- Volume 85, Issue 7, 2004

The deregulation of cellular adhesion molecules by human cytomegalovirus (HCMV) appears to be correlated with the development of vascular disease. In this study, it was investigated whether the expression of Thy-1 (CD90), a member of the immunoglobulin superfamily of adhesion molecules with constitutive expression on fibroblast cells, is modulated following infection with HCMV. It was observed that Thy-1 cell surface expression decreased significantly during the course of infection. Addition of neutralizing antibodies, as well as UV inactivation of virus, prevented Thy-1 downregulation. In contrast, inhibition of virus replication by cidofovir did not alter Thy-1 regulation by HCMV, indicating that immediate-early (IE) and/or early (E) gene products are responsible. Interestingly, after infection of fibroblasts with a recombinant GFP-expressing virus, infected as well as non-infected cells showed a reduced Thy-1 cell surface expression. From these findings, it is concluded that IE or E gene products of HCMV induce a so far unidentified soluble factor that mediates Thy-1 downregulation.

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

Here is described the isolation of a naturally occurring A-type inclusion body (ATI)-negative vaccinia-like virus, Belo Horizonte virus (VBH), obtained from a mousepox-like outbreak in Brazil. The isolated virus was identified and characterized as an orthopoxvirus by conventional methods. Molecular characterization of the virus was done by DNA cross-hybridization using Vaccinia virus (VACV) DNA. In addition, conserved orthopoxvirus genes such as vaccinia growth factor, thymidine kinase and haemagglutinin were amplified by PCR and sequenced. All sequences presented high similarity to VACV genes. Based on the sequences, phenograms were constructed for comparison with other poxviruses; VBH clustered consistently with VACV strains. Attempts to amplify the ATI gene (ati) by PCR, currently used to identify orthopoxviruses, were unsuccessful. Results presented here suggest that most of the ati gene is deleted in the VBH genome.

Full-length sequences of the Epstein–Barr virus (EBV) gene for latent membrane protein (LMP)-1 from 22 nasopharyngeal carcinoma (NPC) biopsy specimens and 18 non-neoplastic counterparts (NPI) were determined. Relative to the B95-8 strain, the amino acid sequences of the toxic-signal and transformation domains were changed variably in NPC and NPI specimens; in contrast, no change was observed in the NF-κB (nuclear factor κB) activation domain. HLA typing revealed that 47 % of NPC and 31 % of NPI specimens were HLA A2-positive. A major A2-restricted epitope within LMP-1 (residues 125–133) was analysed. At residue 126, a change of L→F was detected in 91 % (20/22) of NPC and 67 % (12/18) of NPI specimens. In addition, a deletion at residue 126 was detected in one NPC sample from Taiwan. At residue 129, a change of M→I was observed in all samples, regardless of whether they were NPC or NPI. The changes in this peptide between NPC and NPI specimens, including mutation and deletion, are statistically significant (P<0·05). A recent report indicated that this variant sequence is recognized poorly by epitope-specific T cells. Genotyping results indicated that 96 % of NPC and 67 % of NPI samples carried a type A virus. By scanning the entire sequence of LMP-1, eight distinct patterns were identified. Detailed examination of these patterns revealed that type A strains are more prevalent in NPC than in NPI specimens and are marked by the loss of an XhoI site, the presence of a 30 bp deletion and the presence of a mutated, A2-restricted, T cell target epitope sequence. These results suggest that an EBV strain carrying an HLA A2-restricted ‘epitope-loss variant’ of LMP-1 is prevalent in NPC in southern China and Taiwan.

Thymidylate synthase (TS) (EC 2.1.1.45) is essential for the de novo synthesis of dTMP in prokaryotic and eukaryotic organisms. Within the white spot syndrome virus (WSSV) genome, an open reading frame (WSV067) that encodes a 289 amino acid polypeptide showed significant homology to all known TSs from species including mammals, plants, fungi, protozoa, bacteria and DNA viruses. In this study, WSV067 was expressed in Escherichia coli, and the purified recombinant protein showed TS activity in dUMP−folate-binding assays using ultraviolet difference spectroscopy. RT-PCR and Western blot analyses showed that WSV067 was a genuine and early gene. Phylogenetic analysis revealed that WSSV-TS was more closely related to the TSs of eukaryotes than to those from prokaryotes.

