1 Gag (13, 38), thus linking Gag to components of ESCRT-III that are critical for virus release (13).Other retroviruses, including the human T-cell leukemia virus (HTLV) and the Moloney murine leukemia virus (MoMLV), utilize the PPPY-type L domain to efficiently release virus (7, 26, 51). The PPPY motif binds members of the Nedd4-like ubiquitin ligase family (6, 7, 16, 19, 25, 43), whose normal cellular function is to ubiquitinate cargo proteins and target them into the MVB sorting pathway (11, 12, 20). Members of the Nedd4-like ubiquitin ligase family include Nedd4-1, Nedd4-2 (also known as Nedd4L), WWP-1/2, and Itch. They contain three distinct domains: an N-terminal membrane binding C2 domain (12), a central PPPY-interacting WW domain (43), and a C-terminal HECT domain that contains the ubiquitin ligase active site (42). The functional requirement for the binding of Nedd4-like ubiquitin ligases to the PPPY motif in virus budding has been demonstrated (7, 16, 18, 19, 25, 26, 28, 50, 51). Overexpression of dominant-negative mutants of Nedd4-like ligases, ESCRT-III components, or VPS4 cause a potent inhibition of PPPY-dependent virus release (7, 19, 29, 31, 52) and induce assembly and budding defects similar to those observed after perturbation of the PPPY motif (26, 51). These observations demonstrated that Nedd4-like ligases connect Gag encoding PPPY motif to ESCRT-III and VPS4 proteins to facilitate virus release.Whereas the role of Nedd4-like ubiquitin ligases in virus budding has been established, the protein interactions that link them to the cell''s ESCRT-III pathway are still unknown. Evidence for associations of Nedd4-like ligases with ESCRT proteins have been previously reported and include: the binding of Nedd4-like ubiquitin ligases LD1 and Nedd4-1 to ESCRT-I member Tsg101 (6, 31), the colocalization of multiple Nedd4-like ubiquitin ligases with endosomal compartments (1, 28), the requirement of the cell''s ESCRT pathway for Itch mediated L domain independent stimulation of MoMLV release (23), and the ubiquitination of ESCRT-I components with a shorter isoform, Nedd4-2s (8). Therefore, Nedd4-like ubiquitin ligase interactions with members of the cell''s ESCRT pathway may provide retroviral Gag with access to the host budding machinery required for virus release.Although HIV-1 Gag does not carry the PPPY canonical sequence known to interact with Nedd4-like ubiquitin ligases, both Nedd4-1 and Nedd4-2 were shown to restore the release of the HIV-1 PTAP⁻ mutant, albeit Nedd4-1 with less efficiency than Nedd4-2 (8, 46). These findings suggested that HIV-1 might utilize cellular Nedd4-like ubiquitin ligases to increase virus release. We present here evidence demonstrating that Nedd4-1 interacts with Gag and enhances HIV-1 PTAP⁻ virus release. Furthermore, we show that Nedd4-1''s function in HIV-1 release is distinct from that of Nedd4-2 in both its viral and cellular requirements. Notably, we found that Nedd4-1 enhancement of HIV-1 release requires the Alix-binding LYPX_nL L domain motif in the p6 region and basic residues in the NC domain. In addition, Alix''s facilitation of HIV-1 release requires cellular Nedd4-1, since mutations in NC that prevented Alix-mediated HIV-1 release also eliminated release by overexpression of Nedd4-1. This suggested a Nedd4-1-Alix physical and functional interdependence. In agreement with this, we found Nedd4-1 to bind and ubiquitinate Alix in the cell. Taken together, these results support a model in which Alix recruits Nedd4-1 to facilitate late steps of HIV-1 release through the LYPX_nL L domain motif via a mechanism that involves Alix ubiquitination.     

Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed Z_WT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, Z_WT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.Human immunodeficiency virus type 1 (HIV-1) and other retroviruses hijack the cellular Endosomal Sorting Complex Required for Transport (ESCRT) pathway to promote the detachment of virions from the cell surface and from each other (3, 21, 42, 44, 47). The ESCRT pathway was initially identified based on its requirement for the sorting of ubiquitinated cargo into multivesicular bodies (MVB) (50, 51). During MVB biogenesis, the ESCRT pathway drives the membrane deformation and fission events required for the inward vesiculation of the limiting membrane of this organelle (26, 29, 50, 51). More recently, it emerged that the ESCRT pathway is also essential for the normal abscission of daughter cells during the final stage of cell division (10, 43). Most of the components of the ESCRT pathway are involved in the formation of four heteromeric protein complexes termed ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Additional components include ALIX, which interacts both with ESCRT-I and ESCRT-III, and the AAA ATPase Vps4, which mediates the disassembly of ESCRT-III (29, 42).The deformation and scission of endocytic membranes is thought to be mediated by ESCRT-III, which, together with Vps4, constitutes the most conserved element of the pathway (23, 26, 42). Indeed, it was recently shown that purified yeast ESCRT-III induces membrane deformation (52), and in another study three subunits of yeast ESCRT-III were sufficient to promote the formation of intralumenal vesicles in an in vitro assay (61). In mammals, ESCRT-III is formed by the charged MVB proteins (CHMPs), which are structurally related and tightly regulated through autoinhibition (2, 33, 46, 53, 62). The removal of an inhibitory C-terminal domain induces polymerization and association with endosomal membranes and converts CHMPs into potent inhibitors of retroviral budding (34, 46, 53, 60, 62). Alternatively, CHMPs can be converted into strong inhibitors of the ESCRT pathway and of HIV-1 budding through the addition of a bulky tag such as green fluorescent protein (GFP) or red fluorescent protein (RFP) (27, 36, 39, 54). Retroviral budding in general is also strongly inhibited by catalytically inactive Vps4 (22, 41, 55), or upon Vsp4B depletion (31), confirming the crucial role of ESCRT-III.Retroviruses engage the ESCRT pathway through the activity of so-called late assembly (L) domains in Gag. In the case of HIV-1, the primary L domain maps to a conserved PTAP motif in the C-terminal p6 domain of Gag (24, 28) and interacts with the ESCRT-I component Tsg101 (15, 22, 40, 58). HIV-1 p6 also harbors an auxiliary L domain of the LYPx_nL type, which interacts with the V domain of ALIX (20, 35, 39, 54, 59, 63). Interestingly, Tsg101 binding site mutants of HIV-1 can be fully rescued through the overexpression of ALIX, and this rescue depends on the ALIX binding site in p6 (20, 56). In contrast, the overexpression of a specific splice variant of the ubiquitin ligase Nedd4-2 has been shown to rescue the release and infectivity of HIV-1 mutants lacking all known L domains in p6 (12, 57). Nedd4 family ubiquitin ligases had previously been implicated in the function of PPxY-type L domains, which also depend on an intact ESCRT pathway for function (4, 32, 38). However, HIV-1 Gag lacks PPxY motifs, and the WW domains of Nedd4-2, which mediate its interaction with PPxY motifs, are dispensable for the rescue of HIV-1 L domain mutants (57).ALIX also interacts with the nucleocapsid (NC) region of HIV-1 Gag (18, 49), which is located upstream of p6 and the p1 spacer peptide. ALIX binds HIV-1 NC via its Bro1 domain, and the capacity to interact with NC and to stimulate the release of a minimal HIV-1 Gag construct is shared among widely divergent Bro1 domain proteins (48). Based on these findings and the observation that certain mutations in NC cause a phenotype that resembles that of L domain mutants, it has been proposed that NC cooperates with p6 to recruit the machinery required for normal HIV-1 budding (18, 49).NC also plays a role in Gag polyprotein multimerization, and this function of NC depends on its RNA-binding activity (5-8). It has been proposed that the role of the NC-nucleic acid interaction during assembly is to promote the formation of Gag dimers (37), and HIV-1 assembly in the absence of NC can indeed be efficiently rescued by leucine zipper dimerization domains (65). Surprisingly, in this setting the L domains in p6 also became dispensable, since particle production remained efficient even when the entire NC-p1-p6 region of HIV-1 Gag was replaced by a leucine zipper (1, 65). These findings raised the possibility that the reliance of wild-type (WT) HIV-1 Gag on a functional ESCRT pathway is, at least in part, specified by NC-p1-p6. However, it also remained possible that the chimeric Gag constructs engaged the ESCRT pathway in an alternative manner.In the present report, we provide evidence supporting the first of those two possibilities. Particle production became independent of ESCRT when the entire NC-p1-p6 region was replaced by a leucine zipper, and reversion to ESCRT dependence was shown to occur as a result of restoration of p1-p6 but not of p6 alone. Furthermore, although the deletion of p1 alone had little effect in an authentic HIV-1 Gag context, the additional removal of a portion of NC improved particle production in the presence of an inhibitor of the ESCRT pathway. Together, these data imply that the NC-p1 region plays an important role in the ESCRT-dependence of HIV-1 particle production.     

