Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.     

Horizontal transmission of murine retroviruses.

Both a feral mouse ecotropic virus (WM-E) and Friend ecotropic virus (F-MuLV) were transmitted horizontally among adult mice. Infection resulted in the production of antiviral antibody in the recipients, with no evidence of viremia or clinical disease. However, persistent low-level virus replication was detectable in the spleens of these mice as long as 8 months after initial infection. External secretions, including saliva, semen, and uterine secretions from viremic mice contained high concentrations of infectious virus. Nevertheless, transmission occurred only from viremic males to either males or females. Male-to-male transmission appeared to occur by parenteral inoculation of infectious saliva during fighting behavior. Evidence is presented that infection of females was by the venereal route. Of four mouse strains examined, NFS/N, IRW, and C57L females were all susceptible to venereal infection, whereas AKR mice were not. Since AKR mice are susceptible to infection by WM-E administered parenterally, this resistance appeared to be mediated by local viral interference due to the high-level expression of endogenous Akv gp70 within the female reproductive tract. Although both WM-E and F-MuLV were transmitted from viremic males to females, infection by WM-E was significantly more efficient than that by F-MuLV. This difference correlated with a distinct difference in cellular tropism of WM-E and F-MuLV within the epididymis of viremic males. F-MuLV gp70 was expressed only within stromal elements, whereas WM-E gp70 was seen largely within the epithelial lining cells and luminal contents of the duct. No evidence of virus expression within germ cells was observed. The possible influence of virus expression by epithelial cells of the female reproductive tract on infection of embryos is discussed.     

Herpes simplex virus 1 protein kinase is encoded by open reading frame US3 which is not essential for virus growth in cell culture.

Earlier reports have described a novel protein kinase in cells infected with herpes simplex or pseudorabies viruses. These novel enzymes were characterized by their acceptance of protamine as a substrate and by their differential chromatographic behavior in anion-exchange chromatography. We report that this activity was not present in extracts of uninfected cells or of cells infected with a mutant constructed so as to contain a deletion in the US3 open reading frame mapping in the small component of herpes simplex virus 1 DNA. The activity was present in extracts of cells infected with wild-type virus and with a recombinant in which the US3 open reading frame had been rescued. Our results are consistent with the observation reported earlier that the coding sequences predict an amino acid motif common to protein kinases and lead to the conclusion that the US3 open reading frame encodes a virus-specific protein kinase that is not required for virus growth in cells in culture.     

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the expression of viral RNA and proteins, implying that replication was inhibited at some step between penetration and initiation of genomic functions, perhaps at the stage of uncoating. We therefore examined the possibility that protein kinases and phosphatases, which influence cellular regulation during cAMP-induced differentiation, may be responsible for the phenomenon of coronavirus suppression in oligodendrocytes. Evidence was obtained indicating that normal processing of the phosphorylated nucleocapsid protein is inhibited in differentiated oligodendrocytes, consistent with the notion that JHMV replication might be arrested during uncoating.     

Characterization of capsid and noncapsid proteins of B19 parvovirus propagated in human erythroid bone marrow cell cultures.

The major capsid and noncapsid proteins of the pathogenic parvovirus B19, propagated in vitro, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation, and immunoblot of the erythroid fraction of infected human bone marrow cell cultures. There were two capsid proteins of 58 kilodaltons (kDa; the major species) and 84 kDa (the minor species). Newly synthesized capsid viral proteins were present in the supernatants of infected cultures. The major noncapsid protein of 77 kDa was localized to the nucleus.     

Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.     

Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins.

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.