Vaccinia Virus A6 Is Essential for Virion Membrane Biogenesis and Localization of Virion Membrane Proteins to Sites of Virion Assembly

Abstract

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-␤-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein, A6

“…Consistent with this idea, several membrane proteins destined for the MV envelope are synthesized on the ER (9,23), and a pathway exists for the trafficking of MV membrane proteins from the ER to IVs (9). The trafficking of MV membrane precursors or MV membrane proteins to viral factories appears to be an active, virus-mediated process, as repressing VACV A6 expression results in the accumulation of MV membrane proteins in secretory compartments outside the viral factories (13).…”

“…HeLa cells were infected with VACV (vA6-V5 [14], vA46-V5, or iA6-GFP [13]) at a multiplicity of infection (MOI) of 5 PFU/cell. At 12 h postinfection (hpi), the cells were harvested and resuspended in lysis buffer (0.5% [wt/vol] Triton X-100, 25 mM Tris [pH 7.4], 150 mM NaCl) supplemented with protease inhibitor cocktail tablet (Roche Molecular Biochemicals).…”

“…F10 (30,31), A11 (19), H7 (24), L2 (11), and A6 (13) are required at an early step prior to the appearance of viral membrane precursors in the factories, as repressing the expression of these genes results in the loss of discernible membrane structure in the factories and the accumulation of large viroplasm inclusions. A14 and A17 are required at a later step following the acquisition of viral membrane precursors, because repressing the expression of A14 (22,32) or A17 (21,34) results in the accumulation of vesicular or tubular membrane structures at the boundaries of large viroplasm inclusions.…”

Structure-Function Analysis of Vaccinia Virus H7 Protein Reveals a Novel Phosphoinositide Binding Fold Essential for Poxvirus Replication

“…VMAPs are proteins that were recently discovered, through viral genetics, to be essential for poxvirus membrane biogenesis. VACV mutants lacking any one of the VMAPs have very similar defects (11,17,19,20,32), with viral assembly arrested at similar stages right before the formation of crescent membranes, the precursors of the viral envelope. There are at least 5 proteins in this group, varying in size from 5 to 20 kDa for A30.5, L2, and H7 and around 40 kDa for A11 and A6.…”

“…The origin and biogenesis of the crescent membranes are among the least understood aspects of poxvirus biology, but several viral proteins that are involved in crescent formation were recently identified in vaccinia virus (VACV), the prototypical poxvirus. These proteins, including A11 (17), H7 (18), L2 (19), A6 (20), and A30.5 (21), are collectively referred to as the viral membrane assembly proteins (VMAPs) (21). VMAPs were thought to work together to generate breaks in the ER membrane, to stabilize the open-ended crescent membranes, and to elongate the crescents by membrane fusion (21).…”