Expression and Characterization of RNA-Dependent RNA Polymerase of Dendrolimus Punctatus Tetravirus
Total Page：16
File Type：pdf, Size：1020Kb
Journal of Biochemistry and Molecular Biology, Vol. 39, No. 5, September 2006, pp. 571-577 Expression and Characterization of RNA-dependent RNA Polymerase of Dendrolimus punctatus Tetravirus Liang Zhou, Jiamin Zhang, Xiaochun Wang, Hong Jiang, Fuming Yi and Yuanyang Hu* State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, P R China Received 19 February 2006, Accepted 19 May 2006 Dendrolimus punctatus tetravirus (DpTV) has been identified is divided into the genera Betatetravirus and Omegatetravirus as a new member of the genus Omegatetravirus of the on the basis of capsid appearance and genome organization family Tetraviridae that may be related serologically to (Hendry et al., 1995; Hanzlik and Gordon, 1997; Pringle et Nudaurelia capensis virus (NωV). To establish the function al., 1999, 2003). of DpTV RNA genome and to better understand the Betatetraviruses, such as Nudaurelia capensis β virus mechanism of viral replication, the putative RNA- (NβV), Thosea asigna virus (TaV) and Providence virus dependent RNA polymerase (RdRp) domain has been (PrV), have a monopartite genome with both the replicase and cloned and expressed in Escherichia coli. The recombinant capsid precursor genes on an RNA molecule of ~6.5 kb. The protein was purified on a Ni-chelating HisTrap affinity capsid precursor gene is also present on a subgenomic RNA column and demonstrated to initiate viral RNA synthesis molecule of ~2.5 kb (Gordon et al., 1999; Pringle et al., 1999, in a primer-independent manner but not by terminal 2003). Omegatetraviruses, such as Nudaurelia capensis ω nucleotidyle transferase activity in the presence of Mg2+ virus (NωV) and Helicoverpa armigera stunt virus (HaSV), and RNA template. Mutation of the GDD to GAA have a bipartite genome with the replicase and capsid precursor interferes with the residues at the polymerase active site genes on the ~5.3 and ~2.5 kb RNA molecules, respectively and metal ions, and thus renders the polymerase inactive. (Agrawal and Johnson, 1992; Hanzlik et al., 1993, 1995; Gordon et al., 1995). Capsids of both genera contain two Keywords: Dendrolimus punctatus tetravirus, RNA-dependent proteins: a major protein of ~60 kDa and a minor protein of RNA polymerase, RNA synthesis ~7 kDa. Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia Introduction capensis ω virus (NωV). DpTV particles were isolated from diseased Dendrolimus punctatus, a member of the family The family Tetraviridae comprises small RNA viruses whose Lasiocampidae, collected from Yunnan Province, China. Viral host range is restricted to lepidopteran insects (butterflies and particles are isometric, with a diameter of about 40 nm and a moths) (Hanzlik and Gordon, 1997). The Eighth edition of the buoyant density of 1.281 g cm−3 in CsCl. The virus has two ICTV taxonomy of viruses indicates a different status of capsid proteins (of 62.5 and 6.8 kDa) and two single–stranded tetravirus taxonomy (Fauquet et al., 2004). Viruses in this RNA molecules (RNA1 and RNA2), which are 5492 and family have single-stranded, positive-sense RNA genomes 2490 nt long, respectively. RNA1 (Genbank, AY594352) has encased in unenveloped, icosahedral virions that are 35~41 a large open reading frame (ORF) (nt 37-4986) encoding a nm in diameter, with equilibrium densities in CsCl of <1.30 g polypeptide of 180 kDa; RNA2 (Genbank, AY594353) contains cm−3. The family now comprises 11 confirmed members, two partially overlapping ORFs encoding polypeptides of 17 including two viruses whose complete nucleotide sequences and 70 kDa. The 180 kDa protein, which contains consensus have been reported and show different genomic organization, motifs of a putative methyltransferase, helicase and RNA- and eight unassigned possible members. The family Tetraviridae dependent RNA polymerase, shows significant similarity to those of other tetraviruses (Yi et al., 2005). *To whom correspondence should be addressed. To establish the functions of 180 kDa protein and better Tel: 86-27-68756654; Fax: 86-27-68754921 understand the replication strategy, the RdRp domain predicted E-mail: [email protected] by sequence analysis (NCBI) has been cloned and expressed 572 Liang Zhou et al. solublly in Escherichia coli as a recombinant protein. The NS3Rev. After digestion with BamH/Hind, the mutant fragment three domains were named NS1 (methyltransferase), NS2 was subcloned into vector pET-28a. Transformants were analyzed (helicase), NS3 (RNA-dependent RNA polymerase). Then the by restriction enzyme mapping and confirmed by the dideoxy fusion protein was purified and demonstrated to have the sequencing method. ability to initiate RNA synthesis in a primer-independent manner. This work will be useful for the later study on the Expression and purification of recombinant DpTV