Paramyxovirus budding

Our knowledge about envelope virus budding has been dramatically increased, since L-domain motifs were identified within their matrix and retroviral Gag proteins which drive virus budding. These viral proteins have been shown to interact with host cellular proteins involved in endocytosis and/or multi-vesicular body (MVB) sorting via their L-domains. Since budding of many enveloped viruses have been reported to be dependent on the activity of cellular Vps4, which catalyzes the disassembly of ESCRT machinery in the final step of protein sorting, this cellular function is believed to be utilized for efficient virus budding. However, for many enveloped viruses, L-domain motifs have not yet been identified, and the involvement of MVB sorting machinery in virus budding is still unknown. In this review, we will focus on paramyxoviruses among such viruses, and discuss their budding with the latest information.     

TANK-binding kinase 1 attenuates PTAP-dependent retroviral budding through targeting endosomal sorting complex required for transport-I

Retroviruses need to bud from producer cells to spread infection. To facilitate its budding, some virus hijacks the multivesicular body (MVB) pathway that is normally used to cargo and degrade ubiquitylated cellular proteins, through interaction between the late domain of Gag polyproteins and the components of MVB machinery. In this study, we demonstrated that TANK-binding kinase 1 (TBK1) directly interacted with VPS37C, a subunit of endosomal sorting complex required for transport-I (ESCRT-I) in the MVB pathway, without affecting the ultrastructure or general function of MVB. Interestingly, overexpression of TBK1 attenuated, whereas short hairpin RNA interference of TBK1 enhanced HIV-1 pseudovirus release from Vero cells in type I IFN (IFN-I)-independent manner. Down-regulation of TBK1 by short hairpin RNA in TZM-bl cells also enhanced live HIV-1 NL4-3 or JR-CSF virus budding without involvement of IFN-I induction. Furthermore, infection of TBK1-deficient mouse embryonic fibroblast cells with a chimeric murine leukemia virus/p6, whose PPPY motif was replaced by PTAP motif of HIV-1, showed that lack of TBK1 significantly enhanced PTAP-dependent, but not PPPY-dependent retrovirus budding. Finally, phosphorylation of VPS37C by TBK1 might regulate the viral budding efficiency, because overexpression of the kinase-inactive mutant of TBK1 (TBK1-K38A) in Vero cells accelerated HIV-1 pseudovirus budding. Therefore, through tethering to VPS37C of the ESCRT-I complex, TBK1 controlled the speed of PTAP-dependent retroviral budding through phosphorylation of VPS37C, which would serve as a novel mechanism of host cell defense independent of IFN-I signaling.     

HIV budding and Tsg101

HIV, as well as many enveloped viruses, exits the cells by budding directly from the plasma membrane. HIV budding is dependent on a PTAP motif, which is located within the p6 domain of Gag. Recent studies have shown that the cellular protein Tsg101 binds to the PTAP L-domain motif of HIV p6 and facilitates the final stages of virus release. Tsg101 function in the cellular vacuolar protein sorting pathway, where they play central roles in selecting cargo for incorporation into vesicles that bud into the maturing endosome to create multivesicular bodies (MVBs). Vesicle budding into the MVB and viral budding at the plasma membrane are topologically equivalent, and the same machinery could catalyze both processes. It will be important to understand the mechanism of virus budding in detail, since virus budding may be a potential target for interference with HIV propagation.     

Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.     

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.     

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.     

Budding of PPxY-containing rhabdoviruses is not dependent on host proteins TGS101 and VPS4A

Viral matrix proteins of several enveloped RNA viruses play important roles in virus assembly and budding and are by themselves able to bud from the cell surface in the form of lipid-enveloped, virus-like particles (VLPs). Three motifs (PT/SAP, PPxY, and YxxL) have been identified as late budding domains (L-domains) responsible for efficient budding. L-domains can functionally interact with cellular proteins involved in vacuolar sorting (VPS4A and TSG101) and endocytic pathways (Nedd4), suggesting involvement of these pathways in virus budding. Ebola virus VP40 has overlapping PTAP and PPEY motifs, which can functionally interact with TSG101 and Nedd4, respectively. As for vesicular stomatitis virus (VSV), a PPPY motif within M protein can interact with Nedd4. In addition, M protein has a PSAP sequence downstream of the PPPY motif, but the function of PSAP in budding is not clear. In this study, we compared L-domain functions between Ebola virus and VSV by constructing a chimeric M protein (M40), in which the PPPY motif of VSV M is replaced by the L domains of VP40. The budding efficiency of M40 was 10-fold higher than that of wild-type (wt) M protein. Overexpression of a dominant negative mutant of VPS4A or depletion of cellular TSG101 reduced the budding of only M40-containing VLPs but not that of wt M VLPs or live VSV. These findings suggest that the PSAP motif of M protein is not critical for budding and that there are fundamental differences between PTAP-containing viruses (Ebola virus and human immunodeficiency virus type 1) and PPPY-containing viruses (VSV and rabies virus) regarding their dependence on specific host factors for efficient budding.     

