Loss of the vaccinia virus 35-amino acid hydrophobic O3 protein is partially compensated by mutations in the transmembrane domains of other entry proteins.

Eleven highly conserved proteins comprise the poxvirus entry-fusion complex (EFC). We focused on vaccinia virus (VACV) O3, a 35-amino acid, largely hydrophobic component of unknown specific function. Experimental evolution was carried out by blindly passaging a virus that was severely impaired in entry due to deletion of the gene encoding O3. Large plaque variants that arose spontaneously were discerned by round four and their numbers increased thereafter. Genome sequencing of individual cloned viruses revealed mutations in predicted transmembrane domains of three open reading frames encoding proteins with roles in entry. There were frame-shift mutations in consecutive Ts in open reading frames F9L and D8L and a nonsynonymous base substitution in L5R. F9 and L5 are EFC proteins and D8 is involved in VACV cell attachment. The F9L mutation occurred by round four in each of three independant passages, whereas the L5R and D8L mutations were detected only after nearly all of the genomes already had the F9L mutation. Viruses with deletions of O3L and single or double F9L, L5R and D8L mutations were constructed by homologous recombination. In a single round of infection, viruses with adaptive mutations including F9L alone or in combination exhibited statistically significant higher virus titers than the parental O3L deletion mutant or the L5R or D8L mutants, consistent with the order of selection during the passages. Further analyses indicated that the adaptive F9L mutants also had higher infectivities, entered cells more rapidly and increased EFC assembly, which partially compensated for the loss of O3.