- Volume 84, Issue 2, 2003

There is good evidence that cytotoxic T lymphocytes play an important role in the clearance of equine herpesvirus-1 (EHV1) in horses. We have demonstrated that, in common with other alphaherpesviruses, EHV1 infection can lead to dramatic down-regulation of MHC class I expression at the cell surface, a common strategy for pathogen evasion of the host immune response. This down-regulation is specific for MHC class I and does not reflect a general shut-off of host-cell protein synthesis. The use of monoclonal antibodies that recognize different MHC class I epitopes has demonstrated that the effect may be allele- or locus-specific. Use of the viral DNA synthesis inhibitor phosphonoacetic acid, which prevents late viral gene expression, showed that the effect is mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible. The data indicate that EHV1 infection results in enhanced endocytosis of MHC class I from the cell surface; the only other herpesvirus reported to use this mechanism is human herpesvirus-8. Elucidation of the precise mechanisms used by EHV1 in this process and identification of the genes responsible may lead to improved vaccination strategies.

The genome of bovine herpesvirus type 1 Schönböken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1ΔgE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1ΔgE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1ΔgE. From the mutated BAC, double negative BHV-1ΔgE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1ΔgG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1ΔgE, BHV-1ΔgE-gG and BHV-1ΔgG, however, were reduced by 19 to 55 % compared to parental strain Schönböken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.

The non-classical major histocompatibility complex class I molecule HLA-G is expressed mainly by extravillous trophoblasts at the materno–foetal interface. HLA-G has been found to bind endogenously processed nonameric peptides but its function as a restriction element for a cytotoxic T cell response to viruses with tropism for trophoblastic cells has never been demonstrated. In this study, candidate viral peptides derived from human cytomegalovirus (HCMV) pp65 (UL83), which stabilized the HLA-G molecule on HLA-G-transfected T2 cells, were identified. The specific anti-pp65 cytotoxic T lymphocyte (CTL) response restricted by HLA-G in triple transgenic mice (HLA-G, human β2m, human CD8α) was then investigated by injection of dendritic cells loaded with synthetic pp65-derived peptides or by infection with canarypox virus expressing pp65. Results showed that CTLs from HLA-G mice have the capacity to kill target cells either infected with recombinant vaccinia viruses expressing pp65 or loaded with specific pp65-derived peptides using HLA-G as an antigen-presenting molecule. It was also demonstrated that these HLA-G-restricted pp65-specific T cells are able to kill the human astrocytoma cell line U373, which was transfected with HLA-G and infected with HCMV. Moreover, using HLA-G tetramers refolded with a synthetic pp65-derived peptide, peptide-specific CD8+ cells restricted by HLA-G have been detected in vivo. These findings provide the first evidence that HLA-G can select anti-HCMV-restricted CTLs in vivo, although the potency of this cytolytic response is limited (20–25 %). The weak HLA-G-restricted anti-HCMV response is probably due to HLA-G-mediated inhibitory signals on the development of an antiviral CTL response.

Epstein–Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation. To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells. Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells. (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1. So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain. Potential roles of hERR1 in EBV-induced transformation are discussed.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8-encoded viral interferon regulatory factor (vIRF) transforms NIH3T3 cells, represses interferon signal transduction and regulates the expression of other KSHV genes. Here, we have shown that vIRF is a transcriptional activator and auto-activates its own expression. Ectopic expression of vIRF activated the vIRF promoter in KSHV-negative 293, COS7, HeLa and BJAB cell lines in a dose-dependent fashion in a reporter assay and the expression of vIRF transcripts from endogenous viral genomes in BCBL-1 and BC-1 cells latently infected with KSHV. Deletion analysis identified two cis elements, named Vac1 and Vac2, in the vIRF promoter that were responsive to vIRF activation. vIRF auto-activation via Vac1 but not Vac2 was repressed by Tis, a transcriptional silencer in the vIRF promoter. Neither Vac1 nor Vac2 contain any interferon-stimulated response element (ISRE)-like sequences and are unresponsive to induction with interferon-β and -γ. These results indicate that KSHV uses the mechanism of auto-activation to regulate the expression of a viral transforming protein to efficiently evade host tumour suppressor pathways.

The murine gammaherpesvirus-68 kills I-Ab−/− mice despite the presence of virus-specific CD8+ cytotoxic T lymphocytes (CTL). This has raised the possibility that these CTL are functionally abnormal. Here, no difference was observed between I-Ab−/− mice and I-Ab+/+ controls in virus-specific CTL function, T cell receptor usage, or surface phenotype. Thus CTL immunity was independent of CD4+ T cells in a chronic herpesvirus infection, but was still inadequate to control virus replication.

Nucleopolyhedroviruses (NPVs) (Baculoviridae) produce fatal infections in larval lepidopteran insects. NPVs are designated SNPVs or MNPVs based on whether the occlusion-derived virus (ODV) that initiates primary midgut infections contains single (S) or multiple (M) nucleocapsids. The principal consequence of this ODV packaging is that primary target cells infected with the M phenotype receive multiple nucleocapsids, whereas those infected by the S phenotype receive only one. To determine the biological significance of this difference in the initial infection strategy, a comparison of the primary and secondary infection patterns of the recombinants Helicoverpa zea SNPV (HzSNPV-hsp70/lacZ) and Autographa californica MNPV (AcMNPV-hsp70/lacZ) in orally inoculated larvae of Heliothis virescens was carried out. At dosages yielding similar final mortalities (∼85 %), primary midgut infections by HzSNPV-hsp70/lacZ (indicated by lacZ expression) were observed 6 h earlier and in greater numbers than those generated by AcMNPV-hsp70/lacZ. Infection of secondary target cells in the tracheal epidermis, however, occurred at the same time and at the same rate for both NPVs. A 2 h delay was observed between the onset of primary and secondary AcMNPV-hsp70/lacZ infection, supporting the hypothesis that early tracheal infections were initiated by ODV nucleocapsids repackaged as budded virus. In contrast, an 8 h delay was observed with HzSNPV-hsp70/lacZ, suggesting that systemic infections were established only after virus replication in primary targets. Significant numbers of both MNPV- and SNPV-infected primary target cells were sloughed from the midgut beginning as early as 16 h post-infection. Midgut cell sloughing may be an important host-mediated selection pressure influencing the evolution of NPV morphology and gene regulation, shaping, in part, baculovirus infection strategies.