Role of ESCRT-I in retroviral budding

Retroviral late-budding (L) domains are required for the efficient release of nascent virions. The three known types of L domain, designated according to essential tetrapeptide motifs (PTAP, PPXY, or YPDL), each bind distinct cellular cofactors. We and others have demonstrated that recruitment of an ESCRT-I subunit, Tsg101, a component of the class E vacuolar protein sorting (VPS) machinery, is required for the budding of viruses, such as human immunodeficiency virus type 1 (HIV-1) and Ebola virus, that encode a PTAP-type L domain, but subsequent events remain undefined. In this study, we demonstrate that VPS28, a second component of ESCRT-I, binds to a sequence close to the Tsg101 C terminus and is therefore recruited to the plasma membrane by HIV-1 Gag. In addition, we show that Tsg101 exhibits a multimerization activity. Using a complementation assay in which Tsg101 is artificially recruited to sites of HIV-1 assembly, we demonstrate that the integrity of the VPS28 binding site within Tsg101 is required for particle budding. In addition, mutation of a putative leucine zipper or residues important for Tsg101 multimerization also impairs the ability of Tsg101 to support HIV-1 budding. A minimal multimerizing Tsg101 domain is a dominant negative inhibitor of PTAP-mediated HIV-1 budding but does not inhibit YPDL-type or PPXY-type L-domain function. Nevertheless, YDPL-type L-domain activity is inhibited by expression of a catalytically inactive mutant of the class E VPS ATPase VPS4. These results indicate that all three classes of retroviral L domains require a functioning class E VPS pathway in order to effect budding. However, the PTAP-type L domain appears to be unique in its requirement for an intact, or nearly intact, ESCRT-I complex.     

The protein network of HIV budding

HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.     

201 The ESCRT pathway in HIV budding and cell division

The endosomal sorting complexes required for transport (ESCRT) pathway mediates membrane fission reactions during intraluminal endosomal vesicle formation, budding of HIV-1 and other enveloped viruses, and the final abscission step of cytokinesis in mammals and archaea. Current models hold that ubiquitin-binding ESCRT factors act early in the pathway to regulate factor recruitment and assembly, whereas the late acting ESCRT-III proteins form filaments that draw the membranes together and mediate fission, possibly, in collaboration with VPS4-ATPases. I will discuss our current understanding of the structures and functions of the different ESCRT factors in HIV budding and abscission with a particular focus on our studies aimed at understanding: (1) how ubiquitin regulates ESCRT recruitment during HIV-1 budding and (2) the structures and membrane-binding properties of ESCRT-III subunits and filaments.     

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.     

Interaction of AMSH with ESCRT-III and deubiquitination of endosomal cargo

The "class E" vacuolar protein sorting (VPS) pathway mediates sorting of ubiquitinated cargo into the forming vesicles of the multivesicular bodies (MVB), and it is essential for down-regulation of signaling by growth factors and budding of enveloped viruses such as Ebola and HIV-1. Work in yeast has identified DOA4 as a gene that is recruited by the class E machinery to remove ubiquitin from the endosomal cargo before it is incorporated into MVB vesicles, but the identity of the mammalian counterpart is unclear. Here we report the interaction of AMSH (associated molecule with the SH3 domain of STAM), an endosomal deubiquitinating enzyme, with the endodomal sorting complex required for transport (ESCRT-III) subunits CHMP1A, CHMP1B, CHMP2A, and CHMP3. We also show that a catalytically inactive AMSH inhibits retroviral budding in a dominant-negative manner and induces the accumulation of ubiquitinated forms of an endosomal cargo, namely murine leukemia virus Gag. Finally, VPS4 and AMSH compete for binding to the C-terminal regions of CHMP1A and CHMP1B, revealing a coordinated interaction with ESCRT-III. Taken together, these results are consistent with a role of AMSH in the deubiquitination of the endosomal cargo preceding lysosomal degradation.     

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.     

