- Volume 85, Issue 5, 2004

Yaba-like disease virus (YLDV) is a yatapoxvirus, a group of slow-growing poxviruses from primates. Analysis of the growth cycle of YLDV in tissue culture showed that maximum virus titres were reached 3 days post-infection and at this time only 3·3 % of infectious progeny was extracellular. The intracellular and extracellular virions have different buoyant densities and are separable on CsCl density gradients. They are also distinguishable by electron microscopy with the extracellular virions having an additional lipid envelope. In YLDV-infected cells, thick actin bundles with virions at their tips were seen protruding from the cell surface, despite the fact that YLDV lacks a protein comparable to Vaccinia virus A36R, which is required for VV-induced actin tail formation. In addition to these observations, the YLDV gene Y144R was characterized. This gene is predicted to encode a transmembrane protein containing three short consensus repeat (SCR) motifs common to members of the complement control protein family. Antibody generated against recombinant Y144R recognized products of 36, 41 and 48–55 kDa in YLDV-infected cells and purified extracellular enveloped virus (EEV) but not intracellular mature virus (IMV). Y144R protein is a glycoprotein with type I membrane topology that is synthesized early and late during infection. By immunoblot, indirect immunofluorescence and immuno-cryoelectron microscopy the Y144R protein was detected on the intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and EEV. This represents the first study of a YLDV IEV, CEV and EEV protein at the molecular level.

Poxvirus interleukin (IL)-18 binding proteins (IL-18BPs) are soluble decoys that inhibit the activity of IL-18. The aim of this study was to demonstrate IL-18 binding activity of the Variola virus protein D7L. D7L effectively inhibited the biological activity of IL-18 in a bioassay. We compared the affinity and kinetics of D7L and the Ectromelia virus IL-18BP, p13, for human and murine IL-18 using surface plasmon resonance and no differences were detected, indicating that the differences in amino acid sequence did not affect binding or species specificity. Both proteins had higher affinity for murine than human IL-18. This was similar to human IL-18BP and the Molluscum contagiosum virus IL-18BP, which also demonstrated higher affinity for human IL-18. The host range of Variola virus is limited to humans and thus the affinity of D7L for IL-18 does not correlate with its host range. Furthermore, we demonstrated that D7L is capable of interacting with glycosaminoglycans (GAGs) via the C terminus, while p13 is not. Importantly, D7L interacted with both GAG and IL-18 simultaneously, indicating that the binding sites were distinct.

The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1–UL11, UL105–UL112 and UL120–UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.

Two different betaherpesviruses, the English and Maastricht species of rat cytomegalovirus (CMV), have previously been isolated from Rattus norvegicus. CMVs were isolated from both the brown rat, R. norvegicus, and the black rat, R. rattus, within Australia. The viruses isolated from R. norvegicus appeared to be genetically related to the English species of rat CMV by PCR, RFLP, and sequencing, but the viruses isolated from R. rattus were distinct from both prototype virus species, although more closely genetically related to the Maastricht virus. This is the first genetic characterization of cytomegaloviruses from R. rattus, and the first isolation of CMVs from Australian rats.

The human herpesvirus 6 (HHV-6) immediate-early (IE) 1 protein undergoes SUMOylation events during the infectious process. In the present work, we report that Lys-802 (K-802) of IE1 from HHV-6 variant B is the only target residue capable of conjugation to SUMO-1/SMT3C/Sentrin-1, SUMO-2/SMT3A/Sentrin-3 or SUMO-3/SMT3B/Sentrin-2 as determined by transfection and in vitro SUMOylation experiments. PolySUMOylated forms of IE1 were also observed, suggesting that SUMO branching occurs at the K-802 residue. Overexpression of SUMO-1, -2 and -3 led to an overall increase in IE1 levels, irrespective of K-802. The SUMO residues could be efficiently removed by incubating SUMOylated IE1 with SENP1, a recently identified SUMO peptidase. SUMOylation-deficient mutants of IE1 co-localized with nuclear promyelocytic leukaemia protein (PML) oncogenic domains (PODs) as efficiently as WT IE1, indicating that POD targeting is independent of IE1 SUMOylation status. However, in contrast to infection, PODs did not aggregate in IE1B-transfected cells, suggesting that other viral proteins are involved in the process. Transactivation studies indicated that IE1, in combination with IE2, could efficiently transactivate diverse promoters, independent of its SUMOylation status. Overall, the results presented provide a detailed biochemical characterization of post-translational modifications of the HHV-6 IE1 protein by SUMO peptides, contributing to our understanding of the complex interactions between herpesviruses and the SUMO-conjugation pathway.

Ongoing transcription in vitro of Tomato spotted wilt virus (TSWV) has previously been demonstrated to require the presence of reticulocyte lysate. This dependence was further investigated by testing the occurrence of transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein resulting from translation.