Attenuated vesicular stomatitis viruses as vaccine vectors.
(9/2088)
We showed previously that a single intranasal vaccination of mice with a recombinant vesicular stomatitis virus (VSV) expressing an influenza virus hemagglutinin (HA) protein provided complete protection from lethal challenge with influenza virus (A. Roberts, E. Kretzschmar, A. S. Perkins, J. Forman, R. Price, L. Buonocore, Y. Kawaoka, and J. K. Rose, J. Virol. 72:4704-4711, 1998). Because some pathogenesis was associated with the vector itself, in the present study we generated new VSV vectors expressing HA which are completely attenuated for pathogenesis in the mouse model. The first vector has a truncation of the cytoplasmic domain of the VSV G protein and expresses influenza virus HA (CT1-HA). This nonpathogenic vector provides complete protection from lethal influenza virus challenge after intranasal administration. A second vector with VSV G deleted and expressing HA (DeltaG-HA) is also protective and nonpathogenic and has the advantage of not inducing neutralizing antibodies to the vector itself. (+info)
RNA-protein interactions in the human RNase MRP ribonucleoprotein complex.
(10/2088)
The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5' end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38. (+info)
Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94).
(11/2088)
Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (approximately 100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 M NaCl), although peptide binding was stronger (Ka approximately 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were farther apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 A from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading. (+info)
The NS5A protein of hepatitis C virus partially inhibits the antiviral activity of interferon.
(12/2088)
The non-structural protein 5A (NS5A) of some hepatitis C virus (HCV) isolates has been implicated in the inhibition of the antiviral activity of interferon (IFN). In the present study, the possible inhibitory effects of NS5A from two isolates of HCV subtype 1b, HCV-1bJk and M094AJk, and their chimeric form on the antiviral activity of IFN were examined. HCV-1bJk and M094AJk are categorized as IFN resistant and IFN sensitive, respectively, based on the sequences of the IFN-sensitivity determining region (ISDR). When encephalomyocarditis virus was used as a challenge virus, NS5A was shown to eliminate the antiviral activity of IFN, with inhibition being more prominent with HCV-1bJk NS5A than with M094AJk NS5A. Moreover, the inhibition was significantly weaker in cells expressing a chimeric NS5A that had a short stretch of 49 amino acids (aa 2209-2257), including the ISDR sequence, from M094AJk in the backbone of the HCV-1bJk sequence than in cells expressing the original NS5A from HCV-1bJk. These results suggest an important role for the 49 aa sequence, including the ISDR, in the inhibition of IFN-mediated antiviral activity. On the other hand, only a slight reduction of IFN antiviral activity by HCV-1bJk NS5A was observed when vesicular stomatitis virus was used as a challenge virus, and barely any reduction was observed when Japanese encephalitis virus was used. These results may reflect differential importance of each of the IFN-mediated signalling pathways in conferring resistance against different viruses. (+info)
A novel approach to visualize polyclonal virus-specific CD8 T cells in vivo.
(13/2088)
Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 x 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60-80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells. (+info)
Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus.
(14/2088)
The nonsegmented negative-strand RNA viruses (order Mononegavirales) include many important human pathogens. The order of their genes, which is highly conserved, is the major determinant of the relative levels of gene expression, since genes that are close to the single promoter site at the 3' end of the viral genome are transcribed at higher levels than those that occupy more distal positions. We manipulated an infectious cDNA clone of the prototypic vesicular stomatitis virus (VSV) to rearrange three of the five viral genes, using an approach which left the viral nucleotide sequence otherwise unaltered. The central three genes in the gene order, which encode the phosphoprotein P, the matrix protein M, and the glycoprotein G, were rearranged into all six possible orders. Viable viruses were recovered from each of the rearranged cDNAs. The recovered viruses were examined for their levels of gene expression, growth potential in cell culture, and virulence in mice. Gene rearrangement changed the expression levels of the encoded proteins in concordance with their distance from the 3' promoter. Some of the viruses with rearranged genomes replicated as well or slightly better than wild-type virus in cultured cells, while others showed decreased replication. All of the viruses were lethal for mice, although the time to symptoms and death following inoculation varied. These data show that despite the highly conserved gene order of the Mononegavirales, gene rearrangement is not lethal or necessarily even detrimental to the virus. These findings suggest that the conservation of the gene order observed among the Mononegavirales may result from immobilization of the ancestral gene order due to the lack of a mechanism for homologous recombination in this group of viruses. As a consequence, gene rearrangement should be irreversible and provide an approach for constructing viruses with novel phenotypes. (+info)
Identification of the murine Mx2 gene: interferon-induced expression of the Mx2 protein from the feral mouse gene confers resistance to vesicular stomatitis virus.
(15/2088)
The mouse genome contains two related interferon-regulated genes, Mx1 and Mx2. Whereas Mx1 codes for the nuclear 72-kDa protein that interferes with influenza virus replication after interferon treatment, the Mx2 gene is nonfunctional in all laboratory mouse strains examined, since its open reading frame (ORF) is interrupted by an insertional mutation and a subsequent frameshift mutation. In the present study, we demonstrate that Mx2 mRNA of cells from feral mouse strains NJL (Mus musculus musculus) and SPR (Mus spretus) differs from that of the laboratory mouse strains tested. The Mx2 mRNA of the feral strains contains a single long ORF consisting of 656 amino acids. We further show that Mx2 protein in the feral strains is expressed upon interferon treatment and localizes to the cytoplasm much like the rat Mx2 protein, which inhibits vesicular stomatitis virus replication. Furthermore, transfected 3T3 cell lines of laboratory mouse origin expressing Mx2 from feral strains acquire slight resistance to vesicular stomatitis virus. (+info)
The role of beta7 integrins in CD8 T cell trafficking during an antiviral immune response.
(16/2088)
The requirement of beta7 integrins for lymphocyte migration was examined during an ongoing immune response in vivo. Transgenic mice (OT-I) expressing an ovalbumin-specific major histocompatibility complex class I-restricted T cell receptor for antigen were rendered deficient in expression of all beta7 integrins or only the alphaEbeta7 integrin. To quantitate the relative use of beta7 integrins in migration in vivo, equal numbers of OT-I and OT-I-beta7(-/-) or OT-I-alphaE-/- lymph node (LN) cells were adoptively transferred to normal mice. Although OT-I-beta7(-/-) LN cells migrated to mesenteric LN and peripheral LN as well as wild-type cells, beta7 integrins were required for naive CD8 T cell and B cell migration to Peyer's patch. After infection with a recombinant virus (vesicular stomatitis virus) encoding ovalbumin, beta7 integrins became critical for migration of activated CD8 T cells to the mesenteric LN and Peyer's patch. Naive CD8 T cells did not enter the lamina propria or the intestinal epithelium, and the majority of migration of activated CD8 T cells to the small and large intestinal mucosa, including the epithelium, was beta7 integrin-mediated. The alphaEbeta7 integrin appeared to play no role in migration during a primary CD8 T cell immune response in vivo. Furthermore, despite dramatic upregulation of alphaEbeta7 by CD8 T cells after entry into the epithelium, long-term retention of intestinal intraepithelial lymphocytes was also alphaEbeta7 independent. (+info)