Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Spring viraemia of carp virus modulates p53 expression using two distinct mechanisms.

p53, which regulates cell-cycle arrest and apoptosis, is a crucial target for viruses to release cells from cell-cycle checkpoints or to protect cells from apoptosis for their own benefit. Viral evasion mechanisms of aquatic viruses remain mysterious. Here, we report the spring viremia of carp virus (SVCV) degrading and stabilizing p53 in the ubiquitin-proteasome pathway by the N and P proteins, respectively. Early in an SVCV infection, significant induction was observed in the S phase and p53 was decreased in the protein level. Further experiments demonstrated that p53 interacted with SVCV N protein and was degraded by suppressing the K63-linked ubiquitination. However, the increase of p53 was observed late in the infection and experiments suggested that p53 was bound to SVCV P protein and stabilized by enhancing the K63-linked ubiquitination. Finally, lysine residue 358 was the key site for p53 K63-linked ubiquitination by the N and P proteins. Thus, our findings suggest that fish p53 is modulated by SVCV N and P protein in two distinct mechanisms, which uncovers the strategy for the subversion of p53-mediated host innate immune responses by aquatic viruses.

Phosphatidylinositol-3-kinase-Akt pathway in negative-stranded RNA virus infection: a minireview.

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

The biological features and genetic diversity of novel fish rhabdovirus isolates in China.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses which infects mammals, birds, reptiles, fish, insects and plants. Herein, we reported the isolation and characterization of 6 novel viruses from diseased fish collected from China including SCRV-QY, SCRV-SS, SCRV-GM, CmRV-FS, MsRV-SS, OmbRV-JM. The typical clinical symptom of diseased fish was hemorrhaging. Efficient propagation of these isolates in a Chinese perch brain cell line was determined by means of observation of cytopathic effect, RT-PCR and electron microscopy. Sequence alignment and phylogenetic analysis of the complete G protein sequences revealed that these isolates were clustered into one monophyletic lineage belonging to the species Siniperca chuatsi rhabdovirus.

Molecular cloning, characterization and expression of immunoglobulin D on pathogen challenge and pathogen associated molecular patterns stimulation in freshwater carp, Catla catla.

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.

Three-dimensional morphometric analysis of microglial changes in a mouse model of virus encephalitis: age and environmental influences.

Many RNA virus CNS infections cause neurological disease. Because Piry virus has a limited human pathogenicity and exercise reduces activation of microglia in aged mice, possible influences of environment and aging on microglial morphology and behavior in mice sublethal encephalitis were investigated. Female albino Swiss mice were raised either in standard (S) or in enriched (EE) cages from age 2 to 6 months (young - Y), or from 2 to 16 months (aged - A). After behavioral tests, mice nostrils were instilled with Piry-virus-infected or with normal brain homogenates. Brain sections were immunolabeled for virus antigens or microglia at 8 days post-infection (dpi), when behavioral changes became apparent, and at 20 and 40 dpi, after additional behavioral testing. Young infected mice from standard (SYPy) and enriched (EYPy) groups showed similar transient impairment in burrowing activity and olfactory discrimination, whereas aged infected mice from both environments (EAPy, SAPy) showed permanent reduction in both tasks. The beneficial effects of an enriched environment were smaller in aged than in young mice. Six-hundred and forty microglial cells, 80 from each group were reconstructed. An unbiased, stereological sampling approach and multivariate statistical analysis were used to search for microglial morphological families. This procedure allowed distinguishing between microglial morphology of infected and control subjects. More severe virus-associated microglial changes were observed in young than in aged mice, and EYPy seem to recover microglial homeostatic morphology earlier than SYPy . Because Piry-virus encephalitis outcomes were more severe in aged mice, it is suggested that the reduced inflammatory response in those individuals may aggravate encephalitis outcomes.

Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster.