NS3. E. coli replication strategy of virus. BL21 (DE3), transformed with either pET-NS3 or pET-NS3GAA, was grown in Luria-Bertani (LB) medium, at 37oC, to an attenuance (D) at 600 nm of ≈1.0. Then the temperature was lowed to 30oC, Materials and Methods and protein expression was induced for 4 h by the addition of 1 mM isopropyl thio-β-D-galactoside (IPTG). The cell pellet obtained from 500 ml of culture was resuspended in 20 ml of binding buffer Virus purification and the extract of viral RNA. Frozen insect containing 50 mM sodium phosphate, pH 8.0, 500 mM NaCl, larve (200 g) were thawed and then were put into liquefacient 30 mM imidazole, 10 mM β-mercaptoethanol, 10% glycerol, 1% nitrogen. The frozen particles were triturated with pestle and then Triton-X-100, 1 mM phenylmethanesulfonyl fluoride, 10 µg ml−1 homogenized in 500 ml extration buffer (0.05 M Tris/HCl, 1 mM lysozyme. Then 20 µl of DNase (Takara) was added to the EDTA, pH 7.5, 0.2% 2-mercaptoethanol). The homogenate was suspension for 30 min on ice. The lysates were sonicated on ice to clarified by centrifugation at 10,000 g in a Biofuge Stratos rotor for reduce viscosity, and any insoluble materials were removed by 30 min. The supernatant was precipitated by centrifuging at centrifugation at 13,000 g for 30 min. The clear supernatant was 100,000 g for 2 h in a Beckman Coulter Ti70 rotor. Virus pellets applied to a 2 ml Ni-chelating HisTrap affinity column (Novagen) were resuspended overnight at 4oC in TE buffer (0.05 M Tris/HCl, preequilibrated with the binding buffer. The bound protein was then 1 mM EDTA, pH 7.5). The resuspended virus was centrifuged washed by binding buffer with 50 mM imidazole and eluted with through a 30% (w/v) sucrose cushion at 100,000 g for 3 h. Virus elution buffer (50 mM sodium phosphate, pH 8.0, 500 mM NaCl, pellets were resuspended again, then layered on a 10-40% (w/v) 5mM β-mercaptoethanol, 10% glycerol) containing 300 mM sucrose gradient and centrifuged at 100,000 g for 2 h in a Beckman imidazole. The fractions were monitored by SDS-PAGE and Coulter Ti40 rotor. The band containing virus particles was staining with Coomassie Brilliant Blue R250, then, the His-tagged collected, diluted with TE buffer and pelleted at 100,000 g for 3 h. protein peaks were collected and dialyzed against buffer (20 mM Virus pellets were resuspended in 250 µl TE buffer and frozen for Tris/HCl, pH8.0, 500 mM NaCl and 20% glycerol), followed by long-term storage. For further purification, virus in TE buffer was storage at –80oC in small aliquots. The concentration of purified subjected to a CsCl density gradients according to Scotti (1985). protein was determined by the Bradford method using BSA as a The purity and integrity of the purified virus were verified by standard. negative staining with 2% (w/v) phosphotungstic acid (PTA, pH7.0) and examined under a Hitachi H-8100 transmission electron SDS/PAGE and western blot. Protein fractions from the HisTrap microscope. affinity column were separated by 12% SDS/PAGE and Viral RNA was extracted from capsids by using TRIzol reagent elecrotransferred to a PVDF membrane. The membrane was according to the manufacturer’s instructions (Invitrogen). blocked with 3% BSA in TBS buffer (pH 7.5) and treated with rabbit anti-His. Alkaline phosphatase (ALP)-conjugated goat anti- cDNA synthesis and plasmid constructions. Open reading frames (rabbit IgG) was used as the secondary antibody. After washing (ORFs) and RdRp domain (NS3) were identified by using BLAST three times with TBS buffer containing 0.1% Tween-20, membrane- (htttp://www.ncbi.nlm.nih.gov/BLAST/). The conserved sequences bounded antibodies were detected with Nitro Blue tetrazolium/5- were aligned by using CLUSTAL_W version 1.8. With the result of bromo-4-chloroindol-2-yl phosphate. sequence analysis, the former and reverse primers of NS3 were designed. cDNA fragments of NS3 complementary to DpTV RNA Preparation of RNA template and RNA labeling. RNA templates were synthesized in the presence of primier NS3Rev, dNTPs and were prepared by in vitro transcription using T7 Polymerase ThermoScript reverse transcriptase (Invitrogen) as recommended according to the manufacturer’s instruction (Promega). The DNA by the manufacturer. Then the NS3 fragment was PCR amplified fragments of 278 nt (+) 5'-UTR of viral RNA2 were used as with the following primer pair - NS3For and NS3Rev - from transcription templates. The products were further purified and cDNA. After purification, the PCR products were digested with blocked at the 3-hydroxyl group as described previously (Yi et al., BamH and Hind, and then inserted into the BamH/Hind sites of 2003). vector pET-28a (Novagen). The resulting expression vector, pET- For preparation of digoxin (DIG)-labeled RNA probes NS3, which was driven by the T7 RNA polymerase promoter, was complementary to the synthesized RNAs, the DNA fragments of transformed into E.