The vaccinia virus A14.5L gene encodes a hydrophobic 53-amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses

A short sequence, located between the A14L and A15L open reading frames (OR Fs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapox iruses, and molluscipoxviruses, We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF, Synthesis of the tagged protein occurred at late times and was blocked by a n inhibitor of DNA replication, consistent with regulation by a predicted l ate promoter just upstream of the A14.5L ORF, Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. T he protein was associated with purified vaccinia virus particles and with m embranes of immature and mature virions that were visualized by electron mi croscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing age nt. A mutant virus, in which the A14.5L ORF was largely deleted, produced n ormal-size plaques in several cell lines, and the yields of infectious intr a- and extracellular viruses were similar to those of the parent. In contra st, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus wi th a restored A14.5L gene.