Conserved and Variable Features of Gag Structure and Arrangement in Immature Retrovirus Particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.Retrovirus assembly is driven by the oligomerization of Gag, a multidomain protein, including an N-terminal membrane binding domain (MA), a two-domain structural component (CA), and an RNA binding domain (NC). The Gag proteins of all orthoretroviruses, including the alpha-, beta-, and lentiretroviruses discussed here, share this conserved modular architecture (Fig. ​(Fig.1).1). Despite very weak sequence conservation, the tertiary structures of MA, CA, and NC are conserved among retroviruses. Outside these conserved domains the Gag proteins of different retroviruses exhibit substantial variability. Other domains may be present or absent, and the length and sequence of linker peptides may also vary (12) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Modular architecture of the full-length Gag proteins of HIV, M-PMV, and RSV. White rectangles illustrate Gag polyprotein cleavage products. The extent of the constructs used in the electron microscopic analysis is specified under each protein as a black rectangle. Gray triangles specify cleavage sites. Residue numbers are counted from the beginning of Gag.Oligomerization of Gag in an infected cell leads to the formation of roughly spherical immature virus particles, where Gag is arranged in a radial fashion with the N-terminal MA domain associated with a surrounding lipid bilayer, and the more C-terminal NC pointing toward the center of the particle (15, 44, 46). Subsequent multiple cleavages of Gag by the viral protease lead to a rearrangement of the virus. NC and the RNA condense in the center of the particle, CA assembles into a capsid or shell around the nucleoprotein, and MA remains associated with the viral membrane. This proteolytic maturation is required to generate an infectious virion (2). In contrast to the mature CA lattice, which has been extensively studied (11, 16, 36), the Gag lattice in immature particles is incompletely understood.Gag itself contains all of the necessary determinants for particle assembly. For example, the expression of Gag alone in an insect cell expression system is sufficient to generate viruslike particles (3, 17, 22, 38). Retroviral Gag proteins also can be assembled in vitro in the presence of nucleic acids to form spherical particles (9, 19, 39, 43, 47). The arrangement of Gag within these in vitro-assembled Gag particles is indistinguishable from that found in immature virus particles (6), and the in vitro assembly systems have proved valuable for unraveling the principles of virus assembly (18, 28, 29, 39). Multiple layers of interaction promote the assembly of Gag in vivo, including MA-membrane-MA interactions, CA-CA interactions, and NC-RNA-NC interactions. An extensive body of literature has explored which regions of Gag are required for assembly and which can be replaced or deleted without compromising assembly. MA-membrane-MA interactions contribute but are not essential. NC-RNA-NC interactions appear to function to nonspecifically link Gag molecules together and can be replaced both in vivo and in vitro by other interaction domains such as leucine zippers (4, 13, 20, 32, 48). The C-terminal domain of CA (referred to here as C-CA) and the stretch of amino acids immediately following this domain (termed the spacer peptide [SP] region) are critical for assembly and sensitive to mutation (1, 22, 27, 30).We set out to understand how the substantial sequence variation among Gag proteins in different retroviruses is manifested in structural differences in the immature Gag lattice. To do this, we studied three retroviruses from different genera: the lentivirus human immunodeficiency virus type 1 (HIV-1), the betaretrovirus Mason-Pfizer monkey virus (M-PMV), and the alpharetrovirus Rous sarcoma virus (RSV). These retroviruses are those for which in vitro assembly was first established and has been most extensively studied (6, 19, 24, 28, 29, 35, 43, 47).The domain structures of the three retroviruses differ most substantially upstream of CA. Both M-PMV and RSV have domains located between MA and CA that are absent in HIV (Fig. ​(Fig.1).1). In M-PMV there are 198 residues forming the pp24 and p12 domains; in RSV there are 84 residues forming the p2a, p2b, and p10 domains. The three retroviruses have different requirements for regions upstream of CA during assembly. The C-terminal 25 residues of p10 are essential for proper immature RSV assembly, both in vitro and in vivo, and these residues are inferred to interact directly with