Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase

Abstract

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cellshas been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5B⌬CT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primerindependent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (⌬CT179 and ⌬CT218) that lacked RdRp activities.

Mutational analysis of the structure and functions of hepatitis C virus RNA–dependent RNA polymerase

“…10,11,16 The remaining mutations have not been addressed previously including the so-called fingers and thumb subdomains of NS5B. 31,48 The designed mutations of the constructs were confirmed by nucleotide sequencing.…”

Specific interaction between the classical swine fever virus NS5B protein and the viral genome

“…This mechanism has been observed in BVDV NS5B for initiation of RNA synthesis [21]. It was also found that both the copyback mechanism and the de novo mechanism for initiation of RNA synthesis might be present in the NS5B protein of CSFV and BVDV [7,13]. The fact that the CSFV NS5B has a TNTase activity, together with other data in this research and previous reports, increases this possibility that two mechanisms, copy-back and de novo, are compatible with each other in the CSFV viral genomic replication.…”

“…4B, lanes 2-4), followed by +RNA2 and +RNA3 (lanes 5-10). In contrast, +RNA4 and +RNA5 were poor competitors (lanes [11][12][13][14][15][16]. These results indicated that the CSFV NS5B protein bound the 3¢ UTR mainly through interaction with the 21 nucleotide bases at the terminal.…”

“…3A, the complex formed between the radiolabeled +RNA0 and the NS5B protein was almost completely abolished by 25-fold molar excess of the unlabeled +RNA0 (lane 5). In contrast, the +5¢ UTR, +RNAr1 and +RNAr2 had no effect on the binding (lanes [6][7][8][9][10][11][12][13][14][15]. The fact that the +RNAr1 without 3¢ UTR had no effect on the complex formed between the radiolabeled +RNA0 containing 3¢ UTR and the NS5B protein, showed that the site of interaction between them is 3¢ UTR.…”

De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

“…With sequence analysis and analysis of the hydropathy profile, we found that the C-terminal part of NS5B contains a highly hydrophobic region which is predicted to be an anchoring domain (Lindenbach and Rice, 2001). It has been reported that their deletion increases the solubility of NS5B expressed in E. coli Lai et al, 1999;Tomei et al, 2000;Yamash ita et al, 1998;Zhong et al, 2000). Therefore, the effect of the Cterminal region was examined by constructing the expression plasmids, pET-NS5B 24 and pET-NS5B 24GAA, which lacked the C-terminal 24 amino acid residues.…”