- Volume 80, Issue 10, 1999

A gene of rat cytomegalovirus (RCMV), designated R32, has been identified that encodes a homologue of the human cytomegalovirus (HCMV) pp150 (ppUL32) major tegument phosphoprotein. The R32 ORF has the capacity to encode a 667 amino acid polypeptide (pR32) with a calculated molecular mass of 73 kDa. The predicted amino acid sequence of pR32 shows similarity to that of polypeptides predicted to be encoded by the HCMV UL32, murine cytomegalovirus M32 and human herpesvirus types 6 and 7 U11 genes. The R32 gene is transcribed as a 2·5 kb mRNA during the late phase of RCMV infection in rat embryo fibroblasts in vitro. To study expression of the pR32 protein in vitro and in vivo, a rabbit polyclonal antiserum was raised against a recombinant protein that comprised amino acids 252–522 of pR32. By using this antiserum, pR32 could be detected predominantly in the cytoplasm of RCMV-infected fibroblasts at 24 and 48 h post-infection in vitro. The pR32 protein was also detected within virions isolated from the culture medium of RCMV- infected cells. Expression of pR32 in vivo was observed within the cytoplasm of salivary gland epithelial cells of RCMV-infected rats. In addition, recombinant pR32 was found to react with sera from rats that were previously infected with RCMV, whereas reactivity was not seen with sera from mock-infected rats. Together, these findings indicate that RCMV pR32 represents the homologue of HCMV ppUL32, both in primary structure and in function.

Primary infection with murine gammaherpesvirus-68 (MHV-68), as with other members of the gammaherpesvirus subfamily, is characterized by a lymphoproliferative phase. MHV-68 causes acute splenomegaly and an infectious mononucleosis-like syndrome in which there is expansion of the CD8+ T cell subset. In long-term infections, MHV-68 is associated with lymphoma development. In order to elucidate the mechanisms underlying the proliferative processes, the events following infection of murine splenocytes or purified murine B lymphocytes in vitro have been examined. MHV-68 infection prolonged the viability of murine splenocytes and stimulated cellular proliferation. Unlike Epstein–Barr virus and herpesvirus saimiri, MHV-68 did not cause growth transformation. Growth transformation did not occur even when cells with a predisposition to transformation were infected or when culture conditions were selected to enhance the viability of the cells. Following MHV-68 infection, the latency-associated viral tRNAs were transcribed. However, transcription of the other known latency- associated gene, M2, was not observed. In addition, there was no evidence of productive virus replication either by staining with antibodies specific for late virus antigens or by in situ hybridization for early and late mRNAs. In contrast to Epstein–Barr virus- and herpesvirus saimiri-infected lymphocytes, where episomal genomes are seen, Gardella gel analysis indicated that the primary lymphocytes infected by MHV-68 in vitro contained only linear virus DNA. This DNA was nuclease sensitive, indicating that, while MHV-68 was efficiently uncoated, its circularization in vitro was extremely inefficient. These results are discussed in terms of the host–virus interaction.

The murine gammaherpesvirus-68 (MHV-68) M11 gene encodes a protein predicted to have limited homology to the bcl-2 family of proteins. Unlike most of the other viral bcl-2 homologues, which have both BH1 and BH2 domains conserved with respect to bcl-2, the M11 protein has a BH1 domain, but apparently lacks a BH2 domain. Transfection of HeLa cells with an epitope-tagged MHV-68 M11 construct showed that the protein is predominantly located in the cytoplasm of cells. In HeLa cells, M11 inhibited apoptosis induced by anti-Fas antibody and by TNF-α. Thus, despite its limited conservation with respect to other bcl-2 family members, the MHV-68 M11 protein is a potent inhibitor of apoptosis.

EBNA-1 is the only viral protein consistently expressed in all cells latently infected by Epstein–Barr virus (EBV). There is a high frequency of sequence variation within functionally important domains of EBNA-1, with five subtypes identified. Individuals may be infected with multiple EBV strains (classified according to EBNA-1 subtype), but Burkitt’s lymphoma (BL) tumours carry a single subtype and exhibit some subtype preference. Subtype variation has also been related to geographical location. In the present study EBNA-1 polymorphisms were examined in a series of haematological malignancies from two distinct geographical regions, Brazil and the United Kingdom. Nucleotide sequence analysis of the carboxy-terminal region of EBNA-1 in 34 cases revealed six distinct sequences, some of which are novel. A new subtype, named V-Ala, was identified. EBNA-1 subtype in tumours differed markedly according to geographical location. In contrast to previous studies, we found evidence of EBNA-1 sequence variation within individual BL tumour samples.

