Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Abstract

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr 112 and Tyr 132 of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 m/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.The infectious forms of some enveloped viruses are assembled at the plasma membrane, providing direct access to the extracellular environment. Other viruses, however, are assembled in the nucleus or internal regions of the cytoplasm and require locomotion to reach the periphery of the cell. Such movement is likely to involve the actin or microtubule cytoskeleton (28). Infectious intracellular mature vaccinia virus virions (IMV) form in juxtanuclear factory regions of the cytoplasm (5, 19) and are wrapped by a double membrane derived from trans-Golgi or endosomal cisternae to become intracellular enveloped virions (IEV), which are then translocated to the periphery of the cell where the outer IEV and plasma membranes fuse (12,15,19,26,31). The plasma membrane adherent and the released extracellular virions are called cell-associated enveloped virions (CEV) and extracellular enveloped virions (EEV), respectively. The association of actin filaments with CEV at the tips of specialized microvilli has been appreciated since the early electron and immunofluorescence microscopic studies of Stokes (29) and Hiller e...

Virus trafficking – learning from single-virus tracking

“…Intracellular enveloped Vaccinia virus particles move from their perinuclear assembly site to the plasma membrane along microtubules [58][59][60][61][62][63] . These particles fuse with the plasma membrane, but remain attached as cell-associated enveloped virus (CEV) 64 .…”

Cdc42 and the RhoGEF Intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation

“…Vaccinia is unable to induce actin tail formation in the absence of phosphorylation of tyrosine 112 or in cells lacking Nck, as the latter is essential to recruit WIP-N-WASP to facilitate activation of the Arp2/3 complex (Frischknecht et al, 1999;Ward and Moss, 2001a;Scaplehorn et al, 2002;Weisswange et al, 2009). Once recruited by Nck, the WIP-N-WASP complex is further stabilised by an interaction with Grb2, a downstream response to tyrosine 132 phosphorylation in A36 (Scaplehorn et al, 2002;Weisswange et al, 2009;Donnelly et al, 2013).…”

“…Vaccinia induces actin polymerisation when newly assembled virus particles leaving an infected cell fuse with and remain attached to the plasma membrane (Cudmore et al, 1996;Hollinshead et al, 2001;Ward and Moss, 2001a;Doceul et al, 2010). These adherent extracellular viruses induce an outside-in signal that locally activates Src and Abl family kinases (Frischknecht et al, 1999;Newsome et al, 2004;Reeves et al, 2005;Newsome et al, 2006).…”

“…Clathrinmediated clustering of A36 also functions to stabilise N-WASP and enhance the rate of actin tail formation (Humphries et al, 2012). Ultimately, Arp2/3-induced actin polymerisation propels the virus away from the cell surface towards neighbouring cells, enhancing the spread of infection (Cudmore et al, 1995;Cudmore et al, 1996;Hollinshead et al, 2001;Ward and Moss, 2001a;Doceul et al, 2010).…”

Rho’ing in and out of cells

“…4F). 154,155,[158][159][160][161][162] While extensive information is available about the viral and host factors that initiate actin polymerization, much less is known about the factors that contribute to subsequent virion release. The host phosphoinositide 5-phosphatase SHIP2 and the viral A34 protein were reported to act as gatekeepers to regulate virion release.…”