Vaccinia virus F12L protein is required for actin tail formation, normal plaque size, and virulence

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthe sized early and late during infection and that is not modified by glycosyla tion. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (v Delta F12L) and a revertant virus with the F12L gene reins erted into the deletion mutant (vF12L-rev) were constructed and analyzed. A version of the F12L gene with a C-terminal amino acid tag derived from the influenza virus hemagglutinin and that is recognized by a monoclonal antib ody was also inserted into the F12L locus of v Delta F12L. Loss of the F12L protein reduced the formation of IMV 2-fold, but there was a dramatic (99. 5%) reduction in actin tail formation, and the levels of cell-associated en veloped virus and extracellular enveloped virus were reduced 8- to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, v Delta F12L produced a very small plaque. The v Delta F12L virus was seve rely attenuated in vivo, such that a dose of v Delta F12L 10,000-fold great er than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.