RESUMEN

M protein mutant vesicular stomatitis virus is an attractive candidate oncolytic virus for the treatment of metastatic colorectal cancer due to its ability to kill cancer cells that are defective in their antiviral responses. The oncolytic activity of recombinant wild-type and M protein mutant vesicular stomatitis viruses was determined in RKO, Hct116 and LoVo colorectal cancer cells, as well as in human fibroblast and hepatocyte primary cultures. RKO and Hct116 cells were sensitive to both viruses, whereas LoVo cells were resistant. [(35)S]methionine labeling experiments and viral plaque assays showed that sensitive and resistant colorectal cancer cells supported viral protein and progeny production after infection with either virus. Colorectal cancer cells were pretreated with ß-interferon and infected with vesicular stomatitis virus to evaluate the extent to which interferon signaling is downregulated in colorectal cancer cells. Although colorectal cancer cells retained some degree of interferon signaling, this signaling did not negatively impact the oncolytic effects of either virus in sensitive cells. Murine xenografts of RKO cells were effectively treated by intratumoral injections with M protein mutant virus, whereas LoVo xenografts were resistant to treatment with this virus. These results suggest that M protein mutant vesicular stomatitis virus is a good candidate oncolytic virus for the treatment of selected metastatic colorectal cancers.

Asunto(s)

Neoplasias Colorrectales/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Vesiculovirus/genética , Proteínas de la Matriz Viral/genética , Animales , Línea Celular Tumoral , Supervivencia Celular , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are attractive candidates as oncolytic viruses for tumor therapies because of their capacity to selectively target cancer cells. The effectiveness of rM51R-M virus as an antitumor agent for the treatment of breast cancer was assessed by determining the ability of rM51R-M virus to infect and kill breast cancer cells in vitro and in vivo. Several human- and mouse-derived breast cancer cell lines were susceptible to infection and killing by rM51R-M virus. Importantly, non-tumorigenic cell lines from normal mammary tissues were also sensitive to VSV infection suggesting that oncogenic transformation does not alter the susceptibility of breast cancer cells to oncolytic VSV. In contrast to results obtained in vitro, rM51R-M virus was only partially effective at inducing regression of primary breast tumors in vivo. Furthermore, we were unable to induce complete regression of the primary and metastatic tumors when tumor-bearing mice were treated with a vector expressing interleukin (IL)-12 or a combination of rM51R-M virus and IL-12. Our results indicate that although breast cancer cells may be susceptible to VSV in vitro, more aggressive treatment combinations are required to effectively treat both local and metastatic breast cancers in vivo.

Asunto(s)

Neoplasias de la Mama/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Vesiculovirus/genética , Proteínas de la Matriz Viral/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/virología , Línea Celular Tumoral , Femenino , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Ratones , Proteínas Mutantes/genética , Mutación , Vesiculovirus/patogenicidad , Proteínas de la Matriz Viral/metabolismo , Replicación Viral

RESUMEN

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by tsO82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component. tsO82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.

Asunto(s)

Apoptosis , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/fisiología , Animales , Caspasa 3 , Caspasas/fisiología , Cricetinae , Células HeLa , Humanos , ARN Mensajero/análisis , Proteínas de la Matriz Viral/genética

RESUMEN

The platelet integrin alphaIIbbeta3 exhibits bidirectional signaling, in that intracellular messengers enable adhesive macromolecules to bind to its ectodomain, while ligation promotes the association of cytoskeletal proteins with its cytoplasmic domains. In order to understand the linkage between these distant regions, we investigated the effects of receptor occupancy on the solution structure of both full-length recombinant alphaIIbbeta3 and alphaIIbDelta991beta3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of (35)S-labeled human A549 cells expressing either full-length alphaIIbbeta3 or alphaIIbDelta991beta3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant alphaIIbbeta3 exhibited a weight-average sedimentation coefficient, S(w)=11.3+/-1.4 S with 73% sedimenting as protomers/dimers (9.1+/-1.0 S) and 27% as oligomers (15.4+/-0.4 S). Truncation mutant alphaIIbDelta991beta3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length alphaIIbbeta3 and alphaIIbDelta991beta3 sedimented mainly at >14 S, indicating 2-3-fold increased oligomerization. Thus we have demonstrated that alphaIIb's cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.

