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.

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.The assembly and budding of enveloped negative-strand RNA viruses is a multistep process occurring at the plasma membrane of a host cell. Vesicular stomatitis virus (VSV), a prototype enveloped negative-strand RNA virus belonging to the family Rhabdoviridae, has long been utilized as a model for studying various steps in virus assembly and budding. Assembly is initiated when the matrix protein (M) binds to and condenses the nucleocapsid core into a tightly coiled helical structure in association with the inner leaflet of the plasma membrane. During budding, the condensed core becomes enveloped in a host-derived membrane highly enriched with the viral glycoprotein (G) and selectively depleted of most of the host proteins.Recent studies have shown that while G protein contributes to high levels of virus budding, virus assembly and release can occur in the absence of G protein, but with reduced efficiency (28,36,37,43). Thus, the condensed ribonucleocapsid core (RNP) complexed with M protein is sufficient to initiate and drive rhabdovirus budding. Also, in the absence of other viral proteins, VSV M protein can cause budding of lipid vesicles into the surrounding medium (20). In the case of rabies virus, deletion of M protein dramatically reduced virus yields, more than 10,000 fold. The ⌬M particles that were produced were filamentous instead of having the characteristic bullet shape, confirming the earlier reports of the contribution of M protein to virus morphology (25,30,31).Besides its role in virus assembly and budding, matrix protein also mediates most of the cytopathic effects (CPE) attributed to VSV infection (4,6,11,38). Transient expression of M protein alone can cause cell r...

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The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that isThe nonsegmented, negative-strand RNA viruses have a relatively small genome size, ranging from 11 to 19 kb. To maximize their coding capacities, many of these viruses have evolved different strategies to express additional proteins. Increased coding capacity can occur either at the transcriptional level (mRNA processing or modification) or at the translational level, in which proteins are produced from alternative reading frames or from translation initiation at non-AUG or downstream AUG codons (1,5,7,8,10,12,24). The bestcharacterized examples are the use of multiple overlapping open reading frames (ORFs) within the P mRNAs of several paramyxoviruses (1, 10, 27) and the mRNA encoding the NA and NB glycoproteins of influenza B virus (22).A similar phenomenon has recently been described for Vesicular stomatitis virus (VSV), the prototype member of the Rhabdoviridae family. The VSV genome contains five genes, N, P, M, G, and L, and each, except the P gene, is thought to encode a single unique protein. The VSV P gene, like its counterpart in paramyxoviruses, has been shown to encode two additional proteins, C and CЈ, in a second ORF (14, 24) and a 7,000-molecular-weight (7K) polypeptide in the same ORF that encodes the P protein (12).The VSV (Indiana serotype) M gene is transcribed into a single mRNA which encodes the 229-amino-acid matrix (M) protein. M protein has numerous functions in infected cells. For example, M protein is the driving force behind the assembly and budding of virions. M protein interacts with the viral ribonucleoprotein core (RNP), resulting in the condensation of the RNP and subsequent inhibition of viral transcription (29). A fraction of the M protein (ϳ10%) is also associated with the inner leaflet of the plasma membrane where virus assembly and budding takes place (6). Recent work has shown that a motif (PPPY) located within the first 30 amino acids of M contributes to this budding activity (11,13). M protein is also responsible for most of the cytopathic effects of VSV infection. Expression of M protein by itself can cause inhibition of host gene expression, which occurs mostly at the transcriptional level (2,3,18). This inhibition appears to be mediated via inactivation of the TFIID protein (17). M protein, when expressed alone in the absence of other viral components, also causes cytoskeletal disorganization. Disassembly of microtubules by M protein ultimately leads to cell rounding (4, 23), which is a hallmark of VSV infection in cell culture. Recently it was shown that a fraction of M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim (19). This nuclear fraction of M protein is thought to contribute to the host shutoff function of the protein by inhibiting RNA export from the nucleus.In this study, we show that the M mRNA encodes two additional polypeptides, which we refer to as M2 and M3. These proteins are synthesized from downstream methionines in the same reading ...

A PPPY motif within the M protein of vesicular stomatitis virus (VSV) functions as a late-budding domain (L-domain); however, L-domain activity has yet to be associated with a downstream PSAP motif. VSV recombinants with mutations in the PPPY and/or PSAP motif were recovered by reverse genetics and examined for growth kinetics, plaque size, and budding efficiency by electron microscopy. Results indicate that unlike the PPPY motif, the PSAP motif alone does not possess L-domain activity. Finally, the insertion of the human immunodeficiency virus type 1 p6 L-domain and flanking sequences into the PSAP region of M protein rescued budding of a PPPY mutant of VSV to wild-type levels.

