RESUMEN

The goal of these experiments was to determine the steps in virus assembly that are defective at the nonpermissive temperature in temperature-sensitive (ts) matrix (M) protein mutants of vesicular stomatitis virus. It has been proposed that mutations in M protein either reduce the binding affinity for nucleocapsids or lead to aggregation, reducing the amount of M protein available for virus assembly. Cytosolic or membrane-derived M proteins from wild-type VSV and two ts M protein mutant viruses, tsM301 and tsO23, as well as a revertant of tsO23 virus, O23R1, were analyzed for binding to nucleocapsid-M protein (NCM) complexes and for M protein aggregation. The experiments presented here showed that ts M proteins synthesized at the nonpermissive temperature were capable of binding to nucleocapsids and that aggregation of ts M proteins did not reduce the amount of soluble M protein below the amount required for assembly of the O23R1 virus. Instead, the most pronounced defect in ts M proteins was in the ability of membrane-derived M proteins to be solubilized in the presence of the detergent Triton X-100. It is proposed that this detergent-insoluble form of M protein interferes with a step necessary to initiate assembly of NCM complexes. A similar detergent, Triton X-114, caused aggregation of membrane-derived wild-type M protein, disproving an earlier proposal that membrane-derived M protein behaves like an integral membrane protein in the presence of Triton X-114. Aggregation of wild-type M protein in the presence of Triton X-100 could be induced by incubation at 37 degrees C with a high-molecular-weight fraction isolated from uninfected cells by sucrose gradient centrifugation. These results implicate host components in inducing M protein aggregation.

Asunto(s)

Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/fisiología , Sustitución de Aminoácidos , Animales , Línea Celular , Membrana Celular/virología , Cricetinae , Citosol/virología , Cinética , Leucina , Nucleocápside/química , Nucleocápside/fisiología , Fenilalanina , Temperatura , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Virión/genética , Virión/fisiología

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) condenses the viral nucleoprotein core (nucleocapsid) into a tightly coiled, helical nucleocapsid-M protein (NCM) complex. Using NCM complexes assembled in vivo, the dissociation of M protein was examined by measuring the apparent affinity constants and kinetic constants for M protein binding to NCM complexes immediately after detergent solubilization of the virion envelope. Wild-type VSV strains and viruses with mutations in their M proteins were analyzed using sedimentation and light-scattering assays. At physiological ionic strength, the binding reaction had the characteristics of a dynamic reversible equilibrium. A temperature-sensitive M protein mutant lost the ability of M protein to reversibly dissociate from the nucleocapsid, while a temperature-stable revertant regained the ability to undergo reversible dissociation. In contrast to the results obtained at physiological ionic strength, nucleocapsids stripped of M protein by incubation at high ionic strength (250 mM NaCl) were not able to bind M protein at low ionic strength with the same high affinity seen in NCM complexes assembled in vivo. The effect of incubation at 250 mM NaCl was shown to be due to a change in nucleocapsids rather than a change in soluble M protein. This result supports the idea that nucleocapsids devoid of M protein must undergo a separate step that initiates high-affinity binding of M protein in vivo.

Asunto(s)

Proteínas de la Nucleocápside , Nucleocápside/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Matriz Viral/metabolismo , Análisis de Inyección de Flujo , Cinética , Luz , Modelos Químicos , Concentración Osmolar , Unión Proteica/efectos de los fármacos , Dispersión de Radiación , Cloruro de Sodio/farmacología , Ultracentrifugación , Virus de la Estomatitis Vesicular Indiana/genética

