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. 2001 Dec;75(23):11651-63.
doi: 10.1128/JVI.75.23.11651-11663.2001.

Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails

B M Ward  1 , B Moss
Affiliations

Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails

B M Ward et al. J Virol. 2001 Dec.

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 microm/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.

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Figures

FIG. 1
FIG. 1
Plaque phenotypes. The indicated viruses were plated on monolayers of BS-C-1 cells. After 3 days, plaques were imaged using light and fluorescence microscopy and then stained with crystal violet. Areas of at least 140 individual plaques for each of the five viruses were determined, and the averages and standard deviations (in parentheses) were calculated. BF, bright field.
FIG. 2
FIG. 2
Induction of syncytia. Uninfected or infected HeLa cells were incubated for 15 h, briefly treated with buffer at pH 7.4 or 5.5, and then incubated in regular medium for 3 h. The cells were examined by phase-contrast microscopy. Recombinant viruses used for infection are indicated on the left.
FIG. 3
FIG. 3
Virus replication. BS-C-1 cells were infected with 0.01 PFU of the indicated viruses per cell. Virus titers in the medium (A) and cell lysates (B) were determined by plaque assay.
FIG. 4
FIG. 4
Isolation of IMV and wrapped viruses. RK13 cells were infected at a multiplicity of 10 with the indicated recombinant viruses. From 4 to 18 h after infection, the cells were labeled with 500 μCi of a mixture of [35S]methionine and [35S]cysteine. Particles in the medium and cell lysates were concentrated by sedimentation through a sucrose cushion, applied to CsCl density gradients, and centrifuged. The amounts of radioactive material in the fractions were determined by scintillation counting.
FIG. 5
FIG. 5
Immunoelectron microscopy of infected cells. RK13 cells were infected with 10 PFU of vB5R-GFP (A), vB5R-GFP/ΔA36R (B), or vB5R-GFP/A36R-YdF (C) per cell. After 24 h, the cells were fixed, cryosectioned, and incubated successively with polyclonal anti-GFP antibody and 10-nm-diameter gold particles conjugated to protein A. Bar, 500 nm.
FIG. 6
FIG. 6
Scanning electron microscopy of infected cells. RK13 cells were infected with 10 PFU of vB5R-GFP (A), vB5R-GFP/ΔA36R (B), or vB5R-GFP/A36R-YdF (C) per cell or were uninfected (D). After 16 h, the cells were fixed, coated with gold-palladium alloy, and viewed with an Hitachi S-4700 field emission scanning electron microscope at an accelerating voltage of 3 kV. Thick arrows, virus-tipped specialized microvilli; thin arrows, slender cellular microvilli; arrowheads, virions on the cell surface. Bar, 1 μm.
FIG. 7
FIG. 7
Tyrosine phosphorylation of the A36R protein. Infected or mock-infected HeLa cells were incubated for 18 h, harvested, and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with a P-Tyr-specific MAb (αP-Tyr). After detection of P-Tyr, the blot was stripped and reprobed with polyclonal antibody to the A36R protein (αA36R). The arrow marks the position of the Tyr-phosphorylated A36R protein. Positions and masses (in kilodaltons) of marker proteins are shown on the left.
FIG. 8
FIG. 8
Visualization of actin tails by confocal microscopy. (A to C) HeLa cells were infected with vB5R-GFP (A), vB5R-GFP/ΔA36R (B), or vB5R-GFP/A36R-YdF (C) and stained with rhodamine-phalloidin (red) and DAPI (blue) to visualize F-actin and DNA, respectively. Green is GFP fluorescence. The boxed area in the lower left of panel A is enlarged in the upper right in order to more clearly see actin tails. (D) The percentages of infected cells that contained one or more actin tails were determined.
FIG. 9
FIG. 9
Visualization of IEV by digital video microscopy. HeLa cells were infected with 0.2 PFU of vB5R-GFP/A36R-YdF per cell. After 12 h, images were collected at one frame per second. Selected frames are shown, with the cumulative time elapsed (in seconds) indicated in the upper left corner. The lettered arrows in each frame point to the same IEV particles moving downwards in subsequent frames. Note the intense fluorescence, at the bottom of each panel, where IEV accumulate at the vertex of the cell. Bar, 5 μm. The entire time-lapse video is provided as Supplemental Material no. 2 at http://www.niaid.nih.gov/dir/labs/lvd/moss.htm.
FIG. 10
FIG. 10
Association of extracellular virus with actin tails. HeLa cells, which stably express GFP-actin (green), were infected with wild-type vaccinia virus. Unpermeabilized cells were stained with MAb 19C2 to the B5R membrane protein followed by Cy5-conjugated donkey anti-rat antibody (red) and Hoechst dye to stain DNA (blue). Entire cells were imaged by confocal microscopy as a series of optical sections. Starting from the bottom of one cell, every four sequential sections (1 to 4, 5 to 8, 9 to 12, and 13 to 16) were summed, and the set of four maximum-intensity projections is shown. The extracellular virions appear red at the tips of green actin tails and yellow if overlying an actin tail.
FIG. 11
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.
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