- Volume 84, Issue 5, 2003

Glycoprotein D (gD) of herpes simplex virus type 1 is a type 1 membrane protein in the virus envelope that binds to receptor molecules on the cell surface and which induces cell–cell fusion when co-expressed with gB, gH and gL. A chimeric gD molecule in which the membrane anchor and cytoplasmic tail domains were replaced with analogous regions from the human CD8 molecule was as competent as wild-type gD at mediating membrane fusion and virus entry. However, when gD was tethered to the membrane by means of a glycosylphosphatidylinositol (gpi)-anchor sequence, which binds only to the outer leaflet of the lipid bilayer, it was unable to function in cell–cell fusion assays. This chimera was incorporated into virions as efficiently as wild-type gD and yet virus particles containing gpi-linked gD entered cells more slowly than virions containing wild-type gD in their envelopes, suggesting that gD must be anchored in both leaflets of a lipid bilayer for it to function in both cell fusion and virus entry.

Simian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species Macaca. Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gCSHBV) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46·9 %), HSV-1 (44·5 %) and pseudorabies virus (21·2 %). Highly conserved cysteine residues were also noted. Similar to gCHSV-2, gCSHBV is less glycosylated than gCHSV-1, resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gCSHBV on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gCSHBV isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gCHSV-2 and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gCSHBV and gCHSV-2. Binding of recombinant gCSHBV to polysulphates was observed. This, together with the heparin-sensitivity of the gCSHBV–C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.

Orf virus (ORFV) belongs to the genus Parapoxvirus and induces cutaneous pustular lesions in sheep, goats and humans. ORFV is unusual in that it has the ability to reinfect its host and this suggests that the generation of immunological memory has been impaired, thus exposing the host to subsequent infection. The discovery that ORFV encodes an IL-10-like virokine raises the question of whether this factor adversely affects the cells that initiate the acquired immune response. We examined the effect of ORFV-IL-10 on immature murine bone marrow-derived dendritic cells (BMDC). Immature BMDC are activated on exposure to antigen and undergo maturation. This process is characterized by increased expression of CD80, CD86 and MHC class II and reduced antigen uptake. We found that the maturation of BMDC is impaired in cells treated with ORFV-IL-10 prior to antigen exposure and this was exemplified by the reduced expression of the cell-surface markers described above. We have also shown that the activation of a haemagglutinin peptide (HAT)-specific T cell hybridoma by dendritic cell-mediated presentation of HAT and heat-inactivated influenza virus AP8/34 was markedly reduced following exposure to ORFV-IL-10. Finally, we examined the effect of ORFV-IL-10 on Langerhans' cell (LC) migration using cultured murine skin explant tissue and showed that this virokine impaired the spontaneous migration of LC from the epidermis and induced changes in LC morphology. Our findings suggest that ORFV-IL-10 has the capacity to impair the initiation of an acquired immune response and hence inhibit the generation of immunological memory necessary for immunity on subsequent exposure.

The present study provides for the first time an extended investigation of individual genes located at the near-terminal right end of the genome of parapoxvirus bovis 1, Bovine papular stomatitis virus (BPSV) strain B177 and Orf virus (ORFV). Comparison of the respective DNA sequences of ORFV strain D1701 (9·9 kbp) and BPSV B177 (7·7 kbp) revealed a very similar organization of closely related genes transcribed in a rightward orientation. The most salient findings of this study were: (i) the absence of the ORFV-specific vascular endothelial growth factor (VEGF-E) gene in the BPSV isolate; (ii) the presence of an interleukin-10 (IL-10) orthologue; and (iii) the detection of three new genes encoding ankyrin-repeat-containing polypeptides. These results not only contribute to potential improvements of future molecular differentiation between the parapoxvirus species, but also shed new light on different pathobiologies among parapoxviruses.

Two novel spliced genes (UL131A and UL128) flanking UL130 were predicted from sequence comparisons between human cytomegalovirus (HCMV) and its closest known relative, chimpanzee cytomegalovirus (CCMV), and the splicing patterns were confirmed by mRNA mapping experiments. Both genes were transcribed with late kinetics and shared a polyadenylation site. Comparisons with wild-type HCMV in infected human tissues showed that three of five isolates passaged in cell culture contained disruptions of UL128, one was frameshifted in UL131A and one exhibited a deletion affecting UL131A and UL130. CCMV and the Colburn strain of simian cytomegalovirus, which have been passaged in cell culture, also exhibit disruptions of UL128. These observations indicate that expression of either one of UL128 and UL131A is deleterious to growth of primate cytomegaloviruses in cell culture. Although the functions of these genes are unknown, sequence comparisons suggest that UL128 encodes a β-chemokine.

