Characterization of vaccinia virus A12L protein proteolysis and its participation in virus assembly

Su Jung Yang

doi:10.1186/1743-422x-4-78

Abstract

Vaccinia virus (VV) undergoes a proteolytic processing to evolve from immature virus particles into intracellular mature virus particles. Most of structural core protein precursors such as p4a, p4b, and p25K are assembled into previrions and then proteolytically processed to yield core proteins, 4a, 4b, and 25 K, which become components of a mature virus particle. These structural rearrangements take place at a conserved cleavage motif, Ala-Gly-X (where X is any amino acid) and catalyzed by a VV encoded proteinase, the I7L gene product. The VV A12L gene product, a 25 kDa protein synthesized at late times during infection is cleaved at an N-terminal AG/A site, resulting in a 17 kDa cleavage product. However, due to the distinct characteristics of A12L proteolysis such as the localization of both the A12L full-length protein and its cleavage product in mature virions and two putative cleavage sites (Ala-Gly-Lys) located at internal and C-terminal region of A12L ORF, it was of interest to examine the A12L proteolysis for better understanding of regulation and function of VV proteolysis. Here, we attempted to examine the in vivo A12L processing by: determining the kinetics of the A12L proteolysis, the responsible viral protease, and the function of the A12L protein and its cleavage events. Surprisingly, the A12L precursor was cleaved into multiple peptides not only at an N-terminal AG/A but also at both an N- and a C-terminus. Despite the involvement of I7L proteinase for A12L proteolysis, its incomplete processing with slow kinetics and additional cleavages not at the two AG/K sites demonstrate unique regulation of VV proteolysis. An immunoprecipitation experiment in concert with N-terminal sequencing analyses and mass spectrometry led to the identification of VV core and membrane proteins, which may be associated with the A12L protein and suggested possible involvement of A12L protein and its cleavage products in multiple stages in virus morphogenesis.

Highlights

Vaccinia virus (VV), the prototype member of the Poxviridae family has a large double-stranded DNA genome
To determine if any other A12L-derived protein species were evident within the cytoplasm of VVinfected cells, cytoplasmic extracts were prepared and subjected to immunoblot analysis using A12L antisera directed against the entire A12L protein
The rifampicin was replaced with new infection media with and without the drug at 19 hpi to determine the effects of the drug on A12L protein processing for 12 hours

Summary

Introduction

Vaccinia virus (VV), the prototype member of the Poxviridae family has a large double-stranded DNA genome. Replication and viral assembly occur entirely in the cytoplasm of host cells, in particular, in areas referred as viroplasms or virosomes. Virus assembly initiates at virosomes surrounded by crescent membranes, which subsequently engulf granular materials forming spherical-shaped particles named immature virions (IV). The IVs transform into brick-shaped structures referred to as intracellular mature virions (IMV) where viral DNAs become condensed and packaged in an electron dense area and are covered by a viral envelope membrane. Virology Journal 2007, 4:78 http://www.virologyj.com/content/4/1/78 trans-Golgi network and results in the formation of intracellular enveloped virus (IEV), which becomes fused with the plasma membrane. If the IEVs remain associated with the cells, they are referred to as cell-associated enveloped virus (CEV), or if the IEVs bud through the plasma membrane spreading outside of the cells, they are considered extracellular enveloped virus (EEV)

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