Asunto(s)

Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Línea Celular , Membrana Celular/metabolismo , Centrifugación por Gradiente de Densidad , Clonación Molecular , Citoplasma/química , Citometría de Flujo , Eliminación de Gen , Humanos , Ligandos , Mutación , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Transducción de Señal , Transfección , Virus Vaccinia/genética

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Factores de Transcripción TFII/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Matriz Viral/metabolismo , Células HeLa , Humanos , Fosforilación , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID

RESUMEN

In most eukaryotic cells, mitochondria use the respiratory chain to produce a proton gradient, which is then harnessed for the synthesis of ATP. Recently, mitochondrial roles in regulation of apoptosis have been discovered in many cell types. Eosinophils (Eos) die by apoptosis, but the presence and function of mitochondria in Eos are unknown. This study found that Eos contain mitochondria in small numbers, as shown by labeling with membrane potential-sensitive dyes and in situ PCR for a mitochondrial gene. Eos generate mitochondrial membrane potential from hydrolysis of ATP rather than from respiration, as shown by mitochondrial respiratory inhibitors and mitochondrial uncouplers. The mitochondria provide insignificant respiration but can induce apoptosis, as shown by using the mitochondrial F(1)F(0)-ATPase inhibitor oligomycin and translocation of cytochrome c. Thus during differentiation of Eos, although respiration is lost, the other central role of mitochondria, the induction of apoptosis, is retained.

Asunto(s)

Apoptosis , Complejo IV de Transporte de Electrones/metabolismo , Eosinófilos/citología , Mitocondrias/fisiología , ATPasas de Translocación de Protón/metabolismo , Grupo Citocromo c/metabolismo , Transporte de Electrón/fisiología , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/fisiología , Eosinófilos/metabolismo , Eosinófilos/fisiología , Colorantes Fluorescentes , Humanos , Mitocondrias/metabolismo , Compuestos Orgánicos , Consumo de Oxígeno/fisiología , ATPasas de Translocación de Protón/fisiología , Células Tumorales Cultivadas

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

Many viruses interfere with host cell function in ways that are harmful or pathological. This often results in changes in cell morphology referred to as cytopathic effects. However, pathogenesis of virus infections also involves inhibition of host cell gene expression. Thus the term "cytopathogenesis," or pathogenesis at the cellular level, is meant to be broader than the term "cytopathic effects" and includes other cellular changes that contribute to viral pathogenesis in addition to those changes that are visible at the microscopic level. The goal of this review is to place recent work on the inhibition of host gene expression by RNA viruses in the context of the pathogenesis of virus infections. Three different RNA virus families, picornaviruses, influenza viruses, and rhabdoviruses, are used to illustrate common principles involved in cytopathogenesis. These examples were chosen because viral gene products responsible for inhibiting host gene expression have been identified, as have some of the molecular targets of the host. The argument is made that the role of the virus-induced inhibition of host gene expression is to inhibit the host antiviral response, such as the response to double-stranded RNA. Viral cytopathogenesis is presented as a balance between the host antiviral response and the ability of viruses to inhibit that response through the overall inhibition of host gene expression. This balance is a major determinant of viral tissue tropism in infections of intact animals.

Asunto(s)

Efecto Citopatogénico Viral , Interacciones Huésped-Parásitos/genética , Virus ARN/patogenicidad , Apoptosis , Tamaño de la Célula , Citoesqueleto , Biosíntesis de Proteínas , Transcripción Genética

