Mutation of YMYL in the Nipah virus matrix protein abrogates budding and alters subcellular localization

Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.     

Requirements for budding of paramyxovirus simian virus 5 virus-like particles

Enveloped viruses are released from infected cells after coalescence of viral components at cellular membranes and budding of membranes to release particles. For some negative-strand RNA viruses (e.g., vesicular stomatitis virus and Ebola virus), the viral matrix (M) protein contains all of the information needed for budding, since virus-like particles (VLPs) are efficiently released from cells when the M protein is expressed from cDNA. To investigate the requirements for budding of the paramyxovirus simian virus 5 (SV5), its M protein was expressed in mammalian cells, and it was found that SV5 M protein alone could not induce vesicle budding and was not secreted from cells. Coexpression of M protein with the viral hemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins also failed to result in significant VLP release. It was found that M protein in the form of VLPs was only secreted from cells, with an efficiency comparable to authentic virus budding, when M protein was coexpressed with one of the two glycoproteins, HN or F, together with the nucleocapsid (NP) protein. The VLPs appeared similar morphologically to authentic virions by electron microscopy. CsCl density gradient centrifugation indicated that almost all of the NP protein in the cells had assembled into nucleocapsid-like structures. Deletion of the F and HN cytoplasmic tails indicated an important role of these cytoplasmic tails in VLP budding. Furthermore, truncation of the HN cytoplasmic tail was found to be inhibitory toward budding, since it prevented coexpressed wild-type (wt) F protein from directing VLP budding. Conversely, truncation of the F protein cytoplasmic tail was not inhibitory and did not affect the ability of coexpressed wt HN protein to direct the budding of particles. Taken together, these data suggest that multiple viral components, including assembled nucleocapsids, have important roles in the paramyxovirus budding process.     

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.     

Cellular factors required for Lassa virus budding

It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicular body pathway functionally. Our data may provide a clue to develop an effective antiviral strategy for Lassa virus.     

A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.     

Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.     

Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.     

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.     

Identification and structural characterization of the ALIX-binding late domains of simian immunodeficiency virus SIVmac239 and SIVagmTan-1

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX(n)L (where X(n) can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6(Gag) proteins of SIV(mac239) ((40)SREKPYKEVTEDLLHLNSLF(59)) and SIV(agmTan-1) ((24)AAGAYDPARKLLEQYAKK(41)). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain, revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.     

Parainfluenza virus 5 m protein interaction with host protein 14-3-3 negatively affects virus particle formation

Paramyxovirus matrix (M) proteins organize virus assembly, linking viral glycoproteins and viral ribonucleoproteins together at virus assembly sites on cellular membranes. Using a yeast two-hybrid screening approach, we identified 14-3-3 as a binding partner for the M protein of parainfluenza virus 5 (PIV5). Binding in both transfected and PIV5-infected cells was confirmed by coimmunoprecipitation and was mapped to a C-terminal region within the M protein, namely, 366-KTKSLP-371. This sequence resembles known 14-3-3 binding sites, in which the key residue for binding is a phosphorylated serine residue. Mutation of S369 within the PIV5 M protein disrupted 14-3-3 binding and improved the budding of both virus-like particles (VLPs) and recombinant viruses, suggesting that 14-3-3 binding impairs virus budding. 14-3-3 protein overexpression reduced the budding of VLPs. Using (33)P labeling, phosphorylated M protein was detected in PIV5-infected cells, and this phosphorylation was nearly absent in cells infected with a recombinant virus harboring an S369A mutation within the M protein. Assembly of the M protein into clusters and filaments at infected cell surfaces was enhanced in cells infected with a recombinant virus defective in 14-3-3 binding. These findings support a model in which a portion of M protein within PIV5-infected cells is phosphorylated at residue S369, binds the 14-3-3 protein, and is held away from sites of virus budding.     

Requirement for a non-specific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus.

The cytoplasmic domains of viral glycoproteins are often involved in specific interactions with internal viral components. These interactions can concentrate glycoproteins at virus budding sites and drive efficient virus budding, or can determine virion morphology. To investigate the role of the vesicular stomatitis virus (VSV) glycoprotein (G) cytoplasmic and transmembrane domains in budding, we recovered recombinant VSVs expressing chimeric G proteins with the transmembrane and cytoplasmic domains derived from the human CD4 protein. These unrelated foreign sequences were capable of supporting efficient VSV budding. Further analysis of G protein cytoplasmic domain deletion mutants showed that a cytoplasmic domain of only 1 amino acid did not drive efficient budding, whereas 9 amino acids did. Additional studies in agreement with the CD4-chimera experiments indicated the requirement for a short cytoplasmic domain on VSV G without the requirement for a specific sequence in that domain. We propose a model for VSV budding in which a relatively non-specific interaction of a cytoplasmic domain with a pocket or groove in the viral nucleocapsid or matrix proteins generates a glycoprotein array that promotes viral budding.     

Role of Matrix and Fusion Proteins in Budding of Sendai Virus

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.     

Contribution of ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles

The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i). VP40 L domains are not required for GP-mediated enhancement of budding; (ii). the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii). NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv). the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.     

Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.     

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.     

Interaction of Tsg101 with Marburg virus VP40 depends on the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and Tsg101 plays a critical role in the budding of Marburg virus-like particles induced by VP40, NP, and GP

Marburg virus (MARV) VP40 is a matrix protein that can be released from mammalian cells in the form of virus-like particles (VLPs) and contains the PPPY sequence, which is an L-domain motif. Here, we demonstrate that the PPPY motif is important for VP40-induced VLP budding and that VLP production is significantly enhanced by coexpression of NP and GP. We show that Tsg101 interacts with VP40 depending on the presence of the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and plays an important role in VLP budding. These findings provide new insights into the mechanism of MARV budding.     

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

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

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.     

Interaction of influenza A virus matrix protein with RACK1 is required for virus release

The mechanism of budding of influenza A virus revealed important deviation from the consensus mechanism of budding of retroviruses and of a growing number of negative-strand RNA viruses. This study is focused on the role of the influenza A virus matrix protein M1 in virus release. We found that a mutation of the proline residue at position 16 of the matrix protein induces inhibition of virus detachment from cells. Depletion of the M1-binding protein RACK1 also impairs virus release and RACK1 binding requires the proline residue at position 16 of M1. The impaired M1-RACK1 interaction does not affect the plasma membrane binding of M1; in contrast, RACK1 is recruited to detergent-resistant membranes in a M1-proline-16-dependent manner. The proline-16 mutation in M1 and depletion of RACK1 impairs the pinching-off of the budding virus particles. These findings reveal the active role of the viral matrix protein in the release of influenza A virus particles that involves a cross-talk with a RACK1-mediated pathway.     

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.