CHARACTERIZATION OF THE TWO VACCINIA VIRUS MATURE VIRION-SPECIFIC PROTEINS, A26P AND A25P

Abstract

Poxviruses produce two morphologically distinct infectious particles, mature virions (MVs) and extracellular virions (EVs). During replication, some MVs differentiate and become wrapped with cellular membranes, transported to the periphery, and exported as EVs. Some orthopoxviruses, e.g., cowpox virus (CPXV), form large, discrete cytoplasmic inclusions called A-type inclusion bodies (ATIs) within which MVs are embedded by a process called occlusion. ATIs are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) that fail to form ATIs. VACV does encode a functional A26p, which along with the ATIp is required for occlusion. A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV and regulates occlusion. Additionally, little is known about the formation of ATIs and how MVs become embedded within them. Here, experiments show that A26p is incorporated into MVs by the A27p-A17p complex and interacts with A25p, a truncated form of the CPXV ATIp. Restoration of the full-length ATI gene is sufficient for VACV ATI formation and the occlusion of MVs. A26p directly interacts with ATIp, and this interaction, as well as the A26p-A27p interaction, are required for occlusion. The data demonstrates that ATI mRNAs are transported out of viral factories (VFs) and translated in the cytoplasm. ATIs enlarge both by new protein synthesis and by coalescence, which requires microtubules. ATIs do not nucleate around MVs but rather MVs move along microtubules to embed within ATIs. Taken together, the data suggest a model for occlusion in which MVs move along microtubules to ATIs that are translated from mRNAs in the cytoplasm. At the ATIs, A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane. Although the specificity of A26p for MVs suggested A26p might regulate wrapping, I did not detect an effect of either the deletion of A26 or occlusion on the production of EVs.