Redundancy complicates the definition of essential genes for vaccinia virus

Abstract

Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants.

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

“…We differ from some of the previous studies in that we here observe an extensive rearrangement at the termini that appears to improve [rather than interfere with (Dobson and Tscharke 2015)] the replication of the affected virus. At least one of the point mutations and especially the rearrangement appears to be associated with improved replication of MVA-CR19.…”

“…Heterogeneity and rearrangements in the ITRs of vaccinia viruses has been described previously (Moss et al 1981;Pickup et al 1982;Paez et al 1985;Qin et al 2011). The ITRs of poxviruses, similar to the multigene families in the genome of the distantly related Asfariviruses (Portugal et al 2015), contain partially redundant sets of immunomodulatory factors and determinants for host range Meisinger-Henschel et al 2007;Dobson and Tscharke 2015). The rearrangement of the ITR can therefore impact properties of vaccinia virus vectors:…”

Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool

“…To test if the results above were a function of the site targeted we investigated a second locus in the genome (Fig. 2c ) 39 . In this experiment, the repair plasmid, pBII-ΔR, was used to replace 23 kb at the right arm of the VACV genome, from genes Spi2 ( B13R ) to C11R in the inverted terminal repeat (ITR), with mCherry.…”

Modified Vaccinia Virus Ankara

“…Moreover, due to the extensive loss of genetic information MVA may serve as a particularly useful tool to study VACV host-regulatory genes. Obviously, there are several pathways of the host (cell) defence that are targeted by multiple VACV proteins and the phenotype of a VACV single gene mutant may be masked by the complementary function of (an)other viral gene(s) (Dobson and Tscharke, 2015). There are three obvious strategies using MVA to elucidate on functions of selected VACV host-interaction factors: (i) the few remaining regulatory genes in the MVA genome can be targeted for inactivation, (ii) candidate VACV genes missing in MVA are reinserted in the genome to rescue a host-interaction phenotype, or (iii) a combination of these latter approaches may serve to investigate putatively complementary gene functions.…”