A novel heterogeneous nuclear ribonucleoprotein-like protein interacts with NS1 of the minute virus of mice.

NS1, the major nonstructural parvovirus protein of the minute virus of mice, is a multifunctional protein responsible for several aspects of viral replication. NS1 transactivates the P38 promoter (used to express the structural proteins), as well as its own strong promoter, P4. To study the mechanism of activation and to map regions of NS1 responsible for transactivation, NS1 and various deletions of NS1 were cloned in frame with the GAL4DB and cotransfected into COS-7 and LA9 cells with a synthetic GAL4-responsive reporter plasmid. These studies showed NS1 can directly activate transcription through its 129 carboxyl-terminal amino acid residues. Any deletion from this region of the C terminus, even as few as 8 amino acids, completely abolishes transactivation. A yeast two-hybrid system used to identify protein-protein interactions demonstrated that NS1 is able to dimerize when expressed in yeast cells. However, only an almost complete NS11-638 bait was able to interact with the full-length NS1. A two-hybrid screen identified a HeLa cell cDNA clone (NS1-associated protein 1 [NSAP1]) that interacts with NS11-276 and NS11-638. An additional sequence was predicted from human EST (expressed sequence tag) data, and the cDNA was estimated to be at least 2,221 bp long, potentially encoding a 562-amino-acid protein product. A polyclonal antibody raised to a synthetic peptide within NSAP1 recognizes an approximately 65-kDa cellular protein. This NSAP1 cDNA has not previously been characterized, but the predicted protein sequence is 80% identical to the recently identified heterogeneous nuclear ribonucleoprotein (hnRNP) R (W. Hassfeld et al., Nucleic Acids Res. 26:439-445, 1998). NSAP1 contains four ribonucleoprotein domains, as well as a highly repetitive C-terminal region. A closely related mouse cDNA (deduced from murine EST data) encodes a protein with only a single amino acid residue change from the human protein. NSAP1 is predicted to be a 65-kDa polynucleotide binding protein, and it likely functions in the regulation of splicing and/or transport of mRNAs from the nucleus.