- Volume 94, Issue 5, 2013

Human cytomegalovirus (HCMV)-encoded G protein-coupled-receptor US28 is believed to participate in virus dissemination through modulation of cell migration and immune evasion. US28 binds different CC chemokines and the CX3C chemokine CX3CL1. Membrane-anchored CX3CL1 is expressed by immune-activated endothelial cells, causing redirection of CX3CR1-expressing leukocytes in the blood to sites of infection. Here, we used stable transfected cell lines to examine how US28 expression affects cell migration on immobilized full-length CX3CL1, to model how HCMV-infected leukocytes interact with inflamed endothelium. We observed that US28-expressing cells migrated more than CX3CR1-expressing cells when adhering to immobilized CX3CL1. US28-induced migration was G protein-signalling dependent and was blocked by the phospholipase Cβ inhibitor U73122 and the intracellular calcium chelator BAPTA-AM. In addition, migration was inhibited in a dose-dependent manner by competition from CCL2 and CCL5, whereas CCL3 had little effect. Instead of migrating, CX3CR1-expressing cells performed ‘dancing-on-the-spot’ movements, demonstrating that anchored CX3CL1 acts as a strong tether for these cells. At low receptor expression levels, however, no significant difference in migration potential was observed when comparing the migration of CX3CR1- and US28-expressing cells. Thus, these data showed that, in contrast to CX3CR1, which promotes efficient cell capture upon binding to anchored CX3CL1, US28 acts to increase the migration of cells upon binding to the same ligand. Overall, this indicates that infected cells probably move more than uninfected cells in inflamed tissues with high CX3CL1 expression, with soluble chemokines affecting the final migration.

Vectors based on vaccinia virus (VACV), the vaccine used to eradicate smallpox, are currently popular candidates for the vaccination against numerous infectious diseases including malaria and AIDS. Although VACV induces robust cellular and humoral responses, enhancing the safety and efficacy of these vectors remains an important area of research. Here, we describe the enhanced immunogenicity of a recombinant VACV Western Reserve (WR) strain lacking the immunomodulatory protein C6 (vΔC6). Intradermal infection of mice with vΔC6 was shown previously to induce smaller lesions, indicating viral attenuation, and this was confirmed here using a different inoculation dose. In addition, data presented show that vaccination with vΔC6 provided better protection against challenge with a lethal dose of VACV WR, indicating this virus is a better vaccine. Increased protection was not due to improved humoral responses, but instead enhanced cytotoxic activity of T-cells 1 month post-inoculation in the spleens of vΔC6-vaccinated mice.

Epstein–Barr virus (EBV) latently infected B-cells are the precursors of EBV-associated malignancies. EBV-infection induces the production of pro-survival and anti-inflammatory cytokines that may be important in the transition between latency and malignancy. One EBV protein, LMP2A, can be detected in both latently infected resting B-cells and in EBV-associated malignancies. Therefore, we tested the ability of LMP2A to influence cytokine production using both LMP2A-Tg primary B-cells and LMP2A-expressing B-cell lines. Our data demonstrate that LMP2A does not globally alter B-cell-produced cytokine levels, but specifically targets IL-10. Additional studies using ELISA and real-time-RT-PCR confirm that LMP2A utilizes PI3-kinase to increase IL-10 levels. Finally, the data demonstrate that LMP2A-expressing B-cell lines are more dependent on IL-10 for survival in comparison to LMP2A-negative B-cell lines. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumour survival and anti-tumour responses.

Tranosema rostrale ichnovirus (TrIV) is a polydnavirus (PDV) transmitted by the endoparasitic wasp T. rostrale to its host Choristoneura fumiferana during oviposition. PDV genes are expressed in infected caterpillars, causing physiological disturbances that promote the survival of the developing endoparasite. The previously sequenced genome of TrIV contains ~86 genes organized in multigene families and distributed on multiple segments of circular dsDNA. Among these, the ‘T. rostrale virus’ (TrV) family comprises seven genes that are absent in other PDV genomes examined to date and whose function(s) remain(s) unknown. Here, we initiated a functional analysis of the TrV family using qPCR, transfection and RNAi approaches. TrV family genes were weakly expressed in wasp ovaries, but some displayed high transcript abundance in parasitized caterpillars. Whilst TrV1 was the most highly transcribed TrV gene in infected caterpillars, transcript levels for TrV5 and TrV6 were nearly undetectable, indicating that they may be pseudogenes. Temporal and tissue-specific patterns of transcript abundance were similar for all expressed TrV family genes, indicative of an apparent lack of difference in function or tissue specificity. Infection of Cf-203 and Sf-21 insect cells with TrIV led to a dose-dependent inhibition of cell proliferation with no sign of apoptosis. Whilst similar inhibition was observed following transfection of cells with a cloned genome segment carrying the TrV1 gene, RNA interference targeting TrV1 largely restored cell growth in TrIV-infected cells, indicating that TrV1 expression was responsible for the observed inhibition. We suggest that TrV genes may contribute to host developmental disruption by interfering with host-cell proliferation during parasitism.