Genetic contribution to cytopathic effect in Vaccinia virus

Abstract

Vaccinia virus (VACV) infection induces cell migration, the formation of cytoplasmic extensions, actin polymerisation, cell rounding, detachment and lysis—changes collectively known as cytopathic effects (CPEs). Identification of genes that contribute to CPE phenotypes may highlight important virulence factors or novel signaling processes during infection. This investigation has focused on characterising four VACV genes (A55R, C2L, F3L and F5L) that have been identified as contributors to CPE. VACV protein F5 is a predicted membrane protein that is truncated in modified virus Ankara (MVA), a highly mutated strain with reduced CPE. We have characterised F5 to contribute to the rate of viral plaque expansion through a monolayer of infected cells, a contribution that may be derived from a localisation to cell junctions. The VACV BTB-BACK-Kelch proteins (BBKs) A55, C2 and F3 have been identified as contributors to CPE, with both A55 and C2 completely absent from the MVA genome. Human BBKs are well characterised as Cullin-3 substrate adaptors that facilitate the ubiquitination and subsequent degradation of various protein targets via the ubiquitin-proteasome system (UPS). Ubiquitination also has diverse non-degradative regulatory functions. Poxviruses are the only viruses known to encode BBKs and have been characterised to interact with Cullin-3. Our investigation has revealed functional redundancy between A55 and C2 from BBK-deficient CPE phenotypes. We have also been able to highlight a possible interaction of overexpressed VACV BBKs with ubiquitin-associated autophagy, a link between overexpression of C2 and filopodia formation and an association of endogenous expression levels of A55 and F3 with regulatory processes in the nucleus. Furthermore, we have been able to characterise the interactions of the VACV BBKs with each other, revealing that an A55:C2 heterodimer is most likely responsible for the CPEs generated by the VACV BBKs.