The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.

Section: Resultsmentioning

confidence: 99%

Plasma Membrane Microdomains Containing Vesicular Stomatitis Virus M Protein Are Separate from Microdomains Containing G Protein and Nucleocapsids

Swinteck

Lyles

2008

“…Alternatively, or additionally, more-subtle changes have been considered to signal capsid maturation, such as a change in core protein hyperphosphorylation (characterized by the complex series of core protein species detected upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis ([SDS-PAGE]) that is typically observed with intracellular capsids but is absent in the secreted virion (21,22). We addressed the issues of how hepadnavirus nucleocapsids are selected for secretion using duck HBV (DHBV) and an adapted flotation assay that had been previously used to study other viral systems (1,5). The data we obtained indicate that (i) an initial selection of mature nucleocapsids by membrane attachment does not require an interaction with the large envelope protein and, furthermore, that (ii) extensive dephos-phorylation of capsid protein subunits, although correlating with membrane attachment, is by itself not sufficient to confer membrane affinity to free cytosolic nucleocapsids.…”

mentioning

confidence: 99%

Intracellular Hepadnavirus Nucleocapsids Are Selected for Secretion by Envelope Protein-Independent Membrane Binding

Mabit¹,

Schaller²

2000

Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surfaceexposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBVtransfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.

“…Briefly, transfected cells were fixed and incubated for 5 h at 37°C in a solution containing 0.2% X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside), 10 mM sodium phosphate buffer (pH 7.0), 150 mM NaCl, 1 mM MgCl 2 , 3.3 mM K 4 Fe(CN) 6 ⅐ 3H 2 O, and 3.3 mM K 3 Fe(CN) 6 , and the blue cells were identified under a light microscope.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…The rhabdovirus matrix (M) protein has many different functions in virus replication, the most obvious one being the initiation of virion assembly by forming a bridge between the host plasma membrane and the ribonucleocapsid core (6,12,13). For vesicular stomatitis virus (VSV), the M protein has been shown to be solely responsible for the cytopathic effect typically seen as rounding of polygonal cells in culture (11).…”

mentioning

confidence: 99%

Infectious Hematopoietic Necrosis Virus Matrix Protein Inhibits Host-Directed Gene Expression and Induces Morphological Changes of Apoptosis in Cell Cultures

Chiou

Kim

Ormonde

et al. 2000

Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shownto result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or ␤-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA "laddering" in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.The rhabdovirus matrix (M) protein has many different functions in virus replication, the most obvious one being the initiation of virion assembly by forming a bridge between the host plasma membrane and the ribonucleocapsid core (6, 12, 13). For vesicular stomatitis virus (VSV), the M protein has been shown to be solely responsible for the cytopathic effect typically seen as rounding of polygonal cells in culture (11). VSV M protein is also a potent inhibitor of host-directed transcription in mammalian cells when expressed in the absence of other viral components (1,8,9,17,31,35). It was first shown by double transient-transfection experiments that VSV M protein could inhibit the transcription of a cotransfected plasmid, pS-VCAT (simian virus 40 early-promoter-controlled chloramphenicol acetyltransferase [CAT]), while it stimulated the translation of the CAT mRNA (8, 9). The combined effect was a greater-than-20-fold inhibition of the reporter CAT activity in the M-and CAT-cotransfected cells (9). VSV M protein also inhibited other viral as well as cellular promoters including the human beta interferon (IFN-␤) promoter (17, 35). Most recently, Ahmed and Lyles (1) have shown that VSV M protein is capable of suppressing the transcription directed by each of the three RNA polymerases (RNAP): RNAPI, RNAPII, and RNAPIII. We sought to determine whether the M proteins of a rhabdovirus from an entirely different genus could function in the same manner.We examined the effect of M protein expression on fish cells for a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). This ...