Variation in susceptibility to infectious disease often has a substantial genetic component in animal and plant populations. We have used genome-wide association studies (GWAS) in Drosophila melanogaster to identify the genetic basis of variation in susceptibility to viral infection. We found that there is substantially more genetic variation in susceptibility to two viruses that naturally infect D. melanogaster (DCV and DMelSV) than to two viruses isolated from other insects (FHV and DAffSV). Furthermore, this increased variation is caused by a small number of common polymorphisms that have a major effect on resistance and can individually explain up to 47% of the heritability in disease susceptibility. For two of these polymorphisms, it has previously been shown that they have been driven to a high frequency by natural selection. An advantage of GWAS in Drosophila is that the results can be confirmed experimentally. We verified that a gene called pastrel--which was previously not known to have an antiviral function--is associated with DCV-resistance by knocking down its expression by RNAi. Our data suggest that selection for resistance to infectious disease can increase genetic variation by increasing the frequency of major-effect alleles, and this has resulted in a simple genetic basis to variation in virus resistance.

Spread and morphological-structural properties of plant rhabdoviruses and similar pathogens in Basidiomycetes.

Long-term studies of spread of rhabdoviruses which indicated their harmfulness in different plant species under conditions of environmental factors were first discussed. Their harmfulness to different plant species under environmental conditions was shown. A comparative description of rhabdoviruses with similar pathogens of the mushrooms is carried out. Thus the main focus was on the morphology and structure of the pathogens. These data are extremely important in the study of distribution of the rhabdovirus on crops in different regions.

Non-human viruses developed as therapeutic agent for use in humans.

Viruses usually infect a restricted set of host species, and only in rare cases does productive infection occur outside the natural host range. Infection of a new host species can manifest as a distinct disease. In this respect, the use of non-human viruses in clinical therapy may be a cause for concern. It could provide the opportunity for the viruses to adapt to the new host and be transferred to the recipient's relatives or medical caretakers, or even to the normal host species. Such environmental impact is evidently undesirable. To forecast future clinical use of non-human viruses, a literature study was performed to identify the viruses that are being considered for application as therapeutic agents for use in humans. Twenty-seven non-human virus species were identified that are in (pre)clinical development, mainly as oncolytic agents. For risk management, it is essential that the potential environmental consequences are assessed before initiating clinical use, even if the virus is not formally classified as a genetically modified organism. To aid such assessment, each of these viruses was classified in one of five relative environmental risk categories, ranging from "Negligible" to "Very High". Canary pox virus and the Autographa californica baculovirus were assigned a "Negligible" classification, and Seneca Valley virus, murine leukemia virus, and Maraba virus to the "High" category. A complicating factor in the classification is the scarcity of publicly available information on key aspects of virus biology in some species. In such cases the relative environmental risk score was increased as a precaution.

[Rhabdoviruses].

The family Rhabdoviridae has a non-segmented single stranded negative-sense RNA and its genome ranges in size from approximately 11 kb to almost 16 kb. It is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms. The five structural protein genes are arranged in the same linear order (3'-N-P-M-G-L-5') and may be interspersed with one more additional accessory gene. For many years, a full of knowledge of the rhabdoviridae has been established on extensive studies of two kinds of prototype viruses; vesicular stomatitis virus (VSV) and rabies virus (RABV). Among them, the genus Lyssavirus includes RABV and rabies-related viruses naturally infect mammals and chiropterans via bite-exposure by rabid animals and finally cause fatal encephalitis. In this review, we describe the sketch of the various virological features of the Rhabdoviridae, especially focusing on VSV and RABV.

Genetic diversity of perch rhabdoviruses isolates based on the nucleoprotein and glycoprotein genes.

