The NS2 polypeptide of parvovirus MVM is required for capsid assembly in murine cells.
Mutants of minute virus of mice (MVM) which express truncated forms of the NS2 polypeptide are known to exhibit a host range defect, replicating productively in transformed human cells but not in cells from their normal murine host. To explore this deficiency we generated viruses with translation termination codons at various positions in the second exon of NS2. In human cells these mutants were viable, but showed a late defect in progeny virion release which put them at a selective disadvantage compared to the wildtype. In murine cells, however, duplex viral DNA amplification was reduced to 5% of wildtype levels and single-strand DNA synthesis was undetectable. These deficiencies could not be attributed to a failure to initiate infection or to a generalized defect in viral gene expression, since the viral replicator protein NS1 was expressed to normal or elevated levels early in infection. In contrast, truncated NS2 gene products failed to accumulate, so that each mutant exhibited a similar NS2-null phenotype. Expression of the capsid polypeptides VP1 and VP2 and their subsequent assembly into intact particles were examined in detail. Synchronized infected cell populations labeled under pulse-chase conditions were analyzed by differential immunoprecipitation of native or denatured extracts using antibodies which discriminated between intact particles and isolated polypeptide chains. These analyses showed that at early times in infection, capsid protein synthesis and stability were normal, but particle assembly was impaired. Unassembled VP proteins were retained in the cell for several hours, but as the unprocessed material accumulated, capsid protein synthesis progressively diminished, so that at later times relatively few VP molecules were synthesized. Thus in NS2-null infections of mouse cells there is a major primary defect in the folding or assembly processes required for effective capsid production.
Cotmore
S F
SF
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
D'Abramo
A M
AM
Carbonell
L F
LF
Bratton
J
J
Tattersall
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry; D001483:Base Sequence; D002213:Capsid / Q000648:ultrastructure*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051379:Mice; D008915:Minute Virus of Mice / Q000254:growth & development*; D008969:Molecular Sequence Data; D009024:Morphogenesis; D016297:Mutagenesis, Site-Directed; D013045:Species Specificity; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication
D000956:Antigens, Viral; D004279:DNA, Viral; D017361:Viral Nonstructural Proteins