For the positive strand RNA viruses, the mechanisms of initiating viral RNA synthesis are rather different. Poliovirus has been shown to use a uridylylated protein as primer (protein-primed) to initiate RNA synthesis . Phage Qβ initiated de novo RNA synthesis , whereas rabbit hemorrhagic disease virus (RHDV) initiated RNA synthesis by using a template-primed copy-back mechanism . Besides copy-back synthesis, Dengue virus RdRp was also demonstrated to be capable of de novo initiation of RNA synthesis . Previous work has shown that crude extracts of recombinant CSFV NS5B expressed in insect cells could produce two RNA products using D-RNA (an mRNA of the liver-specific transcription factor DCoH) as template. One product, which was identical in size to the input RNA template, might have resulted from a TNTase activity, while the other was determined to be a double-stranded hairpin dimer RNA synthesized by a copy-back mechanism. However, our work found that the predominant RNA products were template-sized, and thus the question was whether the template-sized RNA products were generated through de novo RNA synthesis. As the template-sized RNA could be detected by complementary probes, and RNA polymerization required all four ribonucleotides as substrates (data not shown), it seemed that the template-sized RNA products probably represented RNA synthesized by a de novo initiation mechanism but should not result from a terminal transferase activity. If RNA products were synthesized by TNTase activity, it would have the same polarity as the input RNA template . To provide further evidence of de novo RNA synthesis, the 3′-hydroxyl group of RNA templates were blocked by treatment with sodium periodate. The migration patterns of RNA products are shown in Fig. 3A, indicating that the template-sized RNA products were truly synthesized by de novo initiation, but not by the 3′-end elongation copy-back synthesis. It should be pointed out that a very small amount of high molecular weight RNA products were still observed with 3′-blocked (–)IRES as template, possibly because the polymerase used the nascent RNA as template for additional rounds of RNA synthesis. Furthermore, we performed RT using strand-specific oligodeoxynucleotides as a primer that could anneal only to the 3′-terminus of either synthesized plus- or minus-strand RNA products, followed by PCR amplification. The amplified fragments were analyzed by agarose gel electrophoresis and stained by ethidium bromide. As shown in Fig. 3B, the expected sizes of DNA fragments (373 nucleotides for new synthesized plus-strand and 228 nucleotides for the minus-strand) were observed, verifying that template-sized RNA products were initiated de novo from the 3′-terminus of the template but not by premature termination or internal initiation, as suggested in reports for tomato bushy stunt virus, cucumber necrosis virus  and hepatitis C virus . Taken together, these results strongly suggest that the purified CSFV NS5BΔ24 could preferentially initiate either plus- or minus-strand viral RNA synthesis de novo in the absence of primers and viral or host factors. This result is contrary to the previous reports for CSFV RdRp , as well as BVDV RdRp , another member of the Pestivirus genus, in which the major RNA products catalyzed by RdRp were shown to be a covalently linked double-stranded molecule generated by a copy-back mechanism. At present it is unknown whether this discrepancy is caused by the various viral enzyme preparations or different templates used. In fact, reports have shown that the RdRp of BVDV could preferentially initiate RNA synthesis by a de novo initiation mechanism with chemically synthesized short RNA (21 nucleotides) as a template , although a primer extension RNA product was also observed . Therefore, de novo initiation of RNA synthesis might represent the preferred mechanism used by Pestivirus RdRps in vitro.
Figure 3. De novo initiation of viral RNA synthesis by classical swine fever virus(CSFV) RNA-dependent RNA polymerase(RdRp). Both viral plus- and minus-strand RNA templates were treated with sodium periodate to block the 3′-OH group and then used as template for RdRp assay. (A) Northern blot assay with (+)3′-UTR and (–)IRES as templates. Lane 1, RdRp assay with NS5BΔ24GAA as a control; lane 2, RdRp assay with NS5BΔ24 and 3′-blocked RNA template. (B) Synthesized RNA was subjected to RT-PCR. RT was performed using a primer complementary to the newly synthesized minus-strand (lanes 2 and 3) or plus-strand RNA (lanes 4 and 5), followed by PCR amplification. Lane 1, 100 bp DNA ladder; lanes 2 and 4, RNA template (T) used as a control; lanes 3 and 5, RT–PCR results of newly synthesized products (P); lane 6, NS5BΔ24 protein as a control. PCR products were electrophoresed through an agarose gel and visualized by ethidium bromide staining. The expected fragments were 228 nucleotides (newly synthesized minus-strand RNA) and 373 nucleotides (synthesized plus-strand RNA) in length.
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