Despite the increasing impact of rhabdoviruses in European percid farming, the diversity of the viral populations is still poorly investigated. To address this issue, we sequenced the partial nucleoprotein (N) and complete glycoprotein (G) genes of nine rhabdoviruses isolated from perch (Perca fluviatilis) between 1999 and 2010, mostly from France, and analyzed six of them by immunofluorescence antibody test (IFAT). Using two rabbit antisera raised against either the reference perch rhabdovirus (PRhV) isolated in 1980 or the perch isolate R6146, two serogroups were distinguished. Meanwhile, based on partial N and complete G gene analysis, perch rhabdoviruses were divided into four genogroups, A-B-D and E, with a maximum of 32.9% divergence (G gene) between isolates. A comparison of the G amino acid sequences of isolates from the two identified serogroups revealed several variable regions that might account for antigenic differences. Comparative analysis of perch isolates with other rhabdoviruses isolated from black bass, pike-perch and pike showed some strong phylogenetic relationships, suggesting cross-host transmission. Similarly, striking genetic similarities were shown between perch rhabdoviruses and isolates from other European countries and various ecological niches, most likely reflecting the circulation of viruses through fish trade as well as putative transfers from marine to freshwater fish. Phylogenetic relationships of the newly characterized viruses were also determined within the family Rhabdoviridae. The analysis revealed a genetic cluster containing only fish viruses, including all rhabdoviruses from perch, as well as siniperca chuatsi rhabdovirus (SCRV) and eel virus X (EVEX). This cluster was distinct from the one represented by spring viraemia of carp vesiculovirus (SVCV), pike fry rhabdovirus (PFRV) and mammalian vesiculoviruses. The new genetic data provided in the present study shed light on the diversity of rhabdoviruses infecting perch in France and support the hypothesis of circulation of these viruses between other hosts and regions within Europe.

Autophagy Delays Apoptotic Cell Death Induced by Siniperca chuatsi Rhabdovirus in Epithelioma Papulosum Cyprinid Cells.

Autophagy and apoptosis are two key cell fate determination pathways, which play vital roles in the interaction between viruses and host cells. Previous research had confirmed that one strain of fish rhabdoviruses, Siniperca chuatsi rhabdovirus (SCRV), could induce apoptosis and autophagy after infection. In the current study, we continued to analyze the interaction of autophagy and apoptosis in SCRV-infected EPC cell lines after treatment with different autophagy or apoptosis inhibitors. We found that SCRV infection could activate the mitochondrial apoptotic pathway by the detection of the activities of the caspase-3 and caspase-9 and by flow cytometry analysis in JC-1-stained cells, respectively. Furthermore, no significant autophagy-related factors were disturbed in SCRV-infected cell after apoptosis inhibitor Z-VAD-FMK treatment, while autophagy inducer rapamycin could obviously delay the occurrence of CPE and cell death. Meanwhile, rapamycin was able to reduce the proportion of apoptotic cells. Besides that, rapamycin could disturb the expression of p62 and LC3B-II, and the transcription level of SCRV nucleoprotein mRNA. The progeny virus titers did not show a big difference between the rapamycin treatment or without it. Collectively, our data preliminarily confirmed that SCRV-activated autophagy could delay apoptosis in EPC cells and may not affect virus production. Further study may need to focus on the crosstalk regulation and its roles on the SCRV infection.

Human Tibroviruses: Commensals or Lethal Pathogens?

Rhabdoviruses are a large and ecologically diverse family of negative-sense RNA viruses (Mononegavirales: Rhabdoviridae). These viruses are capable of infecting an unexpectedly wide variety of plants, vertebrates, and invertebrates distributed over all human-inhabited continents. However, only a few rhabdoviruses are known to infect humans: a ledantevirus (Le Dantec virus), several lyssaviruses (in particular, rabies virus), and several vesiculoviruses (e.g., Chandipura virus, vesicular stomatitis Indiana virus). Recently, several novel rhabdoviruses have been discovered in the blood of both healthy and severely ill individuals living in Central and Western Africa. These viruses-Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2-are members of the little-understood rhabdoviral genus Tibrovirus. Other than the basic genomic architecture, tibroviruses bear little resemblance to well-studied rhabdoviruses such as rabies virus and vesicular stomatitis Indiana virus. These three human tibroviruses are quite divergent from each other, and each of them clusters closely with tibroviruses currently known only from biting midges or healthy cattle. Seroprevalence studies suggest that human tibrovirus infections may be common but are almost entirely unrecognized. The pathogenic potential of this diverse group of viruses remains unknown. Although certain tibroviruses may be benign and well-adapted to humans, others could be newly emerging and produce serious disease. Here, we review the current knowledge of tibroviruses and argue that assessing their impact on human health should be an urgent priority.

Comparative modulation of lncRNAs in wild-type and rag1-heterozygous mutant zebrafish exposed to immune challenge with spring viraemia of carp virus (SVCV).

