Functional analysis of vaccinia virus B5R protein: role of the cytoplasmic tail

Vaccinia extracellular enveloped virus (EEV) is important for cell-to-cell and long-range virus spread both in vitro and in vivo. Six genes have been identified that encode protein constituents of the EEV outer membrane, and some of these proteins are critical for EEV formation. The B5R gene encodes an EEV-specific type I membrane protein, and deletion of this gene markedly decreases EEV formation and results in a small plaque phenotype. Data suggest that the transmembrane domain, cytoplasmic tail, or both contain the EEV localization signals that are required for targeting of the B5R protein to EEV and for EEV formation. Here, we report the construction of mutant vaccinia viruses in which the wild-type B5R gene was replaced with a mutated one that encodes a protein with the putative cytoplasmic tail deleted. The mutated protein showed normal intracellular distribution and was properly incorporated into EEV. Vaccinia viruses expressing the B5R protein lacking the cytoplasmic tail formed plaques that were similar in type and size to those formed by wild-type viruses and produced equivalent amounts of infectious EEV. These results indicate that the B5R cytoplasmic tail is not necessary for EEV formation and points to the transmembrane domain as the major determinant for targeting the B5R protein to the outer membrane of EEV and for supporting EEV formation.