Vaccinia virus utilizes microtubules for movement to the cell surface

Abstract

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 μm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.

Directed Egress of Animal Viruses Promotes Cell-to-Cell Spread

“…Actin tails project CEV at the tips of microvilli into neighboring cells, and this process is apparently required for efficient cell-to-cell spread. This was confirmed in a recent microscopic analysis showing VV atop actin tails projecting across cell junctions (43). However, it is not yet clear whether there is preferential transport of VV particles to sites of epithelial cell contact or specific production of actin tails there.…”

“…Second, confocal z-axis analyses and electron microscopy have provided evidence that actin tails form exclusively at the cell surface and not in the cytoplasm (43,97; E. J. Wolffe and B. Moss, unpublished data). Third, inhibitors of microtubules, but not inhibitors of actin polymerization, reduced movement of VV to the cell surface (43,97). Therefore, it appears that VV moves via microtubules from virus factories to the TGN, where envelopment occurs, and then microtubules ferry IEV to the cell surface (Fig.…”

“…First, A36R is targeted to the outer envelope of the IEV, but it remains in the plasma membrane at one pole of the CEV, orchestrating actin tails (73,94,102). Second, confocal z-axis analyses and electron microscopy have provided evidence that actin tails form exclusively at the cell surface and not in the cytoplasm (43,97; E. J. Wolffe and B. Moss, unpublished data). Third, inhibitors of microtubules, but not inhibitors of actin polymerization, reduced movement of VV to the cell surface (43,97).…”

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 .…”

Existence of an operative pathway from the endoplasmic reticulum to the immature poxvirus membrane

“…Moreover, ER, ERGIC, or Golgi proteins were not detected in the crescent membranes, and continuity between the latter and cytoplasmic tubules has been disputed (2,3). Similarly controversial is the nature of the IV and MV membranes, specifically whether they are composed of one (1)(2)(3)14) or two (8,15,16) sets of lipoprotein bilayers.…”