A fluorescence photobleaching study of vesicular stomatitis virus infected BHK cells. Modulation of G protein mobility by M protein

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.     

Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization.

Twenty-five spontaneous temperature-stable revertants of four different temperature-sensitive (ts) M protein mutants (complementation group III: tsG31, tsG33, tsO23, and tsO89) were sequenced and tested for their ability to inhibit vesicular stomatitis virus RNA polymerase activity in vitro. Consensus sequences of the coding region of each M protein gene were determined, using total viral RNA as template. Fifteen different sequences were found among the 25 revertants; 14 differed from their ts parent by a single amino acid (one nucleotide), and 1 differed by two amino acids (two nucleotides). Amino acids were altered in various positions between residues 64 and 215, representing over 60% of the polypeptide chain. Resequencing of the Glasgow and Orsay wild types and the four ts mutants confirmed previously published differences (Y. Gopalakrishana and J. Lenard, J. Virol., 56:655-659, 1985), and one or two additional differences were found in each. The relative charges of the revertant M proteins, as determined by nonequilibrium pH gradient electrophoresis, were consistent with the deduced sequences in every case. The ability of each revertant M protein to inhibit the RNA polymerase activity of nucleocapsids prepared from its parent ts mutant was also tested. Only 13 of the 25 revertants had M protein with high (wild type-like) polymerase-inhibiting activity, while 5 had low (ts-like) activity, and 7 had intermediate activity, demonstrating that this property is not an essential concomitant of the temperature-stable phenotype. It is concluded that the high reversion frequency observed for these mutants arises from a very high incidence of pseudoreversion, i.e., many different molecular changes can repair the ts phenotype.     

Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity.

Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well.     

Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Immune response in the absence of neurovirulence in mice infected with m protein mutant vesicular stomatitis virus

Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.     

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.     

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

We have previously shown that Phe(120), Glu(216), and Asp(301) in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021-4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353-360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (K(d)) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25-0.50 microm. The amide substitution of Glu(216) or Asp(301) resulted in 30-64-fold increases in the K(d) for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K(d) of 65 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations.     

Characterization of vesicular stomatitis virus mutants by partial proteolysis

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Interactions of wild-type and mutant M protein of vesicular stomatitis virus with viral nucleocapsid and envelope in intact virions. Evidence from [125I]iodonaphthyl azide labeling and specific cross-linking

Four different temperature-sensitive M protein mutants (tsM) of vesicular stomatitis virus (VSV) were characterized with regard to the association of the mutated M protein either with nucleocapsids or with membranes in the intact virions. Virions were labeled with the photoreactive hydrophobic probe [125I]iodonaphthyl azide (INA) to assess interactions between viral proteins and the lipid envelope. In wild type (wt) virions, the three major structural proteins--G, M, and N--were labeled in the ratio ca. 1.0:0.4:0.2. INA labeled only the membrane-associated peptide of G protein, both in the intact virion and in reconstituted G protein--viral lipid vesicles, demonstrating the specificity of INA for lipid bilayer regions. Labeling of tsM virions with INA resulted in a 2--3-fold greater incorporation into M protein than was found for wt virions, suggesting increased M--membrane associations in the mutant virions. Temperature-stable revertants from tsM possessed wt labeling characteristics. Interaction of the M protein with nucleocapsids was assessed from the abundance of disulfide-linked M--N complexes found after disruption of the virions by sodium dodecyl sulfate solution under nonreducing conditions. The abundance of such complexes was 30--80% less from tsM virions than from wt virions, suggesting decreased M--nucleocapsid interactions in tsM virions. Temperature-stable revertants from tsM resembled wt in the abundance of M--N complex formed. We conclude that the mutations alter M protein in such a way as simultaneously to increase its association with membrane and to decrease its affinity for nucleocapsids in the intact virion.     

Oncolytic vesicular stomatitis virus induces apoptosis via signaling through PKR, Fas, and Daxx

Matrix (M) protein mutants of vesicular stomatitis virus (VSV) are promising oncolytic agents for cancer therapy. Previous research has implicated Fas and PKR in apoptosis induced by other viruses. Here, we show that dominant-negative mutants of Fas and PKR inhibit M protein mutant virus-induced apoptosis. Most previous research has focused on the adapter protein FADD as a necessary transducer of Fas-mediated apoptosis. However, the expression of dominant-negative FADD had little effect on the induction of apoptosis by M protein mutant VSV. Instead, virus-induced apoptosis was inhibited by the expression of a dominant-negative mutant of the adapter protein Daxx. These data indicate that Daxx is more important than FADD for apoptosis induced by M protein mutant VSV. These results show that PKR- and Fas-mediated signaling play important roles in cell death during M protein mutant VSV infection and that Daxx has novel functions in the host response to virus infection by mediating virus-induced apoptosis.