RESUMEN

Based on the x-ray crystal structure of lipid-free Delta43 apoA-I, two monomers of apoA-I were suggested to bind to a phospholipid bilayer in an antiparallel paired dimer, or "belt orientation." This hypothesis challenges the currently held model in which each of the two apoA-I monomers fold as antiparallel alpha-helices or "picket fence orientation." When apoA-I is bound to a phospholipid disc, the first model predicts that the glutamine at position 132 on one apoA-I molecule lies within 16 A of glutamine 132 in the second monomer, whereas, the second model predicts glutamines at position 132 to be 104 A apart. To distinguish between these models, glutamine at position 132 was mutated to cysteine in wild-type apoA-I to produce Q132C apoA-I, which were labeled with thiol-reactive fluorescent probes. Q132C apoA-I was labeled with either fluorescein (donor probe) or tetramethylrhodamine (acceptor probe) and then used to make recombinant phospholipid discs (recombinant high density lipoprotein (rHDL)). The rHDL containing donor- and acceptor-labeled Q132C apoA-I were of similar size, composition, and lecithin:cholesterol acyltransferase reactivity when compared to rHDL-containing human plasma apoA-I. Analysis of donor probe fluorescence showed highly efficient quenching in rHDL containing one donor- and one acceptor-labeled Q132C apoA-I. rHDL containing only acceptor probe-labeled Q132C apoA-I showed rhodamine self-quenching. Both of these observations demonstrate that position 132 in two lipid-bound apoA-I monomers were in close proximity, supporting the "belt conformation" hypothesis for apoA-I on rHDL.

Asunto(s)

Apolipoproteína A-I/química , Lípidos , Cristalografía por Rayos X , Polarización de Fluorescencia , Humanos , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.

Asunto(s)

Cápside/fisiología , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/fisiología , Ensamble de Virus , Animales , Células Cultivadas , Citosol/metabolismo

RESUMEN

During infection with vesicular stomatitis virus (VSV), host-cell mRNA synthesis is inhibited due to shut off of host-cell transcription. The transcriptional activity of nuclear extracts prepared from VSV-infected cells was compared to the activity of nuclear extracts from uninfected cells. An exogenous DNA template was used which contained an adenovirus major late promoter (AdMLP) but lacked upstream activating sequences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuclear extracts from infected cells as early as 3 h p.i. and by >90% by 6 h p.i. Mixing nuclear extracts from uninfected and VSV-infected cells revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the presence of an excess of inhibitory factor. To determine which transcription factors were lacking from nuclear extracts of infected cells, host transcription initiation factors isolated from uninfected cells by ion-exchange chromatography were added separately to nuclear extracts inactivated by VSV infection. A phosphocellulose column fraction from uninfected cells eluted with 0. 8 M KCl, which contained transcription factor IID (TFIID), overcame the inhibition. The corresponding fraction from infected cells had no detectable activity in a TFIID-dependent in vitro transcription assay. TATA-binding protein (TBP) is the DNA-binding subunit of TFIID and has been shown previously to substitute for TFIID in basal transcription. Purified recombinant TBP also reconstituted the transcription activity of nuclear extracts from infected cells, supporting the idea that TFIID is the target of virus-induced inhibition. Western blot analysis showed that the level of TBP in nuclear extracts or in the 0.8 M KCl column fraction was not changed by VSV infection. These results indicated that VSV infection leads to an inhibition of host transcription by inactivation of TFIID rather than reduction in the level of TFIID.

Asunto(s)

ARN Polimerasa II/antagonistas & inhibidores , Factores de Transcripción TFII/antagonistas & inhibidores , Transcripción Genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Western Blotting , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Proteínas Recombinantes/metabolismo , Infecciones por Rhabdoviridae/genética , Infecciones por Rhabdoviridae/metabolismo , Ribonucleasas/metabolismo , Estomatitis/genética , Estomatitis/metabolismo , Proteína de Unión a TATA-Box , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo

RESUMEN

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)- RNA synthesis were measured by incorporation of [3H]uridine. v6 and tsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)- RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and alpha-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/metabolismo , Transcripción Genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Línea Celular , Cricetinae , ARN Viral/biosíntesis , Virus de la Estomatitis Vesicular Indiana/genética