Although the modulation of immune-related genes after viral infection has been widely described in vertebrates, the potential implications of non-coding RNAs (ncRNAs), especially long non-coding RNAs (lncRNAs), in immunity are still a nascent research field. The model species zebrafish could serve as a useful organism for studying the functionality of lncRNAs due to the numerous advantages of this teleost, including the existence of numerous mutant lines. In this work, we conducted a whole-transcriptome analysis of wild-type (WT) and heterozygous rag1 mutant (rag1+/-) zebrafish after infection with the pathogen spring viraemia of carp virus (SVCV). WT and rag1+/- zebrafish were infected with SVCV for 24 h. Kidney samples were sampled from infected and uninfected fish for transcriptome sequencing. From a total of 198,540 contigs, 12,165 putative lncRNAs were identified in zebrafish. Most of the putative lncRNAs were shared by the two zebrafish lines. However, by comparing the lncRNA profiles induced after SVCV infection in WT and rag1+/- fish, most of the lncRNAs that were significantly induced after viral challenge were exclusive to each line, reflecting a highly differential response to the virus. Analysis of the neighboring genes of lncRNAs that were exclusively modulated in WT revealed high representation of metabolism-related terms, whereas those from rag1+/- fish showed enrichment in terms related to the adaptive immune response, among others. On the other hand, genes involved in numerous antiviral processes surrounded commonly modulated lncRNAs, as expected. These results clearly indicate that after SVCV infection in zebrafish, the expression of an array of lncRNAs with functions in different aspects of immunity is induced.

Viral Equine Encephalitis, a Growing Threat to the Horse Population in Europe?

Neurological disorders represent an important sanitary and economic threat for the equine industry worldwide. Among nervous diseases, viral encephalitis is of growing concern, due to the emergence of arboviruses and to the high contagiosity of herpesvirus-infected horses. The nature, severity and duration of the clinical signs could be different depending on the etiological agent and its virulence. However, definite diagnosis generally requires the implementation of combinations of direct and/or indirect screening assays in specialized laboratories. The equine practitioner, involved in a mission of prevention and surveillance, plays an important role in the clinical diagnosis of viral encephalitis. The general management of the horse is essentially supportive, focused on controlling pain and inflammation within the central nervous system, preventing injuries and providing supportive care. Despite its high medical relevance and economic impact in the equine industry, vaccines are not always available and there is no specific antiviral therapy. In this review, the major virological, clinical and epidemiological features of the main neuropathogenic viruses inducing encephalitis in equids in Europe, including rabies virus (Rhabdoviridae), Equid herpesviruses (Herpesviridae), Borna disease virus (Bornaviridae) and West Nile virus (Flaviviridae), as well as exotic viruses, will be presented.

Characterization of a novel rhabdovirus isolated from a stranded harbour porpoise (Phocoena phocoena).

An adult male harbour porpoise (Phocoena phocoena) stranded off the coast of Alaska displaying poor body condition, scattered mild ulcerative dermatitis, and necrotizing balanoposthitis. Necropsy findings included severe verminous panniculitis, pneumonia, hepatitis, and enteritis. Histopathological examination of skin lesions revealed a pustular epidermitis and dermatitis, with ballooning degeneration of keratinocytes and occasional amphophilic intracytoplasmic inclusion bodies. A swab sample collected from the ulcerative penile lesions was processed for virus isolation resulting in cytopathic effects observed in primary beluga whale kidney (BWK) cells. Transmission electron microscopy revealed bullet-shaped virions budding from the cell surface of infected BWK cells consistent with a rhabdovirus. A cDNA library was prepared using RNA extracted from infected cell culture supernatant and sequenced on an Illumina MiSeq sequencer. The near-complete genome of a novel rhabdovirus was recovered. Genetic and phylogenetic analyses based on the complete L gene supported the harbour porpoise rhabdovirus (HPRV) as a new species. HPRV clustered phylogenetically with dolphin rhabdovirus (DRV) and this cetacean rhabdovirus clade was found to be the sister group to members of the genus Perhabdovirus that infect fish. A specific nested RT-PCR assay detected HPRV RNA in the epaxial musculature of the harbour porpoise. Our results are consistent with a previous hypothesis that cetacean rhabdoviruses may have arisen following a host jump from fish and suggest that DRV and HPRV represent separate species belonging in a new genus within the family Rhabdoviridae. Further research is needed to determine the health impact of HPRV in harbour porpoise populations, its prevalence, and route of transmission.

