Strain-specific genetic requirements for growth of vaccinia virus in culture

Abstract

Poxviruses, including vaccinia virus (VACV), have linear DNA genomes, containing approximately 200 genes. Many of these genes contribute to virulence but are dispensable for replication in tissue culture. To determine whether interactions between these non-essential genes influence vaccinia virus growth in vitro, this thesis focuses on strains of VACV that lack functional copies of multiple genes. Poxvirus genomes are arranged with the highly conserved genes, which are essential for basic replication, clustered at the centre. Vaccinia virus strains have been isolated with large deletions that eliminate multiple genes from the genomic termini. Although poorly characterized, isolation of these strains has supported the view that all genes at the ends of the genome are non-essential for replication in vitro. To investigate whether the genes at the termini contribute to replication, a set of vaccinia virus strains were generated with large targeted deletions that remove up to 30% of the annotated open reading frames (ORFs) from the commonly used VACV strain, Western Reserve (WR). Analysis of these recombinants has revealed unexpected functional redundancy that safeguards basic viral replication in vitro. Viruses with large deletions from both the left (42 ORFs) and right (25 ORFs) termini of the genome had an extremely severe growth defect on all cell lines tested. In contrast, moderate defects were associated with either one of these deletions if the opposite end of the genome was intact. The gene C11R, which encodes the VACV homologue of the epidermal growth factor, provided an example of redundancy and dependency. C11R was shown to be important for VACV replication in cell culture when 42 genes are missing from the left end of the genome. The effect of C11R was dependent on additional loci at the right terminus. The candidate vaccine vector, Modified Vaccinia virus Ankara (MVA), fails to replicate in most mammalian cells, due to mutations acquired during several hundred rounds of serial passage in vitro. MVA has a genome 13% smaller than its replication-competent parent and has mutations in 60% of the remaining ORFs. A marker rescue experiment previously mapped the mutations important for the attenuation of MVA to the left half of the genome and produced a set of rescued MVA strains that showed improved replication on mammalian cells. I have extended characterization of these rescued MVA strains and identified three repaired regions in two of the rescued MVA strains. These experiments identified two genes that influence plaque formation, but not replication, on the primate cell line, BS-C-1. The previously uncharacterized F5L gene was also shown to influence the plaque morphology of VACV WR on a subset of cell lines. Despite reducing plaque size, the deletion of F5L from VACV WR did not affect either replication in vitro or VACV virulence in mouse models. In summary, this investigation of VACV strains with large deletions revealed that genetic redundancy and dependency between genes considered non-essential contributes to basic viral replication, and that plaque size does not always reflect viral replication.