Vaccinia Virus F12L Protein Is Required for Actin Tail Formation, Normal Plaque Size, and Virulence

Abstract

Vaccinia virus gene F12L is shown to encode a 65-kDa protein that is synthesized early and late during infection and that is not modified by glycosylation. Computational sequence comparison revealed that related proteins are encoded by all sequenced chordopoxviruses. A virus deletion mutant lacking the F12L gene (v⌬F12L) and a revertant virus with the F12L gene reinserted 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 antibody was also inserted into the F12L locus of v⌬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 enveloped virus and extracellular enveloped virus were reduced 8-to 11-fold and 7-fold, respectively. Consistent with the lack of actin tail formation, v⌬F12L produced a very small plaque. The v⌬F12L virus was severely attenuated in vivo, such that a dose of v⌬F12L 10,000-fold greater than the dose of wild-type virus that induced severe disease was unable to induce disease in mice infected intranasally.

Actin-Based Motility of Intracellular Microbial Pathogens

“…Data suggest that A33R may function in part as a chaperone for transfer of A36R from the Golgi membrane to the IEV (192). Deletion of F12L leads to a twofold reduction in IEV formation and a 99% reduction in actin tail formation (198), suggesting that it may play a role in actin assembly. Further studies will be required to definitively determine whether any of these viral proteins are directly required for actin assembly.…”

Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling

“…Nine proteins from which HLA ligands were derived are among the 29 previously described immunogenic early proteins (10), and 4 proteins, B19R, E5R, G5.5R, and B15R, were newly identified in this study to contain relevant human CTL epitopes (Table VI). The proteins bearing HLA ligands functionally belong to two groups: proteins with immunomodulatory or host range and virulence function (B8R, B15R, B19R, C7L, and F12L (51,56,57)), and proteins functionally connected to DNA replication or transcription (E5R, A48R, G5.5R, A23R, D12L, and J6R (51)). The function of O1L is unknown.…”

Directed Egress of Animal Viruses Promotes Cell-to-Cell Spread

“…These proteins loosely fall into two classes: (i) F13L and B5R are required for wrapping of the IEV envelope around the IMV (4, 24, 99), while (ii) F12L, A33R, A34R, A36R, and A56R are not required for this envelopment. F12L, A33R, A34R, and A36R are required for the assembly of actin tails; IEV, CEV, and EEV are formed in the absence of these viral proteins, but CEV do not extend toward other cells on microvilli, and plaques are small (53,72,75,100,101,104). Therefore, CEV and actin tails are crucial for VV cell-to-cell spread.…”