N-CA to stabilize the hexamer by forming contacts between adjacent N-CA domains (35). An equivalent assembly domain has not been described for other retroviruses. Within M-PMV p12 is the so-called internal scaffolding domain that is not essential for assembly in vitro (43) but is required for particle assembly when the precursor is expressed under the control of the M-PMV promoter (41). It is a key domain for the membrane-independent assembly of immature capsids (40).In HIV, five residues upstream of CA must be present for assembly of immature virus-like spherical particles in vitro, although larger upstream extensions, including part of MA, are required for efficient assembly of regular particles, both for HIV and RSV. For HIV, if the entire MA domain is included, in vitro assembly requires the presence of inositol penta- or hexakis phosphate (8). If no sequences upstream of CA are present, the in vitro particles in both of these viruses adopt a mature-type tubular morphology (10, 18). It has been hypothesized that cleavage at the N terminus of N-CA during maturation leads to the N-terminal residues of CA folding back into the N-CA structure to form a β-hairpin. The β-hairpin is important for assembly of the mature CA lattice, whereas its absence is important for immature assembly (23, 42). These requirements explain why, in HIV and RSV, immature Gag lattice-like structures are formed only if regions upstream of CA are present (18). In M-PMV, an immature Gag lattice can be produced when the regions upstream of CA are deleted if this is combined with mutations (such as deleting the initial proline of CA), which prevent β-hairpin formation (43).During maturation, HIV and RSV Gag proteins are cleaved twice between CA and NC to release a small peptide called SP1 or SP. In RSV the most N-terminal of these two cleavages can occur at one of two possible positions such that the released peptide is either 9 or 12 amino acids long (33). In M-PMV only one cleavage occurs between CA and NC, and no short peptide is produced. The region between the final helix of CA and the Zn fingers has been proposed to adopt a helical bundle architecture in HIV and RSV based on bioinformatic prediction, on mutational analysis, and on structural studies (1, 22, 27, 45). In all three viruses, C-CA and the residues immediately downstream are critical for assembly and are sensitive to mutation. C-CA contains the major homology region, a group of residues that are highly conserved across the retroviruses.Cryo-electron tomography (cET) studies of immature virus particles (6, 45) have resolved the electron density of the HIV Gag lattice in three dimensions at low resolution. Using these methods, we have also described the three-dimensional architecture of in vitro-assembled HIV Gag particles (6). In immature viruses and in vitro-assembled particles, Gag is seen to adopt an 8 nm hexameric lattice, as was predicted from previous Fourier analysis of two-dimensional images (7, 46). The hexameric lattice is interrupted by irregularly shaped holes and cracks in the lattice (6, 45). A similar observation has been made using AFM of in vitro-assembled particles of M-PMV Gag (26). These holes and cracks allow an otherwise planar hexameric lattice to form the surface of an approximately spherical particle.The radial positions of the MA, CA, and NC domains had been assigned previously from cryo-electron micrographs (44, 46). Based on these assignments and the shape of the density, the position and relative orientations of CA domains can be modeled into the low-resolution structure of the HIV lattice (6, 45). Density ascribed to the N-terminal domain of CA (N-CA) forms rings around large holes at the 6-fold symmetry positions in the lattice. Below this layer, at the expected radius of the C-CA, are 2-fold densities, interpreted as corresponding to dimers of C-CA. These densities are linked by rodlike densities, which descend into the NC-nucleic acid layer.HIV is the only retrovirus for which the arrangement of Gag in the immature particle has been described in three dimensions. Prior to this work, important open questions were therefore: which features of the arrangement of Gag are conserved between genera and therefore reflect general principles of Gag-Gag interactions, and which features are specific to certain genera? We have applied subtomogram averaging of cryo-electron tomograms to generate reconstructions of in vitro-assembled Gag particles from HIV, M-PMV, and RSV. These allow identification of the general and variable features of the arrangement of Gag and the architecture of immature retroviruses.