A Newly Identified MHV-A59 ORF1a Polypeptide p65 is Temperature Sensitive in Two RNA Negative Mutants (original) (raw)
Related papers
Altered virion proteins of a temperature-sensitive mutant of polyoma virus, ts59
Virology, 1977
The ts59 mutant of polyoma virus is blocked in a late step of infection at the restrictive temperature. Cellular and viral DNA synthesis proceed normally in ts59infected cells at the restrictive temperature, but infectious progeny virus particles are not assembled. The ts59 mutant complements early tsA mutants in mixed infection, and the temperature-sensitive mutation maps in the late region ofthe polyoma genome. The infectivity of ts59 virions is much more heat labile than wild-type polyoma. All three nonhistone capsid proteins of ts59, VP1 (45,000 daltons) and the overlapping proteins VP2 (30,000 daltons) and VP3 (20,000 daltons), show altered mobilities when analyzed by SDS-polyacrylamide gel electrophoresis. The tryptic peptide patterns of all three ts59 virion proteins also differ from the tryptic peptide patterns of wild-type proteins. Analysis of the t&9 proteins synthesized in vitro and in infected cells suggests that the alterations in the t&9 virion proteins are caused by differences in primary structure rather than by post-translational modifications. The capsid proteins of convertant virions produced by marker rescue of the ta59 temperature-sensitive mutation, using various restriction endonuclease fragments of wild-type DNA, have been analyzed. Results of these studies suggest that (i) 26 map units is the furthest point, in a clockwise direction on the genetic map, that the information for the C-terminus of VP1 can be from the Eco. RI cleavage site; (ii) the N-terminal end of VP2 extends beyond the N-terminal end of VP3; (iii) the temperature-sensitive phenotype of ts59 is correlated with a peptide alteration common to VP2 and VP3. The ts59 mutant contains two further peptide alterations not related to the temperature-sensitive phenotype: a C-terminal alteration in VP1 and an alteration unique to VI??. Cells infected by ts59 contain approximately fourfold lower amounts of viral capsid proteins and virus-specific messenger RNA at the restrictive temperature compared to the permissive temperature.
Transcription Program of Murine Gammaherpesvirus 68
Journal of Virology, 2003
Murine gammaherpesvirus 68 (MHV-68 [also referred to as ␥HV68]) is phylogenetically related to Kaposi's sarcoma-associated herpesvirus (KSHV [also referred to as HHV-8]) and Epstein-Barr virus (EBV). However, unlike KSHV or EBV, MHV-68 readily infects fibroblast and epithelial cell lines derived from several mammalian species, providing a system to study productive and latent infections as well as reactivation of gammaherpesviruses in vivo and in vitro. To carry out rapid genome-wide analysis of MHV-68 gene expression, we made DNA arrays containing nearly all of the known and predicted open reading frames (ORFs) of the virus. RNA obtained from an MHV-68 latently infected cell line, from cells lytically infected with MHV-68 in culture, and from the lung tissue of infected mice was used to probe the MHV-68 arrays. Using a tightly latent B-cell line (S11E), the MHV-68 latent transcription program was quantitatively described. Using BHK-21 cells and infected mice, we demonstrated that latent genes are transcribed during lytic replication and are relatively independent of de novo protein synthesis. We determined that the transcription profiles at the peak of lytic gene expression are similar in cultured fibroblast and in the lung of infected mice. Finally, the MHV-68 DNA arrays were used to examine the gene expression profile of a recombinant virus that overexpresses replication and transcription activator (RTA), C-RTA/MHV-68, during lytic replication in cell culture. The recombinant virus replicates faster then the parental strain and the DNA arrays revealed that nearly every MHV-68 ORF examined was activated by RTA overexpression. Examination of the gene expression patterns of C-RTA/ MHV-68 over a time course led to the finding that the M3 promoter is RTA responsive in the absence of other viral factors.
