Interactions between Vaccinia Virus IEV Membrane Proteins and Their Roles in IEV Assembly and Actin Tail Formation

Abstract

The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the ΔB5R and ΔF13L deletion strains, but not ΔA34R, were still able to induce actin tails. The ΔA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from ΔA34R- and ΔA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.

Functional domains and motifs of bacterial type III effector proteins and their roles in infection

“…Nck itself possesses a single SH2 domain and three src homology 3 (SH3) domains, with the former binding to the YDEV Tir motif, while the latter binds to and recruits proteins involved in actin polymerization. Intriguingly, the Nck-binding site of Tir is homologous to that of the vaccinia virus protein A36R that is also tyrosine phosphorylated and is known to recruit Nck (Rottger et al, 1999), implying clear functional similarities between these unrelated proteins. In contrast, the Tir homologue in enterohaemorrhagic E. coli does not possess the Nck-binding site, but instead recruits its own type three effector called Tccp, which substitutes for the Nck protein (Garmendia et al, 2004).…”

Viral transport and the cytoskeleton

“…Vaccinia strains that are unable to induce actin tails have a small plaque phenotype suggesting they have reduced cell to cell spread [46][47][48]. Recent work has begun to identify the proteins involved in vaccinia-induced actin polymerization [49][50][51][52]. The integral IEV membrane protein A36R, which has a cytoplasmic domain of ~195 residues, is responsible for initiating the cascade of events that leads to actin tail formation [51].…”

“…Although the ability to form actin tails is beneficial to the spread of vaccinia, it is not essential. Vaccinia virus particles are able to reach the cell periphery in the absence of actin tail formation (see images in [49]), suggesting that viral particles can also move out on microtubules. Indeed, microtubule-based anterograde movements of vaccinia virus particles do occur and are essential, as actin tails only form at the plasma membrane (JM Rietdorf, M Way, unpublished data).…”

Budding of Marburgvirus is associated with filopodia

“…The actin cortex can also either facilitates or hinders the movement and release of viruses that bud at the plasma membrane. For example, the power of actin polymerization is used to propel vaccinia virus away from the cell surface, thus facilitating spread to uninfected cells (Cudmore et al, 1995;Rottger et al, 1999;Hollinshead et al, 2001). On the other hand, short-term actin depolymerization stimulated the release of equine infectious anaemia virus suggesting that the intact actin cortex can hinder the release of some viral particles (Chen et al, 2004).…”