Canine parvovirus (CPV), a model virus for the study of parvoviral entry, enters host cells by receptormediated 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.

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.

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Enteroviruses induce fundamental changes in cell morphology. The molecular mechanisms of these changes and the ensuing cellular release of viral progeny are mostly unknown. It is generally assumed that enteroviruses, like many other nonenveloped viruses, require cell lysis for infection spread. For example, the release of coxsackievirus B3 (CVB3) virions from infected cells depends on enhancement of cell membrane permeability caused by viral components (53). However, the lytic escape of enteroviruses can also be complemented by nonlytic virus release, as shown for poliovirus using an autophagosomal pathway (27,49). Moreover, direct cell-to-cell spread has been suspected to occur with poliovirus in the central nervous system (40). Nonlytic transmission might be particularly important in persistent infection by enteroviruses and various other picornaviruses, such as Theiler's murine encephalomyelitis virus, foot-and-mouth disease virus, and Nora virus, a picornalike virus (17,25,41,58). Such transmission could be envisioned to provide an important advantage for the virus by helping it to hide inside the cell to avoid immune defense by neutralizing antibodies.CVB3, a nonenveloped RNA virus, is a member of the genus Enterovirus of the family Picornaviridae. It causes myocarditis and pancreatitis in newborns and may play a role in some chronic diseases, such as dilated cardiomyopathy and type 1 diabetes (4,5,26,30,42). The icosahedral viral capsids are 29 nm in diameter and consist of the structural proteins VP1, VP2, VP3, and VP4. The 7,400-nucleotide viral RNA genome is polyadenylated, single stranded, and positive sense (39), encoding 11 proteins from a single open reading frame. CVBs invade the intestinal epithelium and bind to the major cellular receptor, a glycoprotein termed the coxsackie-adenovirus receptor (CAR) (12,15,16,44,61). CVBs enter the cytoplasm by a caveolin-dependent endocytic pathway, with similarities to macropinocytosis (15,16,35). CVB3 replicates on the outer surfaces of virus-induced membranous vesicles (8,12,20,61). Viral assembly is a multistep process, in which cleavage of viral protein (VP0) yields mature capsid proteins VP4 and VP2, which, together with VP1 and VP3, form the mature capsid (6). Later in infection CVB3 induces apoptosis through caspase activation in cultured cells (11,36,60).In this study, alterations in the cellular architecture and intracellular distribution of CVB3 proteins were studied in infected cells. The cells were either inoculated with the virus or transfected with the viral RNA by lipofection or cytoplasmic microinjection. Lipofection of viral RNA was used to synchronize viral infection, and comicroinjection of the viral RNA and fluorescent dextran were used to mark the transfected cells (7,23,24,33,38). Living as well as fixed cells were examined by electron microscopy (EM), wide-field and confocal microscopy, and flow cytometry. CVB3 RNA was shown to induce extensive cellular changes, including extended protrusions connecting adjacent cells. These studies MAT...

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.