Virology, 1997
A strategy for targeted RNA recombination between the spike gene on the genomic RNA of MHV-A59 and a synthetic DI RNA containing the MHV-RI spike gene is described. The MHV-RI spike gene contains several nucleotide differences from the MHV-A59 spike gene that could be used as genetic markers, including a stretch of 156 additional nucleotides starting at nucleotide 1497. The MHV-RI S gene cDNA (from nucleotide 277-termination codon) was inserted in frame into pMIDI, a full-length cDNA clone of an MHV-A59 DI, yielding pDPRIS. Using the vaccinia vTF7.3 system, RNA was transcribed from pDPRIS upon transfection into MHV-A59-infected L cells. DPRIS RNA was shown to be replicated and passaged efficiently. MHV-A59 and the DPRIS DI particle were copassaged several times. Using a highly specific and sensitive RT-PCR, recombinant genomic RNA was detected in intracellular RNA from total lysates of pDPRIS-transfected and MHV-A59infected cells and among genomic RNA that was agarose gel-purified from these lysates. More significantly, specific PCR products were found in virion RNA from progeny virus. PCR products were absent in control mixes of intracellular RNA from MHV-A59-infected cells and in vitro-transcribed DPRIS RNA. PCR products from intracellular RNA and virion RNA were cloned and 11 independent clones were sequenced. Crossovers between A59 and RI RNA were found upstream of nucleotide 1497 and had occurred between 106 nucleotides from the 5-border and 73 nucleotides from the 3-border of sequence homologous between A59 and RI S genes. We conclude that homologous RNA recombination took place between the genomic RNA template and the synthetic DI RNA template at different locations, generating a series of MHV recombinant genomes with chimeric S genes.
Localization of mouse hepatitis virus open reading frame 1A derived proteins
Journal of Neurovirology, 1998
We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immuno¯uorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunouorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immuno¯uorescent labeling of BHK cells expressing the MHV receptor (BHK MHVR1 ) and infected with MHV-A59 with a Golgi-speci®c anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies speci®c for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the ®rst 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immuno¯uorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the ®rst papain-like protease domain (PLP-1) was suf®cient to associate this protein with the Golgi.
Journal of virology, 1996
The reovirus core is a multienzyme complex that contains five different structural proteins and 10 segments of double-stranded RNA. The core is responsible for transcribing mRNA from the enclosed double-stranded RNA. The reovirus transcriptase has an unusual temperature profile, with optimum transcription occurring at approximately 50 degrees C and little activity occurring below 30 or above 60 degrees C. Purified reovirus serotype 1 Lang (T1L) cores transcribed most efficiently at 48 degrees C. The transcriptase temperature optimum of purified reovirus serotype 3 Dearing (T3D) cores was 52 degrees C. In addition, T1L cores produced more mRNA per particle than did T3D cores at their respective temperature optima. Core particles were purified from T1L x T3D reassortants and were used to map these differences. The M1 gene, which encodes minor core protein mu 2, was uniquely associated with the difference in temperature optimum of transcription (P = 0.0003). The L1 gene, which encodes ...
Journal of General Virology, 1984
When chick embryo fibroblasts infected with high multiplicities of a non-revertible temperature-sensitive (ts) mutant (ts53) of Newcastle disease virus (NDV) at the permissive temperature (34 °C) and incubated to allow the appearance of virus-induced proteins were shifted up to and incubated at the non-permissive temperature (42 °C), analysis of [3H]leucine-labelled proteins by SDS-PAGE revealed a marked increase in cellular protein synthesis and a decline in viral synthesis, with an eventual return to an apparently uninfected state. Mutant ts53 apparently ceased to suppress cellular protein synthesis, and translation of viral proteins was markedly inhibited. This was not observed in ts+-infected cells, or in ts53-infected cells maintained at the permissive temperature. Recovery from the infected state was inhibited by actinomycin D at any stage, but was not prevented by cycloheximide present immediately before and during temperature shift-up. Mutant ts53 was shown to be RNA-at 42 °C, although viral mRNAs synthesized at 34 °C are shown to be present throughout host recovery but in an inactive state.