Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus

Abstract

Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.

FIG. 1.

Localization of CPV capsid proteins in NLFK cells during the infection cycle. Confocal immunofluorescence images of infected cells, fixed at time points p.i., are shown in the figure. CPV was detected with polyclonal antibody to CPV capsid and with TRITC-labeled goat anti-rabbit antibody as a secondary antibody. Bar, 10 nm.

FIG. 2.

Immunoelectron microscopy analysis of CPV binding and uptake into the NLFK cells. CPV was detected with polyclonal anti-capsid antibody, which was visualized with protein A-gold conjugate (10 nm) by using the preembedding technique. Cells were fixed after adsorption at 0 min p.i. (A), 5 min p.i. (B), and 5 min p.i. (C). Bar, 100 nm.

FIG. 3.

Localization of CPV capsid proteins and caveolae during early steps of infection. (A) Immunoelectron microscopy (preembedding) at 5 min p.i. Bar, 100 nm. (B) Confocal images. Color: red, caveolae; green, CPV; yellow, colocalization. Bar, 10 μm.

FIG. 4.

Association of CPV-containing vesicle with microtubule. Immunoelectron microscopy (preembedding) analysis of CPV at 1 h p.i. was done. For technical details, see the legend to Fig. 1.

FIG. 5.

Effect of nocodazole on intracellular distribution of tubulin and on localization of CPV. The analysis was carried out by immunofluorescence microscopy at 12 h p.i. Control, without nocodazole; nocodazole, nocodazole at 60 μM. Color: red, CPV; green, microtubules. Bar, 10 μm.

FIG. 6.

Effect of microinjected anti-dynein and anti-kinesin antibody on CPV entry. The antibodies were microinjected into the cytoplasm of NLFK cells, after which the cells were inoculated with CPV. Cells were fixed at 2 h p.i. Microinjected cells are indicated by arrows. Noninjected cells serve as controls. (A) Injected antibody to dynein visualized with fluorescein isothiocyanate-conjugated anti-mouse antibody. (B) Cells injected with anti-dynein antibody, CPV detected with polyclonal anti-capsid antibody and TRITC-conjugated anti-rabbit antibody. (C) Cells microinjected with anti-kinesin antibody indicated by arrows. Injected antibody was detected as in panel B. (D) Cells injected with anti-kinesin antibody indicated by arrows. CPV was detected as described above.

FIG. 7.

Immunoelectron microscopy analysis of CPV-containing vesicles. For technical details, see the legend to Fig. 1. Bars, 100 nm. (A) Preembedding at 1 h p.i.; (B) preembedding at 3 h p.i.; (C) cryosection at 1 h p.i.; (D) cryosection at 3 h p.i.

FIG. 8.

Colocalization of full CPV capsids (H-CPV) and the organelle markers TF, MPR, and LAMP-2. The infected cells were fixed at various time points p.i. as indicated on the left. Left column: TF, green; L-CPV, red. Middle column: MPR, green; L-CPV, red. Right column: L-CPV, green; LAMP-2, red. In all columns, colocalization is yellow. Bars, 10 μm.

FIG. 9.

Localization of CPV full-capsids (H-CPV) compared to TGN-38 and to PDI. Lack of colocalization of H-CPV with trans-Golgi marker TGN-38 and/or with ER marker PDI. Time points p.i. are indicated on the left. CPV, green; organelle marker, red (TGN-38, PDI); colocalization, yellow.

FIG. 10.

Colocalization of empty CPV capsids (L-CPV) and the organelle markers TF, MPR, and LAMP-2. The infected cells were fixed at time points p.i. indicated on the left. Left column: TF, green, L-CPV red. Middle column: MPR, green, L-CPV, red. Right column: L-CPV, green, LAMP-2, red. In all columns, the colocalization is yellow. Bars, 10 μm.

FIG. 11.

Localization of CPV-DNA and capsid proteins during infection. Viral DNA was detected by fluorescence in situ hybridization, and polyclonal antibody was used to stain capsid proteins. The time points p.i. are indicated on the left.