The middle-sized (M) surface proteins of hepatitis B virus (HBV) and other orthohepadnaviruses contain a conserved N-glycan in their pre-S2 domain, which is essential for the secretion of viral particles. Recently, we also found O-glycans in the pre-S2 domain of M protein from woodchuck hepatitis virus (WHV) and HBV genotype D. Since the O-glycosylation motif is not conserved in all genotypes of HBV, the glycosylation patterns of HBV genotypes A and C were analysed. Pre-S2 (glyco)peptides were released from HBV-carrier-derived HBV subviral particles by tryptic digestion, purified by reversed-phase HPLC and identified by amino acid and amino-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion-exchange chromatography, methylation analysis and on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides in all genotypes examined. Pre-S2 O-glycans were characterized by on-target sequential exoglycosidase digestions in combination with MALDI-TOF-MS. The pre-S2 domain of M protein and, to a minor extent, of L (large) protein from HBV genotype C and D was partially O-glycosylated by Neu5Ac(α2–3)Gal(β1–3)GalNAcα- or Gal(β1–3)GalNAcα-units at Thr-37 within a conserved sequence context. Genotype A, containing no Thr at position 37 or 38, was not O-glycosylated. Analytical data further revealed that M protein is mostly amino-terminally acetylated in all examined genotypes and that the terminal methionine is partially oxidized. The findings may be relevant for the secretion and the immunogenicity of HBV.

The role that T cell subsets play in herpetic stromal keratitis (HSK) has been the subject of intense research efforts. While most studies implicate CD4+ T cells as the principal cell type mediating primary corneal disease, recent reports using knockout mice have suggested that both CD4+ and CD8+ T cell subsets may play integral roles in modulating the disease. Furthermore, recent studies suggest that CD8+ T cells are directly involved in maintaining virus latency in infected trigeminal ganglia. This work has addressed these discrepancies by infecting the corneas of mice lacking CD4+ and CD8+ T cells with herpes simplex virus type 1 (HSV-1) and monitoring both corneal disease and latent infection of trigeminal ganglia. Results indicated that mice lacking CD8+ T cells had more severe corneal disease than either BALB/c or B6 parental strains. In contrast, mice lacking CD4+ T cells had a milder disease than parental strains. When mice were evaluated for persistence of infectious virus, only transient differences were observed in periocular tissue and corneas. No significant differences were found in persistence of virus in trigeminal ganglia or virus reactivation from explanted ganglia. These data support the following conclusions. CD4+ T cells are not required for resistance to infection with HSV-1 and probably mediate HSK. Mice lacking CD8+ T cells do not display differences in viral loads or reactivation and thus CD8+ T cells are not absolutely required to maintain latency. Finally, CD8+ T cells probably play a protective role by regulating the immunopathological response that mediates HSK.

In Capsicum, the resistance conferred by the L 2 gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L 4 gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L 2 gene confers resistance to Paprika mild mottle virus (PaMMV), while the L 4 gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L 2 L 2) and Capsicum chacoense (L 4 L 4) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L 2 L 2) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L 2 resistance gene. In C. chacoense (L 4 L 4) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L 4 gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L 2 L 2) leaves and PMMoV CP into C. chacoense (L 4 L 4) leaves by biolistic co-bombardment with a β-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L 2 and L 4 gene-mediated hypersensitive response.

Turnip mosaic virus (TuMV, genus Potyvirus, family Potyviridae) infects mainly cruciferous plants. Isolates Tu-3 and Tu-2R1 of TuMV exhibit different infection phenotypes in cabbage (Brassica oleracea L.) and Japanese radish (Raphanus sativus L.). Infectious full-length cDNA clones, pTuC and pTuR1, were constructed from isolates Tu-3 and Tu-2R1, respectively. Progeny virus derived from infections with pTuC induced systemic chlorotic and ringspot symptoms in infected cabbage, but no systemic infection in radish. Virus derived from plants infected with pTuR1 induced a mild chlorotic mottle in cabbage and infected radish systemically to induce mosaic symptoms. By exchanging genome fragments between the two virus isolates, the P3-coding region was shown to be responsible for systemic infection by TuMV and the symptoms it induces in cabbage and radish. Moreover, exchanges of smaller parts of the P3 region resulted in recombinants that induced complex infection phenotypes, especially the combination of pTuC-derived N-terminal sequence and pTuR1-derived C-terminal sequence. Analysis by tissue immunoblotting of the inoculated leaves showed that the distributions of P3-chimeric viruses differed from those of the parents, and that the origin of the P3 components affected not only virus accumulation, but also long-distance movement. These results suggest that the P3 protein is an important factor in the infection cycle of TuMV and in determining the host range of this and perhaps other potyviruses.

This study reports on the complete genome sequence of Grapevine leafroll-associated virus 3, the type member of the genus Ampelovirus. The genome is 17 919 nt in size and contains 13 open reading frames (ORFs). Previously, the sequence of 13 154 nt of the 3′-terminal of the genome was reported. The newly sequenced portion contains a 158 nt 5′ UTR, a single papain-like protease and a methyltransferase-like (MT) domain. ORF1a encodes a large polypeptide with a molecular mass of 245 kDa. With a predicted +1 frameshift, the large fusion protein generated from ORF1a/1b would produce a 306 kDa polypeptide. Phylogenetic analysis using MT domains further supports the creation of the genus Ampelovirus for mealy-bug-transmitted viruses in the family Closteroviridae.