The product of the vaccinia virus L5R gene is a fourth membrane protein encoded by all poxviruses that is required for cell entry and cell-cell fusion.

The L5R gene of vaccinia virus is conserved among all sequenced members of the Poxviridae but has no predicted function or recognized nonpoxvirus homolog. Here we provide the initial characterization of the L5 protein. L5 is expressed following DNA replication with kinetics typical of a viral late protein, contains a single intramolecular disulfide bond formed by the virus-encoded cytoplasmic redox pathway, and is incorporated into intracellular mature virus particles, where it is exposed on the membrane surface. To determine whether L5 is essential for virus replication, we constructed a mutant that synthesizes L5 only in the presence of an inducer. The mutant exhibited a conditional-lethal phenotype, as cell-to-cell virus spread and formation of infectious progeny were dependent on the inducer. Nevertheless, all stages of replication occurred in the absence of inducer and intracellular and extracellular progeny virions appeared morphologically normal. Noninfectious virions lacking L5 could bind to cells, but the cores did not enter the cytoplasm. In addition, virions lacking L5 were unable to mediate low-pH-triggered cell-cell fusion from within or without. The phenotype of the L5R conditional lethal mutant is identical to that of recently described mutants in which expression of the A21, A28, and H2 genes is repressed. Thus, L5 is the fourth component of the poxvirus cell entry/fusion apparatus that is required for entry of both the intracellular and extracellular infectious forms of vaccinia virus.