NLRX1 of black carp suppresses MAVS-mediated antiviral signaling through its NACHT domain.

NOD-like receptor (NLR) family member X1 (NLRX1) of human localizes on mitochondria and serves as a negative regulator of antiviral signaling. However, the function of NLRX1 in teleost fish still remains elusive. To explore its role in the innate immunity of teleost fish, NLRX1 homologue has been cloned and characterized from black carp (Mylopharyngodon piceus). Black carp NLRX1 (bcNLRX1) consists of 1008 amino acids, which includes a N-terminal mitochondrial targeting sequence, a central NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. bcNLRX1 was identified as a cytosolic protein locating on mitochondria through immunofluorescence (IF) staining. The overlapped subcellular distribution of bcNLRX1 and black carp MAVS (bcMAVS) was detected in IF staining, and the direct interaction between these two molecules in vitro was identified through co-immunoprecipitation assay. When co-expressed with bcMAVS, bcNLRX1 fiercely reduced bcMAVS-mediated IFN induction in reporter assay. Accordingly, the antiviral activity of bcMAVS against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV) was forcefully repressed by bcNLRX1 in plaque assay. Mutagenic analyses further revealed that the NACHT domain of bcNLRX1 was essential for it to interact with bcMAVS and to suppress bcMAVS-mediated antiviral signaling. Taken together, our data support the conclusion that bcNLRX1 negatively regulates bcMAVS-mediated antiviral signaling through its NACHT domain during host innate immune activation.

The negative regulation of piscine CD44c in viral and bacterial infection.

CD44 gene is a cell surface receptor which undergoes complex alternative splicing and extensive post-translational modifications. Although many studies have showed that CD44 is involved in the process of host defense, the function of piscine CD44 in antibacterial or antiviral defense response remains unclear. In the present study, we report the functional characterization of zebrafish CD44c, which is more similar to CD44b antigen isoforms rather than CD44a based on amino acid composition and phylogenetic analysis. The expression of zebrafish CD44c was inducible in response to bacterial and viral infections. During SVCV infection, the in vivo studies revealed that CD44c overexpression led to the increased virus loads and decreased survival rate. The attenuated response by zebrafish CD44c in response to SVCV infection were characterized by the impaired production of inflammatory cytokines and the impaired expressions of IFNs, IFN-stimulated genes, MHC class I and II genes. During Edwardsiella piscicida infection, the overexpression of zebrafish CD44c facilitated bacterial growth and dissemination, but did not impact on larvae survival. The detrimental role of CD44c in host defense against E. piscicida infection was supported by a decreased production of several antibacterial molecules including defbl2, defbl3, NK-lysin and RNase3. All together, these results firstly demonstrate the negative regulation of piscine CD44c in viral and bacterial infection.

Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.

The budding reactions of a number of enveloped viruses use the cellular machinery involved in the formation of the luminal vesicles of endosomal multivesicular bodies (MVB). Budding of these viruses is dependent on the presence of specific late-domain motifs in membrane-associated viral proteins. Such budding reactions usually involve ubiquitin and are blocked by expression of an ATPase-deficient form of VPS4, a cellular AAA+ ATPase believed to be required late in the MVB pathway for the disassembly/release of the MVB machinery. Here we examined the role of the MVB pathway in the budding of the late-domain-containing rhabdovirus vesicular stomatitis virus (VSV) and the alphavirus Semliki Forest virus (SFV). We tested early and late steps in the MVB pathway by depleting ubiquitin with the proteasome inhibitor MG-132 and by using cell lines inducibly expressing VPS4A or VPS4B protein. As previously shown, VSV budding was strongly dependent on ubiquitin. In contrast to the findings of previous studies with VPS4A, expression of ATPase-deficient mutants of either VPS4A or VPS4B inhibited VSV budding. Inhibition by VPS4 required the presence of the PPPY late domain on the VSV matrix protein and resulted in the accumulation of nonreleased VSV particles at the plasma membrane. In contrast, SFV budding was independent of both ubiquitin and the activity of VPS4, perhaps reflecting the important role of the highly organized envelope protein lattice during alphavirus budding.