We investigated the abilities of synthetic peptides mimicking the potential nuclear localization signal of canine parvovirus (CPV) capsid proteins to translocate a carrier protein to the nucleus following microinjection into the cytoplasm of A72 cells. Possible nuclear localization sequences were chosen for synthesis from CPV capsid protein sequences (VP1, VP2) on the basis of the presence of clustered basic residues, which is a common theme in most of the previously identified targeting peptides. Nuclear targeting activity was found within the N-terminal residues 4Ϫ13 (PAKRARRGYK) of the VP1 capsid protein. While replacement of Arg10 with glycine did not affect the activity, replacement of Lys6, Arg7, or Arg9 with glycine abolished it. The targeting activity was found to residue in a cluster of basic residues, Lys5, Arg7, and Arg9. Nuclear import was saturated by excess of unlabelled peptide conjugates (showing that it was a receptor-mediated process). Transport into the nucleus was an energy-dependent and temperature-dependent process actively mediated by the nuclear pores and inhibited by wheat germ agglutinin.Keywords : nuclear location signal; canine parvovirus ; synthetic peptide; microinjection.Canine parvovirus (CPV) is a nonenveloped DNA virus of (BSA) to the nucleus, following microinjection into the cytoplasm of the A72 cell, and nuclear transport was evaluated by the autonomous Parvoviridae family that causes enteritis and myocarditis in canidae (Cotmore and Tattersall, 1987; Reed et immunofluorescence at various times after microinjection. Peptides were chosen on the basis of the presence of clustered basic al., 1988). CPV contains three structural proteins in the mature virion (VP1ϪVP3). The early phase of the CPV life cycle in-residues. The results demonstrated that the N-terminal residues 4Ϫ13 of VP1 can function as a nuclear localization signal. To volves a series of sequential events starting with the attachment of a virion to receptors on the cell surface (Basak et al., 1994). define further the identified potential NLS region of VP1, we synthesized a set of mutated peptides by changing one or two Viruses are then internalized by a pH-dependent endocytic pathway (Basak and Turner, 1992;Marsh and Helenius, 1989). At basic amino acids with glycine to scan the significance of specific amino acid residues for nuclear localization. To determine present, the mechanism involved in the release of the virus from acidified endosomes is still unknown. Upon penetration of the whether the nuclear import is a receptor-mediated process, i.e.if it can be saturated by excess of peptide conjugates, we coinvirus from endosomes into cytoplasm, the viral DNA and associated proteins are transported to the nucleus, where viral tran-jected 10-fold or 100-fold excess of unlabelled peptide conjugates with labelled peptide conjugates and monitored the nuclear scription and replication occur. It has been suggested that the uncoating process and release of viral DNA takes place mostly import. We also investigated the nucl...

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18°C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.