The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.

Recent studies demonstrated the ability of the recombinant autonomous parvoviruses MVMp (fibrotropic variant of the minute virus of mice) and H-1 to transduce therapeutic genes in tumor cells. However, recombinant vector stocks are contaminated by replication-competent viruses (RCVs) generated during the production procedure. To reduce the levels of RCVs, chimeric recombinant vector genomes were designed by replacing the right-hand region of H-1 virus DNA with that of the closely related MVMp virus DNA and conversely. Recombinant H-1 and MVMp virus pseudotypes were also produced with this aim. In both cases, the levels of RCVs contaminating the virus stocks were considerably reduced (virus was not detected in pseudotyped virus stocks, even after two amplification steps), while the yields of vector viruses produced were not affected. H-1 virus could be distinguished from MVMp virus by its restriction in mouse cells at an early stage of infection prior to detectable viral DNA replication and gene expression. The analysis of the composite viruses showed that this restriction could be assigned to a specific genomic determinant(s). Unlike MVMp virus, H-1 virus capsids were found to be a major determinant of the greater permissiveness of various human cell lines for this virus.

The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus , showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors.