NIH Research Festival
Poxviruses generally encode multiple ankyrin-like proteins with incompletely understood roles in host interactions. Modified vaccinia virus Ankara (MVA) is exceptional as all but one of the parental vaccinia virus (VACV) ankyrin-like protein genes was lost during multiple passages in chick embryo fibroblasts. However, deletion of the remaining ankyrin-like 68-kDa protein gene 186R was shown by Sperling et al. (2009) to abort MVA replication in murine and human cells prior to viral genome replication and late expression. We confirmed this result by constructing a 68-kDa ankyrin deletion mutant (MVA-¿68k) and pinpointed its defect in genome uncoating and DNA replication. But a similar defect did not occur when the homologous gene B18R was deleted from a replication-competent strain of VACV, suggesting the expression of compensating proteins that enable viral genome replication. To locate the compensating genes, the 186R gene was deleted from a panel of human replication-competent recombinant MVAs with varying length segments of DNA derived from a replication-competent VACV. We discovered that compensating genes were present in a ~27-kb fragment derived from the left end of the VACV genome. Finer mapping by construction of additional recombinant viruses revealed addition of either the VACV C5L or M2L gene to MVA-¿68k was sufficient to restore DNA replication and late expression in human cells. The C5 and M2 proteins are not homologous to each other or to the B18/186 protein, nor do they have ankyrin-like motifs. Nevertheless, the three proteins appear to act at a common pathway at an early stage of virus replication.
Scientific Focus Area: Virology
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