Vaccinia virus infection disrupts microtubule organization and centrosome function

Abstract

We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein-dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.

Figure 1

Vaccinia virus localization in the vicinity of the MTOC depends on microtubules. HeLa cells infected with vaccinia virus in the absence (A and C) or presence of nocodazole (B and D) fixed 6 h post‐infection. Depolymerization of microtubules results in dispersed cytoplasmic viral assembly and loss of localization at the MTOC area (B and D). The microtubule cytoskeleton (A and B) and vaccinia virus (C and D) are visualized with anti‐α‐tubulin and anti‐A27L antibodies respectively. Scale bar = 10 μm.

Figure 2

Disruption of the dynein–dynactin complex results in dispersed cytoplasmic viral localization. p50/dynamitin is overexpressed in the left cell as judged by detection of the myc epitope tag (A) although the anti‐A27L antibody labelling shows that both cells are infected (B). Scale bar = 10 μm.

Figure 3

Vaccinia virions do not co‐distribute with disrupted Golgi markers. Golgi and vaccinia localization in control (A–C and J–L), nocodazole‐ (D–F and M–O) and brefeldin A‐ (G–I and P–R) treated cells. Vaccinia virus particles are visualized with the anti‐A27L antibody, while the cis‐Golgi and trans‐Golgi network are labelled with the anti‐gp27 and anti‐TGN‐46 antibodies, respectively. Scale bar = 10 μm.

Figure 4

IMV but not IEV particles assemble in the absence of microtubules. Thin section electron microscope micrographs of HeLa cells infected with vaccinia virus in the absence (A and C) or presence of nocodazole (B and D) fixed 8 h post‐infection. White arrows point to IMV particles, white arrowheads to IEVs, black arrows to IV particles, black arrowheads to IMVs in the process of wrapping with the trans‐Golgi network to become IEVs. Asterisks indicate aberrant virus particles. Scale bar = 500 nm.

Figure 5

IEV formation is microtubule and Golgi dependent. IEV particles, which are identified by co‐labelling with antibodies against A27L and the A36R IEV membrane protein (A–C), are not formed in the presence of nocodazole (D–F) or brefeldin A (G–I). Actin tails normally induced by IEV (J–L) are also absent in nocodazole‐ (M–O) or brefeldin A‐ (P–R) treated cells. Scale bar = 10 μm.

Figure 6

Vaccinia infection induces disruption of the microtubule cytoskeleton and the Golgi apparatus. Vaccinia‐infected HeLa cells labelled 6 h post‐infection with anti‐gp27 and anti‐α‐tubulin antibodies to visualize the Golgi apparatus (A and B) and the microtubule cytoskeleton (C and D), respectively. The Golgi apparatus is dispersed in infected cells whose microtubule cytoskeleton is also disrupted (B, D) but not in cells with normal microtubule morphology (A, C). Scale bar = 10 μm.

Figure 7

Vaccinia virus infection induces severe changes in microtubule organization. Examples of the four different classes of microtubule cytoskeleton morphologies observed in infected cells are shown (B, E, H and K) together with their corresponding actin cytoskeletons (A, D, G and J). Quantification of the relative amounts of these different microtubule cytoskeleton morphologies at 5, 8 and 12 h post‐infection is indicated (C, F, I and L). Scale bar = 10 μm.

Figure 8

Vaccinia virus infection stabilizes the microtubule cytoskeleton. In uninfected cells, microtubules are depolymerized by treatment with 10 μM nocodazole for 1 h (A and B) or by cold treatment for 1 h (E and F) while in vaccinia virus‐infected cells a subpopulation of microtubules is resistant to nocodazole (C and D) or cold (G and H) depolymerization. Scale bar = 10 μm.

Figure 9

Vaccinia encodes proteins that co‐sediment with microtubules. Analysis of pellets from in vitro microtubule co‐sedimentation assays performed with protein extracts from vaccinia‐infected (inf.) and uninfected (uninf.) cells. Twice the amount of pellet has been loaded in control assays performed in the absence of microtubules (nocodazole or 4°C). Proteins co‐sedimenting with microtubules that were only present in extracts from infected cells are indicated by an asterisk. The identity of proteins determined by in‐gel proteolysis MALDI mass spectrometry is indicated (arrowheads).

Figure 10

A10L and L4R associate with a subset of microtubules in infected cells. HeLa cells 24 h post‐infection with vaccinia virus are labelled with anti‐A10L (A and E) or anti‐L4R (C and G) and anti‐α‐tubulin (B and D) or anti‐acetylated α‐tubulin (F and H). Scale bar = 10 μm.

Figure 11

Vaccinia cores bind directly to microtubules in vitro. Purified viral cores labelled by DAPI (green) bind to rhodamine‐labelled micro tubules (red) in the absence of fixation (A). Binding to microtubules is not observed if cores are pre‐treated with protease (B) or pre‐incubated with antibodies against the A10L (C) or L4R (D) proteins. In contrast, pre‐incubation of purified viral cores with control IgG (E) or antibody against the A3L protein (F) does not inhibit their interaction with microtubules. Scale bar = 5 μm.

Figure 12

Vaccinia infection dramatically reduces levels of centrosomal components. Immunofluorescent γ‐tubulin labelling of centrosomes in uninfected control cells (A and B) and in cells 2 h post‐infection with vaccinia (C and D) at similar stages of the cell cycle. All images from the same experiment were collected with identical camera settings, to allow comparison of fluorescence intensity. Inserts show the corresponding images with a 5‐fold increase in brightness and 3‐fold decrease in midtones, to facilitate visualization of the weak γ‐tubulin centrosomal labelling in infected cells. The effects of a 2 h vaccinia infection on centrosomal levels of pericentrin (E and F), C‐Nap 1 (G and H), Nek 2 (I and J) and centrin (K and L) are also shown. Arrowheads indicate the position of weakly labelled centrosomes in infected cells. Scale bar = 10 μm.

Figure 13

Vaccinia infection reduces centrosome microtubule nucleation efficiency. In uninfected cells, microtubules (A, E and I) nucleate from centrosomes (B, F and J) after nocodazole washout for the times indicated. In contrast, 2 h after infection with vaccinia, microtubules (C, G and K) are nucleated inefficiently from centrosomes (D, H and L). All images were collected with identical camera settings, to allow comparison of fluorescence intensity between centrosomes. Inserts (B, D, F, H, J and L) are adjusted as in Figure 12 to facilitate visualization of the weak γ‐tubulin centrosomal labelling. Arrowheads indicate the position of the centrosome. Scale bar = 10 μm.