Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

Abstract

Vaccinia virus A6L is a previously uncharacterized gene that is conserved in all sequenced vertebrate poxviruses. Here, we constructed a recombinant vaccinia virus encoding A6 with an epitope tag and showed that A6 was expressed in infected cells after viral DNA replication and packaged in the core of the mature virion. Furthermore, we showed that A6 was essential for vaccinia virus replication by performing clustered charge-to-alanine mutagenesis on A6, which resulted in two vaccinia virus mutants (vA6L-mut1 and vA6L-mut2) that displayed a temperature-sensitive phenotype. At 31°C, both mutants replicated efficiently; however, at 40°C, vA6L-mut1 grew to a low titer, while vA6L-mut2 failed to replicate. The A6 protein expressed by vA6L-mut2 exhibited temperature-dependent instability. At the nonpermissive temperature, vA6L-mut2 was normal at viral gene expression and viral factory formation, but it was defective for proteolytic processing of the precursors of several major virion proteins, a defect that is characteristic of a block in virion morphogenesis. Electron microscopy further showed that the morphogenesis of vA6L-mut2 was arrested before the formation of immature virion with nucleoid and mature virion. Taken together, our data show that A6 is a virion core protein that plays an essential role in virion morphogenesis.Poxviruses are a family of large, complex, double-stranded DNA viruses that replicate entirely in the cytoplasm of infected cells ( (VACWR125) is one of about 20 conserved open reading frames of WR that has not been previously characterized. The amino acid sequence of A6 gives no hint to its function as it has no recognizable motif or any homolog outside the poxvirus family. To determine the role that A6 plays in viral life cycle, we constructed recombinant VACV encoding A6 with an epitope tag and temperature-sensitive (ts) mutants with a lesion in A6. We characterized these recombinant viruses and report here that A6 is a virion core protein playing an essential role in virion morphogenesis.The morphogenesis of the VACV virion goes through a series of stages that can be distinguished by electron microscopy (recently reviewed in reference 2). Electron-dense virosomes consisting of viral proteins appear first. Then, crescentshaped precursors of the virion membrane develop at the periphery of the virosome and subsequently circularize to form the spherical immature virions (IVs). As IVs encapsidate the viral genome, they appear as IVs with an electron-dense nucleoid (IVNs). Eventually, IVNs evolve into the brick-shaped intracellular mature virions (MVs), which represent the majority of infectious particles produced during VACV infection. Concomitant with this change, precursor forms of several virion proteins, including p4a and p4b, were proteolytically processed into mature proteins (12, 21). We present data here suggesting that A6 is essential for the transition from IV to IVN and MV. MATERIALS AND METHODSCells and viruses. BS-C-1 cells were maintained in minimum essential medium with Earle's s...

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

“…The interaction between A6 and A11, however, is either weak or transient, as only a small amount of A11 coprecipitated with A6, and the cellular localization of A6 and A11 did not overlap extensively. During normal VACV replication, A11 localized to viral factories while A6 localized diffusely in the entire cytoplasm, including the factories (14). It is possible that a weak or transient interaction between A6 and A11 may be sufficient to allow A6 to facilitate A11 membrane association.…”

“…As a specificity control, we performed in parallel the precipitation of another VACV protein, A46, which is similar to A6 in its expression level and distribution in infected cells (data not shown). We infected HeLa cells with VACV encoding either V5 epitope-tagged A46 or A6 (14). We then used anti-V5-conjugated agarose beads to precipitate proteins from the cells and analyzed the precipitates with mass spectrometry (Fig.…”

“…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).…”

“…It associates with membranes during infection and binds some lipids in vitro (30). Both A6 and H7 localize to the cytoplasm (14,24). A11 localizes specifically to viral factories, but it is not incorporated by virions (19).…”

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

“…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. A6 and H7 are localized predominantly in the cytosol (18,32,39), while L2 and A30.5 are localized in the ER (19,21), and A11 associates with viral membranes (40,41). How these diverse proteins function together to facilitate acquisition of membranes from the ER is an intriguing question.…”