Following wrapping with trans-Golgi or endosomal cisternae, vaccinia virus virions must travel from the interior of the cell to the plasma membrane. In a recent review, Sodeik (28
) pointed out that molecular crowding due to the presence of organelles, cytoskeleton, and high protein concentrations effectively restricts free diffusion of molecules larger than 500 kDa. For IEV, she calculated that it would take 5.7 h to travel 10 μm. A possible transport mechanism, used by a number of intracellular pathogens, involves rapid actin polymerization (2
). Indeed, an actin polymerization mechanism to explain vaccinia virus virion movement was proposed by Cudmore et al. (3
) based on video microscopic studies. However, our video microscopy experiments carried out with a recombinant vaccinia virus expressing GFP fused to the B5R membrane protein revealed movement that was similar to vesicle transport along microtubules and which could be inhibited by a microtubule-depolymerizing agent (34
). Because interpretations based on drug inhibition are always risky, we have now extended these observations by specifically preventing the nucleation of actin tails on vaccinia virus particles. To accomplish this, we used data of Frischknecht et al. (9
) indicating that actin nucleation occurs by phosphorylation of the A36R protein on Tyr112
alone or with Tyr132
. These residues had been identified by transcomplementation, rather than by constructing a vaccinia virus mutant with point mutations of the Tyr residues. Thus, our objectives were to construct and use recombinant vaccinia viruses to (i) confirm the site of A36R tyrosine phosphorylation, (ii) confirm the role of A36R Tyr112
in actin nucleation, (iii) analyze the effects of the Tyr mutations on the assembly and movement of IEV and cell-to-cell virus spread, (iv) compare the phenotype of the A36R Tyr mutant with that of an A36R deletion mutant, and (v) determine whether intra- or extracellular virions are associated with actin tails.
The recombinant viruses containing a deleted or mutated A36R gene expressed B5R-GFP in place of the normal B5R IEV membrane protein in order to visualize virion movement. As previously shown (34
) and further validated here, fusion of GFP was without discernible effect on B5R function so that the plaque size of vB5R-GFP was the same as that of standard vaccinia virus. Results obtained with vB5R-GFP/A36R-YdF, which contains conservative Phe substitutions for Tyr112
, will be discussed first. Even though the mutated A36R was synthesized and still had four remaining Tyr residues, Tyr phosphorylation was not detected and both actin tail and specialized microvillus formation failed to occur. Thus, the conclusions regarding the regulatory role of Tyr phosphorylation in actin nucleation were corroborated. In addition, we found that the mutant exhibited no discernible defect in wrapping as determined by electron microscopy and that the outer membranes of the abundant IEV reacted with antibody to GFP that was fused to the B5R protein. Therefore, vB5R-GFP/A36R-YdF was ideal for assessing the putative role of actin tails in intracellular IEV movement. The directional, saltatory movement with maximal speeds of >2 μm per s, first demonstrated with vB5R-GFP, was reproduced with vB5R-GFP/A36R-YdF, even though actin tail formation was prevented. A movie demonstrating this movement is available as Supplemental Material no. 2 at http://www.niaid.nih.gov/dir/labs/lvd/moss.htm
. These results should eliminate from further consideration any significant role for actin tails in intracellular movement of IEV. The type of movement exhibited by IEV was similar to that of vesicles moving along microtubules arranged parallel to the long axis of the cell (14
). Moreover, the movement was inhibited by the microtubule-depolymerizing drug nocodazole. Although nocodazole has also been reported to prevent movement of IMV and the formation of IEV (21
), we previously showed that the effect on IEV movement was rapid and reversible (34
). Microtubules also may be involved in early postentry stages of vaccinia virus replication (21
) and in transport of other viruses (17
Previous studies had not clearly defined whether virions associated with actin tails are intra- or extracellular. To resolve this issue, a cell line that expressed GFP-actin was infected with vaccinia virus and the unpermeabilized cells were stained with a MAb to the B5R membrane protein. By examination of Z-sections by confocal microscopy, we could find an association of all actin tails with virions on the cell surface. The surface location fits well with evidence, obtained by following the movement of individual GFP-labeled virions, that actin tails form at or near the plasma membrane (34
). In addition, van Eijl et al. (32
) suggested that Tyr phosphorylation of A36R occurred after fusion of the IEV and plasma membranes. Moreover, this model fits with evidence that Tyr phosphorylation of A36R occurs by a Src family kinase (9
), which is presumably plasma membrane associated.
Since actin tails are not involved in the intracellular transport of vaccinia virions, unlike the situation with Listeria
, what is their role? Actin tails are unnecessary for virions to exit the cell, as the presence of vB5R-GFP/A36R-YdF virions on the cell surface was demonstrated by low-pH-induced cell-to-cell fusion and by scanning electron microscopy. Nevertheless, the mutant plaques were one-third of the size of those formed by viruses with an intact A36R gene, and a lag in virus production occurred following a low-multiplicity spreading infection. These data are consistent with a primary role for actin tails in producing long motile microvilli that promote efficient cell-to-cell virus spread. A movie showing movement of virus-induced actin tails protruding from cells stably expressing GFP-actin is provided as Supplemental Material no. 4 at http://www.niaid.nih.gov/dir/labs/lvd/moss.htm
. The extracellular location of virions at the ends of actin tails means that they are unlikely to entirely escape the immune system during cell-to-cell spread.
The mutant vB5R-GFP/ΔA36R, with a deleted A36R ORF, also failed to make actin tails. However, this mutant was much more severely impaired than vB5R-GFP/A36R-YdF. Compared to vB5R-GFP/A36R-YdF, the following were all reduced: plaque size, virus replication and spread, low-pH-induced cell fusion (an indicator of virus on the cell surface), wrapped virus isolated by CsCl centrifugation, and IEV seen by transmission electron microscopy. Although previous studies with an A36R deletion mutant had attributed the severe restriction on virus spread to the failure of actin tail formation (24
), our data suggested that the deletion mutant has additional defects. Since the A36R protein has been shown to interact with other IEV membrane proteins (23
), its absence may adversely affect the structure of the protein complex forming the wrapping membrane. Although some virion movement was detected in cells infected with vB5R-GFP/ΔA36R, it was extremely infrequent and therefore could not be characterized in detail. Thus, the mutant containing the specific Tyr substitutions was superior to the deletion mutant for studying actin tail-independent IEV movement and virus spread.
A scheme depicting directional IEV movement along microtubules and actin tail formation at the plasma membrane is shown in Fig. . Because microtubules tend to be arranged parallel to the long axis of the cell, the IEV accumulate at the plus ends near the plasma membrane. The direction of IEV movement could be explained by interaction with a kinesin family microtubule motor, either directly or through additional cell protein adapters.
FIG. 11 Diagram representing the movement of enveloped virions. After wrapping of IMV in the juxtanuclear region (JNR), IEV move out to the cell periphery along microtubules (MT). Actin polymerization occurs at the plasma membrane, forming motile CEV-tipped microvilli. (more ...)