BackgroundA number of studies have revealed that Francisella tularensis (FT) suppresses innate immune responses such as chemokine/cytokine production and neutrophil recruitment in the lungs following pulmonary infection via an unidentified mechanism. The ability of FT to evade early innate immune responses could be a very important virulence mechanism for this highly infectious bacterial pathogen.ResultsHere we describe the characterization of a galU mutant strain of FT live vaccine strain (LVS). We show that the galU mutant was highly attenuated in a murine model of tularemia and elicited more robust innate immune responses than the wild-type (WT) strain. These studies document that the kinetics of chemokine expression and neutrophil recruitment into the lungs of mice challenged with the galU mutant strain are significantly more rapid than observed with WT FT, despite the fact that there were no observed differences in TLR2 or TLR4 signaling or replication/dissemination kinetics during the early stages of infection. We also show that the galU mutant had a hypercytotoxic phenotype and more rapidly induced the production of IL-1β following infection either in vitro or in vivo, indicating that attenuation of the galU mutant strain may be due (in part) to more rapid activation of the inflammasome and/or earlier death of FT infected cells. Furthermore, we show that infection of mice with the galU mutant strain elicits protective immunity to subsequent challenge with WT FT.ConclusionsDisruption of the galU gene of FTLVS has little (if any) effect on in vivo infectivity, replication, or dissemination characteristics, but is highly attenuating for virulence. The attenuated phenotype of this mutant strain of FT appears to be related to its increased ability to induce innate inflammatory responsiveness, resulting in more rapid recruitment of neutrophils to the lungs following pneumonic infection, and/or to its ability to kill infected cells in an accelerated fashion. These results have identified two potentially important virulence mechanisms used by FT. These findings could also have implications for design of a live attenuated vaccine strain of FT because sublethal infection of mice with the galU mutant strain of FTLVS promoted development of protective immunity to WT FTLVS.

Data in this study provide in vitro proof-of-principle that rVSV-deltaG is an effective oncolytic agent that has minimal toxic side effects to neurons compared with rVSV-wt and therefore should be considered for development as an adjuvant to surgery in the treatment of glioma.

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. BDV cell entry occurs via receptor-mediated endocytosis, a process initiated by the recognition of an as yet unidentified receptor at the cell surface by the BDV surface glycoprotein (G). The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of cellular receptors and detailed mechanisms involved in BDV cell entry. To overcome this problem, we generated and characterized a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSV⌬G*/BDVG). Cells infected with rVSV⌬G*/BDVG produced high titers (10 7 PFU/ml) of cell-free virus progeny, but this virus exhibited a highly attenuated phenotype both in cell culture and in vivo. Attenuation of rVSV⌬G*/BDVG was associated with a delayed kinetics of viral RNA replication and altered genome/N mRNA ratios compared to results for rVSV⌬G*/VSVG. Likewise, incorporation of BDV G into virions appeared to be restricted despite its high levels of expression and efficient processing in rVSV⌬G*/BDVG-infected cells. Notably, rVSV⌬G*/ BDVG recreated the cell tropism and entry pathway of bona fide BDV. Our results indicate that rVSV⌬G*/ BDVG represents a unique tool for the investigation of BDV G-mediated cell entry, as well as the roles of BDV G in host immune responses and pathogenesis associated with BDV infection.Borna disease virus (BDV) causes central nervous system (CNS) disease in a variety of vertebrate species, frequently manifested by behavioral abnormalities (31). BDV was originally identified as the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep within regions of endemicity in central Europe (31). Current epidemiological data, however, indicate that the natural host range, prevalence, and geographic distribution of BDV are broader than originally thought (8,16,20,29). Experimentally, BDV has a wide host range, from birds to rodents and nonhuman primates (8,10,16,20,29). Both host and viral factors contribute to a variable period of incubation and significant heterogeneity in the symptoms and pathology associated with infection (8,10,16,20,29).BDV is an enveloped virus with a nonsegmented negativestrand RNA genome with a characteristic mononegavirales organization (4, 34, 35). However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales. The BDV genome (ca 8.9 kb) contains six major open reading frames (ORFs) in the order 3Ј-N-p10/P-M-G-L-5Ј (4, 34, 35). These ORFs code for the virus nucleoprotein (N), phosphoprotein (P) transcriptional activator, matrix (M), surface glyc...