RESUMEN

In addition to its role in virus assembly, the matrix (M) protein of vesicular stomatitis virus (VSV) is involved in virus-induced cell rounding and inhibition of host-directed gene expression. Previous experiments have shown that two M protein mutants genetically dissociate the ability of M protein to inhibit host-directed gene expression from its function in virus assembly. M protein from tsO82 virus is fully functional in virus assembly but defective in the inhibition of host-directed gene expression, while the MN1 deletion mutant, which lacks amino acids 4-21, inhibits host-directed gene expression but cannot function in virus assembly. Experiments presented here compared cell rounding induced by these two mutant M proteins to that of wt M protein. BHK cells were transfected with M protein mRNA transcribed in vitro, and the extent of cell rounding was evaluated at 24 hr posttransfection. The MN1 protein was nearly as effective as wt M protein in the induction of cell rounding, while tsO82 M protein expressed from transfected RNA was not able to induce cell rounding above that observed in negative controls without M protein, although it did cause BHK cells to have a less elongated shape. These results indicate that the ability of MN1 and tsO82 M proteins to induce cell rounding is not correlated with their virus assembly function. Instead the cell rounding activity of these mutants is correlated with their ability to inhibit host-directed gene expression. Previous data suggesting that these two cytopathic activities could be dissociated can be readily accounted for by quantitative differences in M protein expression required. Infection of either BHK cells or L cells with tsO82 virus induced cell rounding, although cell rounding was delayed relative to that following infection with wt VSV, suggesting that tsO82 M protein retains some cytopathic activity. The distribution of actin, vimentin, and tubulin in transfected cells was determined by fluorescence microscopy. In cells transfected with tsO82 M mRNA, these cytoskeletal elements were indistinguishable from those of negative control transfected cells. In cells rounded as a result of transfection with wt M or MN1 mRNA, actin-containing filaments were reorganized into a thick perinuclear ring but were not depolymerized. In contrast, tubulin and vimentin appeared to be diffusely distributed throughout the cytoplasm of rounded cells. These results support the idea that cell rounding induced by M protein results from the depolymerization of microtubules and/or intermediate filaments.

Asunto(s)

Regulación Viral de la Expresión Génica , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/genética , Ensamble de Virus , Animales , Línea Celular , Cricetinae , Citoesqueleto , Mutación , ARN Mensajero/genética , Transfección , Virus de la Estomatitis Vesicular Indiana/genética

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts of in vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700-11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature.

Asunto(s)

Regulación Viral de la Expresión Génica/fisiología , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas de la Matriz Viral/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cricetinae , Regulación de la Expresión Génica/fisiología , Mutación , Factores de Crecimiento Nervioso/farmacología , Células PC12 , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa II , ARN Mensajero/genética , ARN Viral/genética , Ratas , Temperatura , Transcripción Genética/fisiología , Activación Transcripcional , Transfección , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética

RESUMEN

During the process of assembly of enveloped viruses, binding of the nucleoprotein core of the virus (nucleocapsid) to the host membrane is mediated by the viral matrix (M) protein. Light scattering properties of vesicular stomatitis virus (VSV) nucleocapsids and nucleocapsid-M protein (NCM) complexes assembled in vivo were determined following solubilizaton of the virion envelope with detergents at varying ionic strength to vary the extent of M protein binding. Three factors were found to contribute to the light scattering properties of VSV nucleocapsids: their conformation, extent of self-association, and amount of bound M protein. All three were affected by changes in ionic strength but could be distinguished by several parameters. Conformational changes in nucleocapsids and NCM complexes occurred rapidly (millisecond time scale) upon changing salt concentration and were reflected in changes in the angular dependence of light scattering intensity (i.e., changes in radius of gyration, RG). Changes in extent of self-association occurred relatively slowly (seconds to minutes time scale) and could be distinguished by the concentration dependence of the apparent molecular mass and diffusion coefficient of the NCM complex. Changes in M protein binding occurred on an intermediate time scale (t1/2 approximately one s) and reflected changes in both molecular mass and RG. The data presented here provide criteria for assessing binding of M protein to nucleocapsids under conditions of minimal perturbation of the NCM complex assembled in vivo and at low protein concentrations so that self-association of the NCM complex was minimal and reversible.

Asunto(s)

Cápside/química , Virus de la Estomatitis Vesicular Indiana/química , Proteínas de la Matriz Viral/química , Animales , Cápside/metabolismo , Línea Celular , Cricetinae , Cinética , Luz , Sustancias Macromoleculares , Peso Molecular , Unión Proteica , Conformación Proteica , Dispersión de Radiación , Cloruro de Sodio , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Matriz Viral/metabolismo

