Congregation of Orthopoxvirus Virions in Cytoplasmic A-Type Inclusions Is Mediated by Interactions of a Bridging Protein (A26p) with a Matrix Protein (ATIp) and a Virion Membrane-Associated Protein (A27p)

Abstract

Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.

Formation of Orthopoxvirus Cytoplasmic A-Type Inclusion Bodies and Embedding of Virions Are Dynamic Processes Requiring Microtubules

“…As much more is known about the molecular biology of VACV than about that of CPXV or other OPXVs that make ATIs naturally, we primarily used the reconstructed VACV system to study ATI forma-tion and the mechanism of occlusion. We previously demonstrated that the direct association of A26 with both the A27-A17 complex and the ATIp is required to mediate occlusion (14). In the present study, we showed that ATI enlargement was dependent on both continued protein synthesis and coalescence of smaller ATIs, which was documented by live imaging and confirmed with CPXV.…”

“…We demonstrated previously that VACV could be enabled to make ATIs and occlude MVs if the CPXV ATI gene were substituted for the shorter VACV A25 homolog (14). Thus, VACV encodes all of the necessary proteins for this process except for the full-length ATIp.…”

“…ATIp is a large protein (160 kDa in CPXV) containing C-terminal, hydrophobic repeats (10,26) that are required for assembly of inclusion bodies (14). ATIs are localized throughout the cytoplasm and increase in size during infection.…”

“…ATIs are localized throughout the cytoplasm and increase in size during infection. The embedding of MVs within ATIs that leads to their occlusion requires the A26 protein (14,22). A26 is an MV-specific protein anchored to the MV membrane by interactions with the A27-A17 complex (3,4,13).…”

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole

“…The ATI matrix is formed by multiple copies of the A25L polypeptide, which corresponds to the CPXV158 protein (30). A26L, also named p4c or CPXV159 protein, is required to direct intracellular mature virions (IMVs) into ATIs (33); the protein has a bridging function between the A25L matrix protein and IMVs containing the membrane-associated A27L (CPXV162 protein) (34). In addition, the capability to embed mature virions in the ATIs was recently shown to influence the virulence of CPXV (35).…”