Multiple interactions between the ESCRT machinery and arrestin-related proteins: implications for PPXY-dependent budding

Late domains are short peptide sequences encoded by enveloped viruses to promote the final separation of the nascent virus from the infected cell. These amino acid motifs facilitate viral egress by interacting with components of the ESCRT (endosomal sorting complex required for transport) machinery, ultimately leading to membrane scission by recruiting ESCRT-III to the site of viral budding. PPXY late (L) domains present in viruses such as murine leukemia virus (MLV) or human T-cell leukemia virus type 1 (HTLV-1) access the ESCRT pathway via interaction with HECT ubiquitin ligases (WWP1, WWP2, and Itch). However, the mechanism of ESCRT-III recruitment in this context remains elusive. In this study, we tested the arrestin-related trafficking (ART) proteins, namely, ARRDC1 (arrestin domain-containing protein 1) to ARRDC4 and TXNIP (thioredoxin-interacting protein), for their ability to function as adaptors between HECT ubiquitin ligases and the core ESCRT machinery in PPXY-dependent budding. We present several lines of evidence in support of such a role: ARTs interact with HECT ubiquitin ligases, and they also exhibit multiple interactions with components of the ESCRT pathway, namely, ALIX and Tsg101, and perhaps with an as yet unidentified factor. Additionally, the ARTs can be recruited to the site of viral budding, and their overexpression results in a PPXY-specific inhibition of MLV budding. Lastly, we show that WWP1 changes the ubiquitination status of ARRDC1, suggesting that the ARTs may provide a platform for ubiquitination in PPXY-dependent budding. Taken together, our results support a model whereby ARTs are involved in PPXY-mediated budding by interacting with HECT ubiquitin ligases and providing several alternative routes for ESCRT-III recruitment.     

The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.     

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.     

The endosomal Na(+)/H(+) exchanger contributes to multivesicular body formation by regulating the recruitment of ESCRT-0 Vps27p to the endosomal membrane

Multivesicular bodies (MVBs) are late endosomal compartments containing luminal vesicles (MVB vesicles) that are formed by inward budding of the endosomal membrane. In budding yeast, MVBs are an important cellular mechanism for the transport of membrane proteins to the vacuolar lumen. This process requires a class E subset of vacuolar protein sorting (VPS) genes. VPS44 (allelic to NHX1) encodes an endosome-localized Na(+)/H(+) exchanger. The function of the VPS44 exchanger in the context of vacuolar protein transport is largely unknown. Using a cell-free MVB formation assay system, we demonstrated that Nhx1p is required for the efficient formation of MVB vesicles in the late endosome. The recruitment of Vps27p, a class E Vps protein, to the endosomal membrane was dependent on Nhx1p activity and was enhanced by an acidic pH at the endosomal surface. Taken together, we propose that Nhx1p contributes to MVB formation by the recruitment of Vps27p to the endosomal membrane, possibly through Nhx1p antiporter activity.     

NEDD4L overexpression rescues the release and infectivity of human immunodeficiency virus type 1 constructs lacking PTAP and YPXL late domains

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6(Gag) bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding.     

Context-dependent effects of L domains and ubiquitination on viral budding

Many enveloped viruses encode late assembly domains, or L domains, that facilitate virion egress. PTAP-type L domains act by recruiting the ESCRT-I (endosomal sorting complex required for transport I) component Tsg101, and YPXL/LXXLF-type L domains recruit AIP-1/ALIX, both of which are class E vacuolar protein sorting (VPS) factors, normally required for the generation of vesicles within endosomes. The binding cofactors for PPXY-type L domains have not been unambiguously resolved but may include Nedd4-like ubiquitin ligases. Largely because they act as autonomous binding sites for host factors, L domains are generally transferable and active in a context-independent manner. Ebola virus matrix protein (EbVP40) contains two overlapping L-domain motifs within the sequence ILPTAPPEYMEA. Here, we show that both motifs are required for efficient EbVP40 budding. However, upon transplantation into two different retroviral contexts, the relative contributions of the PTAP and PPEY motifs differ markedly. In a murine leukemia virus carrying the EbVP40 sequence, both motifs contributed to overall L domain activity, and budding proceeded in a partly Tsg101-independent manner. Conversely, when transplanted into the context of human immunodeficiency virus type 1 (HIV-1), EbVP40 L-domain activity was entirely due to a PTAP-Tsg101 interaction. In fact, a number of PPXY-type L domains were inactive in the context of HIV-1. Surprisingly, PTAP and YPXL-type L domains that simulated HIV-1 budding reduced the amount of ubiquitin conjugated to Gag, while inactive PPXY-type L domains increased Gag ubiquitination. These observations suggest that active L domains recruit deubiquitinating enzymes as a consequence of class E VPS factor recruitment. Moreover, context-dependent L-domain function may reflect distinct requirements for host functions during the morphogenesis of different viral particles or the underlying presence of additional, as yet undiscovered L domains.     

Live-cell visualization of dynamics of HIV budding site interactions with an ESCRT component

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ～35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.