BALF2, which encodes the major DNA-binding protein of Epstein–Barr virus (EBV), is expressed during the early stage of the lytic cycle. The location of the BALF2 promoter was identified by primer extension, which indicated that the transcription start is located at nucleotide 164,782 of the EBV genome. Transfection analyses revealed that, similar to other EBV early promoters, the BALF2 promoter is activated by the EBV-encoded transcription factors Rta and Zta. The promoter is also synergistically activated if both transcription factors are present in B lymphocytes and in epithelial cells. Deletion analysis and electrophoretic mobility-shift assay revealed that the region between nucleotides −134 and −64 contains Zta- response elements and the region between nucleotides −287 and −254 contains Rta-response elements. This study demonstrates the importance of Rta and Zta in regulating the transcription of EBV early genes.

Vaccinia virus (VV) proteins that interfere with the host response to infection are of interest because they provide insight into virus–host relationships and may affect the safety and immunogenicity of recombinant VV (rVV) vaccines. Such vaccines need assessment in animal models and with this aim a model of VV infection based on intradermal injection of BALB/c ear pinnae was developed and characterized. In this model, the outcome of infection is affected by the dose of virus inoculated but virus spread is minimal and the mice suffer no signs of systemic illness. Cellular and humoral immune responses to these infections were measured readily and were independent of virus dose over a 100-fold range. Thus the model seems suitable for the analysis of the safety and immunogenicity of VV mutants lacking specific immunomodulatory proteins or bearing foreign antigens.

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5′-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genusPomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5′ and 3′ termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3′-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3′-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3′-UTR of RNA 3, which confirms its presence in the 3′-UTRs of all three RNAs of PMTV.

Capsicum annuum cv. Avelar plants resist systemic infection by the Florida isolate of pepper mottle potyvirus (PepMoV-FL). Immuno-tissue blot analysis for detection of PepMoV-FL infection in selected stem segments revealed that virus moved down the stem in external phloem, and, over time, accumulated to detectable levels throughout stem sections (appearing to accumulate in external and internal phloem) taken from below the inoculated leaf. At 21 days post-inoculation, PepMoV-FL was detected in stem segments one or two internodes above the inoculated leaf; however, no virus was observed in internal phloem in stem segments beyond these internodes. In contrast to these observations, PepMoV-FL was detected in the internal phloem of all internodes of the stem located above the inoculated leaf, with subsequent movement into non-inoculated leaves, in Avelar plants co-infected with PepMoV-FL and cucumber mosaic cucumovirus (CMV-KM). No apparent enhancement of PepMoV-FL accumulation occurred in protoplasts inoculated with PepMoV-FL alone versus a mixed inoculum of PepMoV-FL and CMV-KM. These findings confirm earlier observations that potyvirus movement up the stem of Capsicum species occurs via internal phloem. It is also shown that PepMoV-FL does not accumulate to detectable levels in internal phloem in the stem of Avelar plants, thereby limiting its movement to within the inoculated leaf and lower portions of the stem; however, co-infection of Avelar plants with CMV-KM alleviates this restricted movement, allowing PepMoV-FL to invade young tissues systemically.

The PrP gene encodes the putative causative agent of the transmissible spongiform encephalopathies (TSEs), a heterogeneous group of fatal, neurodegenerative disorders including human Creutzfeldt–Jakob disease, bovine spongiform encephalopathy, ovine scrapie and chronic wasting disease (CWD) of North American deer and elk. Polymorphisms in the PrP gene are associated with variations in relative susceptibility, pathological lesion patterns, incubation times and clinical course of TSEs of humans, mice and sheep. Sequence analysis of the PrP gene from Rocky Mountain elk showed only one amino acid change (Met to Leu at cervid codon 132). Homozygosity for Met at the corresponding polymorphic site (Met to Val) in humans (human codon 129) predisposes exposed individuals to some forms of Creutzfeldt–Jakob disease. In this study, Rocky Mountain elk homozygous for PrP codon 132 Met were over-represented in both free- ranging and farm-raised CWD-affected elk when compared to unaffected control groups.