Exploitation of microtubule cytoskeleton and dynein during parvoviral traffic toward the nucleus

Abstract

Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptor-mediated endocytosis, escapes from endosomal vesicles to the cytosol, and then replicates in the nucleus. We examined the role of the microtubule (MT)-mediated cytoplasmic trafficking of viral particles toward the nucleus. Immunofluorescence and immunoelectron microscopy showed that capsids were transported through the cytoplasm into the nucleus after cytoplasmic microinjection but that in the presence of MT-depolymerizing agents, viral capsids were unable to reach the nucleus. The nuclear accumulation of capsids was also reduced by microinjection of an anti-dynein antibody. Moreover, electron microscopy and light microscopy experiments demonstrated that viral capsids associate with tubulin and dynein in vitro. Coprecipitation studies indicated that viral capsids interact with dynein. When the cytoplasmic transport process was studied in living cells by microinjecting fluorescently labeled capsids into the cytoplasm of cells containing fluorescent tubulin, capsids were found in close contact with MTs. These results suggest that intact MTs and the motor protein dynein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the nucleus.

FIG. 1.

Intracellular localization of CPV capsids 10 h after injection into cytoplasm of cells in the presence or absence of drugs affecting the MT cytoskeleton. (A) Localization of capsids after cytoplasmic injection. Bars, 10 μm. (B) Percentages of cells injected with capsids showing substantial nuclear localization (n, ≈300). All experiments were performed in the presence of 0.2 mM cycloheximide, and 2.5 mg of virus/ml was used for injections. The injected capsids were detected with a specific rabbit anti-capsid IgG followed by Alexa-488-labeled anti-rabbit IgG (green), and MTs were detected with an antibody against tubulin followed by an Alexa-546-conjugated anti-mouse antibody (red).

FIG. 2.

Intracellular expression of the viral NS1 protein in infected cells or in cells microinjected with capsids. Expression of NS1 was detected with a TxR-labeled MAb against NS1 protein. (A and B) Percentage of cells showing substantial NS1 expression 10 h after viral inoculation (n, ≈300) (B) or cytoplasmic microinjection (A) with CPV capsids (n, ≈300) in the presence or absence of MT-affecting drugs. (C) Kinetics of NS1 expression in cells inoculated with virus or microinjected with capsids. The percentage of cells showing detectable amounts of NS1 protein in the nucleus or cytoplasm was determined at various times between 0 and 12 h (n, ≈300).

FIG. 3.

Intracellular localization of CPV capsids microinjected into cytoplasm along with antibodies against the motor protein dynein or kinesin. The cells were incubated for 6 h after injection, after which they were fixed and stained for capsids with rabbit polyclonal anti-capsid IgG followed by Alexa-546-conjugated goat anti-rabbit IgG (red). The presence of the injected anti-dynein MAb, the anti-kinesin MAb, and control mouse IgG was detected using Alexa-488-conjugated goat anti-mouse IgG (green).

FIG. 4.

Intracellular distribution of CPV capsids in cytoplasmically microinjected or in normally infected cells. Capsids were detected by use of a preembedding immunolabeling technique where labeling with MAb 8, an antibody generated against intact capsids, was followed by silver-enhanced nanogold (1.4-nm-diameter particles) and gold toning treatments. (A) Cells injected with capsids (2.5 mg/ml) and then incubated for 2 h. (B) Close-up shows intranuclear capsids (arrowheads) and a nuclear membrane with a viral capsid attached to it (arrow). (C) In infected cells fixed at 10 h postinfection, viral capsids were detected on the nuclear membrane (arrowheads). (D) Infected cells fixed at 12 h postinfection showed cytoplasmic localization of capsids in addition to trace amount of nuclear capsids (arrowheads). All experiments were performed in the presence of cycloheximide (0.2 mM). Bars, 200 nm.

FIG. 5.

In vitro binding of CPV capsids to MTs. Shown is the interaction of OG-labeled purified viral capsids with TxR-labeled MTs stabilized with taxol in the presence or in the absence of PNS.

FIG. 6.

Interaction between the CPV viral capsids, MTs, and dynein in vitro. (A) EM image of negatively stained preparation containing taxol-stabilized MTs (arrowhead) and purified capsids (arrows). (B) Immunogold-labeled viral capsids (5-nm-diameter gold particles) containing the intermediate chain of dynein (10-nm-diameter gold particles) (arrowheads) bound to MT. Bars, 50 nm.

FIG. 7.

Localization of CPV capsids in living cells. Cells were microinjected with rhodamine-labeled tubulin and incubated for 4 h at 37°C to label MTs; then they were injected with OG-labeled capsids and incubated for 2 h at 37°C. Cells were visualized with LSM without fixation.

FIG. 8.

Coimmunoprecipitation of CPV capsids with an anti-dynein antibody. Viruses concentrated from cells infected with CPV (lane 2) and a cell extract from noninfected cells (lane 3) were immunoprecipitated with MAbs against dynein. A control precipitation (lane 1) was performed with a MAb against trout Ig, and all samples were immunoblotted with an antibody against viral capsid protein. The migration positions of CPV capsid proteins VP1 (M_r, 83,000) and VP2 (M_r, 67,000) are shown in lane 4.