ORF50, an immediate-early gene of human herpesvirus-8 (HHV-8), encodes a transactivating protein necessary for virus reactivation and lytic replication. ORF50 was reported recently to synergize with human immunodeficiency virus type 1 (HIV-1) tat at a post-transcriptional level. To study the effects of these molecular interactions on HIV replication and biology, cellular clones stably transformed with ORF50 were obtained by transfection of cell lines of different origin. These clones were infected subsequently with HIV. Experiments showed that ORF50 enhances HIV replication in T and B cells (Jurkat and BC-3 cells) and induces susceptibility and transient permissiveness in non-susceptible glial (A172) cells. Upregulation of viral receptors and co-receptors did not account for increased sensitivity to HIV infection and therefore the action of ORF50 might be modulated by the intracellular environment. Interestingly, non-susceptible cells transformed with ORF50 showed transient production of HIV particles that could spread to adjacent cells by direct contact. These findings show that HHV-8 ORF50 has an enhancing effect on HIV replication in vitro and suggest that the two viruses might interact in co-infected patients.

Chronic active Epstein–Barr virus infection (CAEBV) has been considered to be a non-neoplastic T-cell lymphoproliferative disease associated with Epstein–Barr virus (EBV) infection. In EBV-associated diseases, the cell phenotype-dependent differences in EBV latent gene expression may reflect the strategy of the virus in relation to latent infection. We previously reported that EBV latent gene expression was restricted; EBV nuclear antigen 1 (EBNA1) transcripts were consistently detected in all spleen samples from five CAEBV patients, but EBNA2 transcripts were detected in only one sample. EBV latent gene expression is controlled by distinct usage of three EBNA promoters (Cp, Wp and Qp). In this study, we examined the EBNA promoter usage by RT-PCR and the methylation status in the Cp and Wp regions using bisulfite PCR analysis in spleen samples from CAEBV patients. EBNA1 transcripts were unexpectedly initiated not from Qp but from Cp in all samples in spite of the restricted form of latency. Furthermore, while Cp was active, Cp was heavily methylated, indicating that CAEBV has unique EBV latent gene expression, EBNA promoter usage and EBNA promoter methylation status, in part due to unique splicing of Cp-initiated transcripts and an activation mechanism in hypermethylated Cp.

Polydnaviruses are unique symbiotic viruses that are replicated in the calyx cells of the ovary of some parasitic wasps. They have a segmented genome of circular double-stranded DNA and are injected along with the wasp's egg into the host, where they are essential for successful parasitism. Polydnaviruses replicate from integrated proviral DNA, and after excision of viral segments, flanking DNA is rejoined. Little is known about ovarian morphogenesis, the mode of amplification of the viral DNA and the involvement of ecdysteroids. Here we have analysed these parameters in the course of pupal–adult development in the braconid wasp Chelonus inanitus. Immediately after pupation, ovarian cells proliferated and calyx cells began to differentiate; at this stage ecdysteroids, in particular 20-hydroxyecdysone, were highest. Thereafter, calyx cells began to increase in size and DNA content and eventually became gigantic. Amplification of non-viral DNA (actin) and viral DNA in its integrated and excised form and of corresponding rejoined flanking regions was measured by quantitative real-time PCR. In the early phase of calyx cell differentiation, copy numbers of actin and integrated viral DNA increased to a similar extent. This, along with the increase in nuclear volume and DNA content in the absence of extensive cell proliferation, suggested polyploidization of the early stage calyx cells. In the following phase, integrated viral DNA was selectively and intensively amplified and eventually excised and circularized. As copy numbers of excised circular viral DNA and rejoined flanking DNA reached similarly high levels, excised viral DNA appeared not to replicate. After adult eclosion, amplification of viral DNA declined.

