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 cells has 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, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (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 (DeltaCT179 and DeltaCT218) that lacked RdRp activities.

FIG. 1

(A) Hydropathy profile comparison between BVDV and HCV NS5B proteins. Both NS5Bs contained a highly hydrophobic region at the C terminus. (B) Motif organization of BVDV NS5B depicting the positions of six conserved motifs (nc, A, B, C, D, and cc) as well as the C-terminal hydrophobic domain (solid bar containing 24 amino acids). The position of each motif is labeled with a number according to its amino acid position in HCV NS5B. (C) Motif alignments among various members of the Flaviviridae family. Amino acids which are 100% conserved are in bold type with a larger font. A short dash represents an identical amino acid compared to the lead sequence derived from HCV-1b BK strain. Motif cc is likely to be motif E, based on secondary structure prediction. The overall homology between viruses of different genera is rather poor, about 20%. CSFV, classical swine fever virus; HGV, hepatitis G virus.

FIG. 2

(A) Expression and purification of the full-length and C-terminally truncated BVDV NS5Bs. Lanes 2 to 5 contain samples from the full-length NS5B-His; lanes 6 to 10 represent those from the C-terminally truncated NS5BΔCT24-His. Lane 1, molecular weight (mw) markers; lanes 2 and 6, total cell (TC) lysates; lanes 3 and 7, soluble fractions of the cell lysates; lanes 4 and 8, flowthrough (FT) unbound fractions; lanes 5 and 9, eluate (E) from Ni-NTA column; lane 10, the purified protein after passage through a gel filtration column. (B) Western blot analysis of protein expression levels in total cell lysates from full-length (NS5B-His; lane 2) and C-terminally truncated (NS5BΔCT24-His; lane 3) NS5Bs.

FIG. 3

Optimization of assay conditions for RNA synthesis by the BVDV NS5B. The C-terminally truncated NS5B, NS5BΔCT24-His (50 nM), was used in the optimization experiments. Poly(C) was the template, and oligo(G) was used as a primer. (A) Effect of temperatures; (B) effect of pH; (C) effect of glycerol; (D) effect of a zwitterionic detergent, CHAPS.

FIG. 4

Effects of salts on BVDV NS5B RdRp activity. (A) Effects of different concentrations of NaCl; (B) effects of different concentrations of divalent salts, MnCl₂ and MgCl₂; (C) inhibition of NS5B RNA synthesis by Zn²⁺ ions.

FIG. 5

RNA templates preferred by the BVDV NS5B for RNA synthesis and binding. (A) Template preference for RNA synthesis. Four RNA template-primer pairs, poly(U)-oligo(dA), poly(A)-oligo(dT), poly(C)-oligo(G), and poly(G)-oligo(dC), were used to measure primer-dependent RNA synthesis by SPA. (B) RNA bandshift assay to measure the relative affinities of different RNAs bound by BVDV NS5B.

FIG. 6

RNA synthesis from circular single-stranded MP19 templates, using the wild-type NS5BΔCT24-His. All reactions were performed as described in Materials and Methods for de novo synthesis. (A) Comparison of RdRp products synthesized from double-stranded pUC18 and single-stranded MP19 templates. (B) Effects on RNA synthesis upon addition of 100 μM actinomycin D (ActD), 100 μM rifampin (Rif), 300 μM novobiocin (Nov), 600 ng of poly(U), or 600 ng of poly(C) to 40 μl of RdRp reaction. Lane ø, minus-template control; lane 2, RdRp reaction mixture from which ATP and UTP were omitted. The total RNA products were quantified by using a PhosphorImager, and the relative percentage of synthesis normalized to the wild-type level is shown at the bottom.

FIG. 7

Schematic diagram depicting all alanine substitutions and deletion mutations in BVDV NS5B. Underlined letters represent residues that were changed to alanine by site-directed mutagenesis (vertical arrows); numbers next to the letters represent the positions of these amino acids in unmodified NS5B. Horizontal arrows represent deletions at either the N- or C-terminal end of NS5B.

FIG. 8

Effects of various alanine substitutions and deletions on de novo RNA synthesis. The results shown are representative; each was reproduced a minimal of three independent times with two repetitions. The product of de novo RNA synthesis is 21 bases. Lane ø represents reaction without template (−)21g. WT, wild type.