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 function in virus assembly, the viral matrix (M) protein of vesicular stomatitis virus (VSV) inhibits host-directed gene expression. The goal of this study was to determine whether sequence changes in M protein contribute to a reduced shut off of host gene expression in cells persistently infected with VSV. Viruses isolated from L cells persistently infected with VSV inhibited host RNA synthesis more slowly than wild-type (wt) VSV. M genes of the persistent viral population were cloned and sequenced. One mutation, an N to D change at position 163 of the protein sequence (N163D), was common to all the molecular clones. The N163D M protein was synthesized from transfected mRNA at a rate that was 30% of that of wt M protein, but was turned over at a rate that was similar to that of wt M protein. Transfection of mRNA encoding N163D M protein inhibited expression of a cotransfected target gene encoding chloramphenicol acetyl transferase (CAT), but the inhibition was 6 to 10 times less effective than transfection of equivalent amounts of wt M mRNA. This difference could not be accounted for by differences in translation of CAT mRNA. Thus, when the differences in M protein expression were taken into account, N163D M protein was 2 to 3 times less effective than wt M protein in the inhibition of host-directed gene expression, similar to the differences in host transcription observed in virus-infected cells. Point mutations in addition to the N163D mutation were found in about half of the M gene molecular clones. The M gene of an independently isolated molecular clone, N163D.2, contained two additional point mutations in its carboxy terminal region. N163D.2 M protein was highly defective in inhibition of host gene expression and was turned over more rapidly than wt M protein. These results support the idea that M gene mutations contribute to a reduced cytopathic effect in cells persistently infected with VSV.

Asunto(s)

Regulación Viral de la Expresión Génica , Infecciones por Rhabdoviridae/genética , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas de la Matriz Viral/genética , Línea Celular , Mutación , Infecciones por Rhabdoviridae/virología

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 use of different viral promoters for the expression of the EBNA1 gene product appears to be a critical step in the regulation of Epstein-Barr virus latent gene expression and may reflect the extent of differentiation of B-cell hosts. Low-passage Burkitt lymphoma cell lines resemble immature B cells in that they express CD10 (CALLA) and do not express B-cell activation antigens. In these cells, transcription from a promoter located in the BamHI F fragment of the viral genome results in the exclusive expression of EBNA1, referred to as the latency I pattern of viral gene expression. In contrast, high-passage Burkitt lymphoma cells and lymphoblastoid cell lines resemble activated B cells in that they do not express CD10 but do express activation antigens such as CD23. In these cells, the use of two promoters located in the BamHI W and C fragments of the viral genome leads to the expression of all six EBNA gene products (latency III). We have found that four human B-cell lines, DB, LBW2, LBW14, and Josh 7, stably express a pattern of B-cell differentiation antigens intermediate between those found in latency I and latency III cell lines and characterized by the coexpression of CD10 and CD23. The pattern of EBNA1 promoter usage in these cell lines was examined to determine whether their intermediate cellular phenotype was reflected in their patterns of viral gene expression. DB, LBW2, and LBW14 utilize both the BamHI F promoter region and BamHI W promoter region to transcribe the EBNA1 gene. This stable pattern of mixed promoter usage for the expression of the EBNA gene products in B cells has not previously been described. In addition, these three B-cell lines expressed lower levels of the viral latent gene product EBNA2 than those typically observed in latency III cells. The lower levels of activation of viral and cellular promoters known to be regulated by EBNA2 also correlated with the reduced levels of EBNA2 expression in these cells. These included the viral LMP1 and LMP2A promoters and the cellular CD23B promoter. The fourth B-cell line, Josh 7, expressed EBNA1 mRNAs derived from both the BamHI W promoter and BamHI C promoter, similar to latency III cells. The intermediate cellular phenotype in Josh 7 cells appeared to be due, in part, to a deficiency in the expression of viral LMP1.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Antígenos CD/análisis , Antígenos Virales/genética , Linfocitos B/inmunología , Proteínas de Unión al ADN/genética , Herpesvirus Humano 4/genética , Regiones Promotoras Genéticas , Antígenos CD/biosíntesis , Antígenos Virales/biosíntesis , Linfocitos B/microbiología , Línea Celular , ADN Viral/aislamiento & purificación , Proteínas de Unión al ADN/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr , Técnica del Anticuerpo Fluorescente , Expresión Génica , Herpesvirus Humano 4/inmunología , Humanos , Immunoblotting , Síndromes de Inmunodeficiencia/inmunología , Linfoma de Células B , Reacción en Cadena de la Polimerasa , ARN Viral/aislamiento & purificación , Transcripción Genética , Células Tumorales Cultivadas