Characterization of a Nuclear Localization Signal of Canine Parvovirus Capsid Proteins

Abstract

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...

Classic Nuclear Localization Signals and a Novel Nuclear Localization Motif Are Required for Nuclear Transport of Porcine Parvovirus Capsid Proteins

“…4). These results showed that PPV minor capsid protein contained one more active NLS than the VP1up of the closely related parvoviruses MVM and CPV (32,33). Transfection of different mutant fusion proteins always resulted in some cells where the protein was present in both the cytoplasm and the nucleus (Fig.…”

“…Several parvoviruses are known to contain active NLSs in the unique region of VP1 (32,33). Sequence analysis of PPV VP1up revealed five potential NLSs.…”

“…For some viruses, these regions can be found in all structural proteins (for example, adeno-associated virus type 2 [AAV-2]) (31), while for others, such as minute virus of mice (MVM) (32) and canine parvovirus (CPV) (33), active NLSs are found only in the VP1 minor protein. Sequence analysis of PPV showed that there are five BRs that could potentially act as NLSs; all of them are located in VP1up (Fig.…”

RanBP3 Contains an Unusual Nuclear Localization Signal That Is Imported Preferentially by Importin-α3

“…However, mutational analysis has shown that the loss of any one of the basic residues, except the last, resulted in cytoplasmic localization, again suggesting that the basic residues are more central to VP1 NLS function than is the case for the RanBP3 NLS (77). The receptor for VP1 transport is unknown.…”

Pathways of Cell Infection by Parvoviruses and Adeno-Associated Viruses

“…Only two basic sequences that might be classical NLSs are present in the VP2 capsid protein sequence of CPV, while the VP1-unique region contains several such sequences. A VP1 sequence (PAKRARRGYK) between residues at amino acid positions 4 to 13 functions for nuclear transport when conjugated to bovine serum albumin (77). That N-terminal unique sequence was detected on capsids between 1 and 4 h after injection of the capsids into cells, and it was also exposed on incoming infectious virions, since antibodies specific for the VP1-unique region blocked infection when injected into cells before inoculation of the virus (78).…”