Polydnaviruses are unique symbiotic viruses that are formed only in calyx cells in the ovary of parasitic wasps in the families Braconidae and Ichneumonidae; accordingly, two genera, Bracovirus and Ichnovirus are recognized. We have presented a detailed ultrastructural analysis of ovary and calyx cell differentiation and virion morphogenesis, together with the first data on virion release in a bracovirus. Differentiation of the ovary into germarium/vitellarium and the calyx region begins immediately after pupation. In the periphery and central part of the calyx, some cells and their nuclei begin to enlarge and the DNA content increases. The calyx cell nuclei then further increase and become highly lobulated, nuclear pores become very abundant and the cytoplasm is rich in ribosomes. This suggests synthesis and import of viral envelope proteins as viral envelopes appear in the nuclei shortly later. The appearance of viral envelopes is accompanied by a swelling of the nucleus and a change in electron density. Thereafter, the calyx cells reach the final stage with a highly swollen nucleus containing virogenic stroma and mature virions with nucleocapsids. Up to this stage, the DNA content of nuclei increases 120-fold and the volume 45-fold. The mature calyx cells are positioned in the vicinity of the oviduct lumen; for release of virions first the nuclear and then the plasma membrane disintegrate. On the border of the oviduct lumen, cells of an epithelial layer become phagocytic and remove debris, leading to a calyx fluid that contains only densely packed virions.

Multipartite nucleic acid-containing virus-like particles, known as polydnaviruses, are special structures produced by female parasitoid wasps to deliver wasp components into the body of their host at oviposition. The particles confer protection for the developing parasitoid by passive and active means. Although several genes expressed from the circular DNA of these particles have been identified from various host–parasitoid systems, there is not much known about the structural proteins of these particles. Here we report on two genes encoding Cotesia rubecula particle proteins with similarities to molecular chaperones, calreticulin and heat-shock protein 70.

Five fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a recombinant baculovirus that is capable of expressing a β-galactosidase reporter gene from the CAG promoter (consisting of a cytomegalovirus enhancer element, a chicken actin promoter and rabbit β-globin termination sequences). TO (Tilapia ovary), EPC (carp), CHH-1 (Chum salmon heart fibroblast) and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ β-galactosidase assay, whereas RTG-2 (rainbow trout gonad) cells were not. The EPC cell line was used for more detailed studies on baculovirus transduction. The transduction frequency was found to be higher at 28 °C than at 21 °C. Addition of the histone deacetylase inhibitor sodium butyrate increased the number of blue cells detected 5- to 7-fold. The m.o.i. was positively correlated with transduction frequency, although the relationship did not appear to be strictly linear, as has been observed with mammalian cells. The temperature at which baculoviruses were adsorbed to EPC cells did not affect levels of β-galactosidase expression. We also examined expression levels of β-galactosidase in EPC cells after infection with a baculovirus construct that overexpresses the vesicular stomatitis virus G protein and displays it on the virion surface. Expression levels with this virus were approximately 15-fold higher than were observed with Ac-CAlacZ.

Previous studies have shown that the patterns of disease-specific prion protein (PrPd) accumulation in the brain (the ‘PrPd profile’) of scrapie-affected sheep are mainly influenced by the source of scrapie agent. We have now extended those studies to investigate the effect of different PrP antibodies on the PrPd profile of scrapie- and bovine spongiform encephalopathy (BSE)-affected sheep. Immunohistochemical examination of brains of 20 sheep was performed with four different PrP antibodies (P4, 521.7, 505.2 and R486), and the animals were allocated to four groups of five sheep each depending on the transmissible spongiform encephalopathy (TSE) agent source (two natural scrapie sources, SSBP/1 and BSE). Although the PrPd profiles depended on the antibody used, the four TSE sources could always be differentiated. Natural Suffolk scrapie showed the highest levels of glia-associated PrPd, natural Welsh Mountain scrapie uniquely had consistent vascular PrPd plaques, SSBP/1 produced the highest intracellular accumulations of PrPd and BSE led to moderate accumulation of all PrPd patterns except for vascular plaques. The variations in PrPd profile between TSE sources appeared to be the result of variations in cell tropism and in PrP processing. These processing differences are possibly associated with changes in PrPd conformation, and are manifest as differences in intracellular truncation and in release to the extracellular space of the abnormal protein. Moreover, variations in PrPd conformation would appear to be also influenced by the cell type supporting infection, arguing that it is modulated by the interaction between the infectious agent and the host.