The Final Hurdle: The role of envelope proteins in poxvirus release

Abstract

Increasing incidence of antimicrobial resistance requires identification of novel drugs, and the human genome presents thousands of potential targets. Intracellular pathogens rely on host proteins and pathways during their entry, replication and egress.

Poxviruses are reliant on host Abl tyrosine kinases for release of enveloped virus, the last step in their egress. Release can be inhibited by Abl kinase targeting drug imatinib, with treatment shown to rescue mice infected with a lethal dose of vaccinia virus (VACV). It is speculated that host-targeted antimicrobials are less likely to select for resistance. This thesis uses VACV as a model to study the effects of treatment by a host- targeted antimicrobial.

Following treatment by imatinib, a mutation in VACV outer envelope protein A34 was identified that elicits high viral release. This study characterises the novel A34 mutation alongside published mutations in envelope proteins A34 and B5, which each lead to higher virus release from cells than the parental strain. Mutations in A34 demonstrate resistance to treatment with imatinib, suggesting that antimicrobial resistance may still arise against host-targeted antimicrobials via compensatory mutations in parallel pathways. This thesis further explores the potential mechanisms of viral release by expressing the A34 ectodomain for structural analysis.

The biological significance of release of enveloped virus during infection is an open question. Ectromelia virus (ECTV) provides a model of disease in a native murine host. Actin nucleation driving enveloped virus out of cells prior to release is highly conserved among poxviruses. Actin nucleation has been mapped to two tyrosine residues on envelope protein A36 in VACV. Here, the ECTV A36 homologue is examined and actin nucleation mapped to phosphorylation of a single tyrosine. Further, while actin-based motility is shown to play a critical role in the release of ECTV from infected cells, its loss was not critical in vivo.