Structural characterization of H-1 parvovirus: comparison of infectious virions to empty capsids.

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 ß-barrel with large loop regions between the strands. The ß-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.

Production, purification, crystallization and structure determination of H-1 Parvovirus.

Crystals of H-1 Parvovirus (H-1PV), an antitumor gene-delivery vector, were obtained for DNA-containing capsids and diffracted X-rays to 2.7 Šresolution using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=255.4, b=350.4, c=271.6 Å, ß=90.34°. The unit cell contained two capsids, with one capsid per crystallographic asymmetric unit. The H-1PV structure has been determined by molecular replacement and is currently being refined.

Effect of the parvovirus H-1 non-structural protein NS1 on the tumorigenicity of human gastric cancer cells.

OBJECTIVE: To investigate the in vivo oncosuppressive effect of the non-structural protein NS1 of parvovirus H-1 on human gastric cancer cell lines. METHODS: Recombinant plasmid pcDNA3.1-NS1 containing the complete NS1 gene of parvovirus H-1 was constructed and characterized by restriction enzyme digestion and sequence analysis. The human gastric cancer cell lines MKN28, SGC7901 and MKN45 were stably transfected with empty or recombinant plasmids. NS1 gene transcription and protein expression in the latter transfectants were verified by reverse transcriptase polymerase chain reaction and Western blot, respectively. The oncosuppressive effect of the parvoviral protein NS1 on the gastric cancer cell lines was tested by comparing the tumorigenicity of empty and recombinant vector-transfected cells in nude mice. RESULTS: Well differentiated gastric cancer cells (MKN28) transfected with either empty plasmid or pcDNA3.1-NS1 were tumorigenic in nude mice. Moderately (SGC7901) and poorly (MKN45) differentiated gastric cancer cells transfected with empty plasmid were also tumorigenic, but no tumor resulted from the injection when they were transfected with pcDNA3.1-NS1. This NS1-associated suppression of SGC7901 and MKN45 tumors correlated with the decreased percentage of CD44 positive cells. CONCLUSIONS: NS1 expression in poorly differentiated gastric cancer cells prevents them from forming tumors, perhaps by impairing the stem-like phenotype. The parvoviral NS1 protein warrants further investigation for its therapeutic potential against cancer.