Identification And Characterization Of Dendrolimus Punctatus Tetravirus Helicase Domain

Dendrolimus punctatus tetravirus (DpTV) genome RNA1has a large ORF encoding the viral replicase of180kDa, which contains three putative domains indicative of methyltransferase (MT), RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp). Based on sequence comparisons, the putative helicase domain of DpTV contains all of the seven conserved segments typical of the SF-1helicase. So far there is no report about the RNA helicase in Tetraviridae. Here, we describe the biochemical properties of DpTV helicase for the first time.Firstly, a cDNA fragment of the putative DpTV helicase domain (amino acids523to1042of RNA1ORF) was prepared from the extract DpTV genome RNA by reverse transcription-PCR. For expression in Rosetta (DE3) pLysS, the DNA fragment was inserted into the BamHI/HindⅢ sites of vector pET-32a plasmid. The putative DpTV helicase domain was expressed with a6X His tag at its N-terminus (His-Hel) and then purified by Ni-NTA affinity chromatography for producing rabbit polyclonal antibody. Affinity and specificity of the antibody were tested by Western-blot analysis.Because the putative DpTV helicase domain expressed in Rosetta (DE3) pLysS was insoluble and had no activity, we next expressed the MBP tagged DpTV helicase domain (MBP-Hel) using the Bac-to-Bac baculovirus expression system and purified it using amylose affinity chromatography. In order to confirm the significance of the conversed motifs in helicase, we also made three conservative substitutions, K606E, DE670AA and TR830AA within the putative helicase motifs Ⅰ, Ⅱ and VI by standard PCR-mediated site-directed mutagenesis. Three mutants were expressed and purified as MBP-Hel.The biochemical characterization of DpTV MBP-Hel and its mutants revealed that MBP-Hel exhibited efficient hydrolysis of ATP in the absence of nucleic acids. The ATPase activity of MBP-Hel could be stimulated by ssRNA and dsRNA. Unexpectedly, ssDNA and dsDNA almost had no effect on the ATPase activity of MBP-Hel. The mutants did not show any ATPase activities in the absence of nucleic acids or in the presence of ssDNA or dsDNA, indicating the importance of conserved amino acids in motifs; however the presence of ssRNA or dsRNA recovered the ATPase activities of these mutants. In addition, MBP-Hel exhibited efficient hydrolysis of all dNTPs and NTPs in the order of dTTP> CTP> UTP> dGTP> ATP> GTP> dATP> dCTP.Next we investigated the nucleic acid-binding characteristics of MBP-Hel and its mutants. For dsDNA and ssDNA, mobility-shifted bands were observed with MBP-Hel and its mutants, and MBP-Hel showed higher affinity for dsDNA and ssDNA compared with its mutants. The shifted bands were all ATP-independent. However, MBP-Hel and its mutants had the same weak affinity for dsRNA, and the formation of RNA-protein complex band was also ATP-independent. A weak mobility-shifted band was observed with MBP-Hel and ssRNA, and the shifted band was ATP-dependent. No ssRNA binding property was observed with MBP-Hel mutants.Helicases utilize the energy derived from nucleoside triphosphate hydrolysis to translocate along nucleic acid strands and separate the helical structure of double-stranded nucleic acid. The biochemical characterization of MBP-Hel RNA and DNA duplex-unwinding activities indicated that MBP-Hel was a5’to3’helicase and it was only able to unwind oligonucleotides from an RNA template. For effective unwinding of MBP-Hel, it must be supplied with more than three un-base-paired ribonucleotides on the5’single-stranded region of the template strand. All NTPs and dNTPs tested were able to be utilized as energy materials for MBP-Hel RNA duplex-unwinding in the order of dGTP> dCTP> dATP> ATP> GTP> dTTP> CTP> UTP. The MBP-Hel mutants still retained weak RNA duplex-unwinding activities.Taken together, it seems that MBP-Hel, a functional helicase, has some properties distinct from other RNA virus helicase. In order to definitely understand the specific cellular functions of MBP-Hel, our future experiments will focus on the structure study of MBP-Hel and its relationship with other virus and host factors.