Endonuclease Activity Of Periplaneta Fuliginosa Densovirus Nonstructural Protein NS1is Modulated By Tyrosine Phosphorylation

The non-structural protein NS1of Periplaneta fuliginosa densovirus (PfDNV) is a multifunctional protein which locates in the nucleus that has previously been shown to possess ATPase, site-specific DNA-binding, helicase, transcription activation activities and cytopathogenicity. Here, we describe the site-specific endonuclease activity and phosphorylation of NS1of PfDNV.In the beginning, we expressed and purified MBP-NS1using E. coli and analyzed its endonuclease activity. The purified recombinant NS1protein couldn’t make a nick in the substrate which was located in the ITR region of PfDNV genome. Then, we turned to Bac-to-Bac baculovirus expression system. The MBP-NS1protein was purified from Sf9cells and possessed site-specific endonuclease activity requiring ATP and either Mg2+or Mn2+as a cofactor. It could become covalently attached to the5’phosphate of a T residue at the nicking site. The cut occurred near the end of the PfDNV terminal palindrome on the strand containing the sequence5’-CTTGG/TGCTT-3’between nucleotides G and T. The NS1protein could become covalently attached to the5’phosphate of a T residue at the nicking site and it is believed that this linkage involves a tyrosine residue.Since Sf9cells produced NS1possessed site-specific endonuclease activity while bacterial produced NS1didn’t, we speculate that site-specific endonuclease activity of NS1depend on posttranslational modifications, in particular phosphorylation. We dephosphorylated the NS1expressed in a baculovirus system with Alkaline Phosphatase, Calf intestine (CIP), T-Cell Protein Tyrosine Phosphatase (TC PTP) and Lambda Protein Phosphatase (Lambda PP). Then we compared the endonuclease activity of the purified NS1(CIP), NS1(TC PTP) and NS1(Lambda PP) with the native NS1polypeptides. Biochemical analysis of NS1(CIP) and NS1(TC PTP) revealed a severe reduction of nickase activity while NS1(Lambda PP) remained part of the function. It suggests that phosphorylation of the tyrosine residues is important for site specific endonuclease activity of NS1. Genistein is a broad-spectrum tyrosine kinase inhibitor. We found that NS1proteins expressed from Sf9cells which were treated with different concentration of genistein revealed varying degrees of deficiency of endonuclease activity. It was consistent with the result of dephosphorylation assay.To identify which tyrosine residues were phosphorylated and identify the active-site tyrosine residue of NS1, tyrosine residues were site specifically mutated to phenylalanine residues by overlap extension PCR. The mutant NS1proteins were expressed in a baculovirus system, purified with amylose resin, and assayed in vitro for NSI-specific activities. Although mutation Y178F and Y345F both disrupted the endonuclease activity, only the mutation of tyrosine178abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. It suggests that Y178is the active-site tyrosine residue of endonuclease activity. Interestingly, endonuclease activity of the mutant Y86F decreased and lost site specific in some degree. In addition, we performed a tandom mass spectrometry. It turned out that tyrosine345of NS1was phosphorylated in Sf9cells. Phosphorylation of tyrosine345might be related with fold of the helicase domain, and effect helicase activity which was necessary for site-specific endonuclease activity of NS1.