Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1.
The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins.
Yang
Bo
B
State Key Laboratory of Virology, Department of Biotechnology, Wuhan University, Wuhan 430072, China.
Zhang
Jiamin
J
Cai
Dawei
D
Li
Doulin
D
Chen
Wuguo
W
Jiang
Hong
H
Hu
Yuanyang
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D017990:Densovirus / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010526:Periplaneta / Q000821:virology*; D011485:Protein Binding; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
D004279:DNA, Viral; D017361:Viral Nonstructural Proteins