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) binds the nucleocapsid to the cytoplasmic surface of the host plasma membrane during virus assembly by budding. It also condenses the nucleocapsid into a tightly coiled nucleocapsid-M protein complex that appears to give the virion its bullet-like shape. As described here, temperature-sensitive (ts) M mutants produced two classes of membrane-containing extracellular particles at the nonpermissive temperature. These could be distinguished by sedimentation in sucrose gradients and by electron microscopy. One class contained nucleocapsids and envelope glycoprotein, but very little M protein. The other class was devoid of nucleocapsids. Most of these particles were spherical or pleiomorphic in shape as determined by electron microscopy. Expression of wild-type (wt) M protein from plasmid DNA using the vaccinia/T7 virus system did not enhance the incorporation of nucleocapsids into extracellular particles from cells coinfected with the ts M mutants but did enhance the incorporation of M protein into these particles. Electron microscopy showed that wt M protein served to impart the bullet-like shape typical of VSV virions to what would otherwise be spherical or pleiomorphic virus-like particles. These data suggest that there are two distinct processes in VSV envelope biogenesis. One process involves envelopment of the nucleocapsid and can be accomplished by the ts M mutants at the nonpermissive temperature, albeit at a low level compared to wt VSV. The other process involves conversion of virion components into the bullet-like shape and requires a function provided by wt M protein.

Asunto(s)

Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/fisiología , Animales , Cápside/fisiología , Cápside/ultraestructura , Línea Celular , Centrifugación por Gradiente de Densidad , Cricetinae , Prueba de Complementación Genética , Mutación , Plásmidos , Temperatura , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/ultraestructura , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/ultraestructura , Virión/ultraestructura , Replicación Viral

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a central role in virus assembly by binding the nucleocapsid core to the viral envelope during the budding process. A small percentage of M protein molecules are phosphorylated in vivo, but the role of phosphorylation in M protein function is unknown. Using limited proteolysis, we previously determined the sites of in vivo phosphorylation for VSV M protein to be Thr 31 (and possibly Ser 32) and a site N-terminal to position 19 (Ser 2, Ser 3, or Ser 17) (P. E. Kaptur et al., J. Virol. 66, 5384-5392, 1992). M protein mutants were constructed using site-directed mutagenesis by substituting Ala for Ser or Thr at these sites in the M gene of the San Juan strain of VSV. One mutant had substitutions at the major in vivo phosphorylation site(s) at positions 31 and 32 (M31.32) while two others had additional substitutions at positions 2 and 3 (M2.3.31.32) or at position 17 (M17.31.32). Mutant M proteins were expressed in BHK cells using the vaccinia/T7 system, radiolabeled with 32Pi, and then analyzed for 32P content by PAGE and autoradiography. The data show that the site of phosphorylation near the N-terminus is at Ser 2 or 3 and not Ser 17. Further, Ser 38 was not phosphorylated. Mutation of the major phosphorylation site enhanced phosphorylation at alternative sites in the M protein C-terminal to amino acid 43 and at Ser residues 2 and 3. Mutant M proteins were tested for their ability to complement growth of the temperature-sensitive M protein mutant virus tsO23 at the nonpermissive temperature. Mutant M2.3.31.32 was further tested for its ability to assemble into VSV-defective interfering (DI) particles, using a replication system in which the DI genome and all five VSV proteins were expressed from plasmid DNA. Assembly of tsO23 virions or DI particles in the presence of mutant M proteins was similar to that observed for wild-type M proteins. These data indicate that phosphorylation of M protein at the major in vivo sites is not essential for virus assembly.

Asunto(s)

Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Matriz Viral/biosíntesis , Secuencia de Aminoácidos , Animales , Autorradiografía , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Cricetinae , Cartilla de ADN , Virus Defectuosos/crecimiento & desarrollo , Virus Defectuosos/metabolismo , Electroforesis en Gel de Poliacrilamida , Prueba de Complementación Genética , Riñón , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Radioisótopos de Fósforo , Fosforilación , Plásmidos , Reacción en Cadena de la Polimerasa , Serina , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética

RESUMEN

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Glicoproteínas/química , Glicoproteínas de Membrana , Virus de la Estomatitis Vesicular Indiana/química , Proteínas del Envoltorio Viral/química , Reactivos de Enlaces Cruzados , Detergentes , Colorantes Fluorescentes , Glucósidos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos , Micelas , Octoxinol , Polietilenglicoles , Termodinámica