Initiation Of Genomic RNA Replication By A Recombinant Classical Swine Fever Virus RNA-dependent RNA Polymerase

Classical swine fever virus (CSFV) is a small enveloped positive-strand RNA virus classified in the genus of Pestivirus, together with the genera Flavivirus and Hepacivirus, they form the family Flavivirdae. The genomic RNA contains a single long open reading frame (ORF) encoding a polyprotein of about 3898 amino acids which is flanked by 5' and 3' untranslated regions (UTR). This polyprotein is processed co- and/or posttranslationally into four structural proteins and eight nonstructural proteins (NS) by viral and cellular proteases. One of the nonstructural proteins, NS5B, which encodes an RNA-dependent RNA polymerase, is a key component of viral replicase responsible for the replication of viral RNA genome.The replication of CSFV genome is generally thought to be similar to other positive strand RNA viruses: synthesis of complementary minus-strand RNA with the plus-strand genomic RNA as template and subsequent synthesis of the progeny RNA with the minus-strand RNA as template. Thus, 3' end of both plus- and minus-strand RNAs may contain the cw-acting elements, such as promoter or enhancer, involved in the initiation of viral RNA synthesis by RdRp. At present, it has been shown that positive strand RNA viruses initiate RNA synthesis by either of two major mechanisms: primer-dependent RNA synthesis and primer-independent de novo RNA synthesis. Previous reports have shown that recombinant CSFV NS5B (expressed in insect cells) catalyzed RNA synthesis was strictly primer-dependent and that intramolecular priming copy-back synthesis represented the preferred mechanism for initiation of RNA synthesis. However, the activity of cellular terminal nucleotidyle transferase (TNTase) was also demonstrated to present in the cytoplasmic extracts of insect cells. Since cellular TNTase could add extras nucleotide to the 3'terminus of the RNA template and might serve as primer for template-primed copy-back synthesis, it might not represent the original initiation of viral RNA synthesis in vitro.To better understand the initiating of CSFV RNA replication and to establish an in vitro RdRp replication system, we first cloned the NS5B gene by RT-PCR from the pig serum infected with CSFV Shimen strain, and then expressed this protein in Escherichia coli BL21 (DE3). To improve the solubility of the NS5B, 24 amino acids of the C-terminal was deleted, and expression was performed at low temperature. The truncated fusion protein (NS5BA24) was purified on a Ni-chelating HisTrap affinitycolumn and demonstrated to possess RNA-dependent RNA polymerase by RT-PCRand real-time quantitative PCR.Using the plus-strand RNA (+)3'UTR and minus-strand RNA (-)IRES as templates, we performed Northern blot and RT-PCR to study the mechanism of initiating CSFV RNA synthesis by RNA-dependent RNA polymerase. The results showed that CSFV RdRp preferred to initiate either plus- or minus-strand viral RNA synthesis by a primer-independent de novo manner but not primer-dependent RNA synthesis in vitro. Besides de novo RNA synthesis, high molecular RNA products, which were produced by template-switch mechanism were also observed with the minus-strand RNA as template. Using (-)IRES as template, the optimal reaction conditions were found. Both divalent cations Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with high concentration of NTP, and a 3'-terminal cytidylate on the viral plus- and minus-strand RNA templates was preferred for de novo initiation. Mutations of the 3'-terminal cytidylate on the viral RNA templates to guanidylate, adenylate or uridylate results in dramatically decreasing RNA synthesis. These results suggested that the presence of a 3'terminus cytidylate at viral RNA templates was necessary for efficient de novo RNA synthesis by CSFV RdRp.Initiation of RNA synthesis required the initiating recognition and specific binding between RNA-dependent RNA polymerase and template RNA. T...