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

Abstract

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

Analysis of Viral Membranes Formed in Cells Infected by a Vaccinia Virus L2-Deletion Mutant Suggests Their Origin from the Endoplasmic Reticulum

“…As expected, antibodies to the viral A17 and A14 membrane proteins decorated the short crescents. We also detected the A11 protein primarily at the edges of the short crescents, as previously found for L2 during a wild-type virus infection (37), consistent with recent evidence for lipid association of A11 (33). The majority of the D13 scaffold protein was present in intermediate-density occlusions.…”

“…Thus far, we have been unable to demonstrate an association of L2 with these other proteins by coimmunopurification. However, a weak association of A6 and A11 was recently reported (33).…”

“…Interestingly, repression or mutation of several other viral proteins that are not components of the viral membrane or present at a low level also interrupts crescent and IV formation. These include the protein kinase F10 (29)(30)(31) and A11 (32,33), H7 (34), L2 (35), and A6 (36) proteins.…”

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.…”

Direct Formation of Vaccinia Virus Membranes from the Endoplasmic Reticulum in the Absence of the Newly Characterized L2-Interacting Protein A30.5

“…Proteins that appear to be involved in the same or a similar step in morphogenesis as L2, based on the phenotypes of null mutants, will be referred to as viral-membrane assembly proteins (VMAPs). The VMAPS include L2 (16)(17)(18), A11 (19)(20)(21), H7 (22,23), A6 (24,25), and, as will be shown here, A30.5.…”