- Volume 80, Issue 5, 1999

Purified porcine monocytes, the natural carrier cells of pseudorabies virus (PrV) in the pig, were inoculated in vitro with PrV. At different time-points post-inoculation (p.i.) (from 7 to 17 h p.i.), the cells were washed and incubated with fluorescein isothiocyanate-labelled porcine PrV-specific polyclonal antibodies (IgG) at 37 degrees C. At all time-points tested p.i., 1 h of antibody incubation induced passive patching and subsequent internalization of the plasma membrane-anchored viral glycoproteins in approximately 65% of the infected monocytes. This endocytosis process is antibody-dependent, since biotinylated glycoproteins did not undergo spontaneous endocytosis. The process is fast and efficient, since only very low amounts of viral glycoproteins on the plasma membrane (7 h p.i.) and a minimal concentration of antibodies (0.04 mg IgG/ml) were needed to induce endocytosis. Experiments with PrV strains carrying deletions in the genes encoding the 11 different viral glycoproteins showed that viral glycoproteins gB and gD play a very important role in endocytosis (80% reduction with deletion mutants, P < 0.001), while the gE:gI Fc receptor complex, but not gE or gI alone, has a significant but lesser effect (45% reduction, P < 0.05). Double staining of viral glycoproteins and major histocompatibility complex class I (MHC I) showed a clear co-localization and co-endocytosis of MHC I with the viral glycoproteins, suggesting a possible role of the process in immune evasion of the virus.

Our previous work demonstrated that following human cytomegalovirus (HCMV) infection of fibroblasts, there was a protein-protein interaction between the HCMV IE1-72 immediate-early (IE) protein and the cellular p107 protein which resulted in the alleviation of p107-mediated transcriptional repression of E2F-responsive promoters. In a further characterization of this interaction, we now show that IE1-72 binds to the N-terminal portion of p107, not the C-terminal ‘pocket’ region that binds E2F-4, and where a number of other viral gene products bind. Additionally, we show that exons 2 and 3 of IE1-72 are required for binding to p107. After mapping the binding domains, we next wanted to address the additional functional consequences of this interaction. It is well known that p107 can negatively regulate cell growth. To examine whether IE1-72 can also overcome this growth suppression, we transfected and infected or cotransfected various constructs into SAOS-2 cells. We showed that infection of SAOS-2 cells was capable of alleviating p107-mediated growth suppression. Additionally, we showed that IE1-72 alone is capable of overcoming p107-mediated growth arrest. Alleviation of this repression by IE1-72 is dependent on the protein-protein interaction between p107 and IE1-72 as deletion mutants of either protein which lack the identified binding domains fail to achieve this effect. These data indicate that the IE1-72 protein is capable of overcoming p107-mediated blocks in cellular proliferation, events that occur in both productive and non-productive HCMV infections.

The complete ORF54 of the Kaposi’s sarcoma-associated herpesvirus (KSHV) (human herpesvirus-8; HHV-8) was cloned and expressed in E. coli. The results show that KSHV/HHV-8 ORF54 encodes a functional dUTPase which specifically hydrolyses dUTP to dUMP. Monoclonal antibodies against the HHV-8 dUTPase detected a protein with the expected molecular mass of 35 kDa in HHV-8-infected BCBL-1 cells. Induction of the lytic replication cycle of HHV-8 by treatment of BCBL-1 cells with the phorbol ester TPA resulted in an increased expression of the protein which was not inhibited by phosphonoacetic acid, indicating that the protein is expressed early in the lytic replication cycle. Moreover, the sporadic expression of the HHV-8 dUTPase in tissue sections of Kaposi’s sarcoma was detected by immunohistochemistry.

The herpesvirus saimiri (HVS) gene product encoded by ORF 57 shares limited C-terminal similarity with herpes simplex virus 1 ICP27, a protein that has been demonstrated to be involved in the inhibition of host-cell splicing and is responsible for the redistribution of components of the spliceosome. It has previously been shown that ORF 57 can either activate or repress viral gene expression by a post-transcriptional mechanism. Furthermore, repression of gene expression by ORF 57 is dependent on the presence of an intron within the target gene coding region. In this report, it is shown that HVS infection results in the redistribution of the SC-35 splicing factor in the infected cell nucleus. Furthermore, the redistributed SC-35 colocalized with the ORF 57 protein product and expression of the protein alone was sufficient to cause the redistribution of the spliceosome components. These results suggest that the mechanism by which ORF 57 down-regulates expression of intron-containing genes involves the redistribution of the spliceosome complex.

First observed in 1972 in Crassostrea virginica, herpes-like viruses of bivalves were more recently found to be associated with high mortality rates in other cultured oyster species, such as Crassostrea gigas and Ostrea edulis. The diagnosis of herpes-like virus infections is performed currently by laborious histological and transmission electron microscope examinations. Preparation of specific reagents for use in more amenable diagnostic techniques prompted purification of virus particles and investigation of the viral genome. This paper is the first description of the purification of a virus pathogen from a bivalve mollusc. A procedure was developed which facilitated purification of large amounts of virus particles on the 40-50% interface of sucrose gradients. Transmission electron microscopy showed that a purified virus suspension contained capsids and enveloped virus particles. High molecular mass viral DNA was extracted, and the genome size was estimated by the summation of the sizes of restriction endonuclease fragments to be approximately 180 kbp. Partial cloning of the virus genome was achieved and the specificity of certain cloned fragments was established by dot blot hybridization.

In order to facilitate an analysis of the pattern of herpes simplex virus gene expression during latency establishment and reactivation, recombinant viruses containing the lacZ reporter gene under control of either the immediate early 110 (IE110) promoter or the latency-associated promoter have been constructed. Histochemical staining of ganglia taken from mice infected with these viruses allows for the rapid identification and quantification of sensory neurones in which these two promoters are active. Using the mouse ear model, this study demonstrates that, during the establishment of latency in vivo, IE110 promoter activity is only detectable in ganglia which provide innervation to the site of virus inoculation. Latency, however, is efficiently established not only in these ganglia, but also in adjacent ganglia whose neurones do not innervate the ear, and in which there was no evidence of IE110 expression during the acute phase of infection. This implies that replication-competent virus can efficiently establish latency in the absence of detectable IE110 expression. In addition, it has been possible to investigate viral gene expression in neurones following ganglionic explant culture by monitoring IE110 promoter-driven lacZ expression within reactivating neurones. This study shows that virus can be reactivated from all latently infected ganglia, but that reactivation appears to be more efficient from ganglia which provide innervation to the site of infection. The implications of these results for the mechanisms involved in latency establishment and reactivation are discussed.