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. 2001 Sep;75(18):8615-23.
doi: 10.1128/jvi.75.18.8615-8623.2001.

Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis

C T Ranjith-Kumar  1 , J Gajewski, L Gutshall, D Maley, R T Sarisky, C C Kao
Affiliations

Terminal nucleotidyl transferase activity of recombinant Flaviviridae RNA-dependent RNA polymerases: implication for viral RNA synthesis

C T Ranjith-Kumar et al. J Virol. 2001 Sep.

Abstract

Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.

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Figures

FIG. 1
FIG. 1
HCV NS5B has TNTase activity. (A) SDS-PAGE profile of different RdRps used in this study. BacFL is a full-length protein expressed using baculovirus. Proteins HΔ21 and HΔ51 are HCV NS5B with 21- and 51-amino-acid deletions from the C terminus. mΔ21 has a change of the GDD motif to GAA. Lane M, molecular size markers. (B) Predicted secondary structure of template LE19. (C) RNA synthesis assay (lanes 1 and 8) and TNTase assay (lanes 2 to 7 and 9 to 11) performed with RNA LE19. The nucleotide substrates used are listed above the autoradiogram. The asterisk (∗) indicates that the nucleotide is radiolabeled with α-32P. r3 denotes the presence of GTP, ATP, and UTP. The reactions in lanes 1 to 7 were performed with HΔ21, and those in 8 to 11 were performed with mΔ21. Sizes of reaction products are shown (in nt) on the side of the autoradiogram. (D) RNA synthesis (lane 1) and TNTase (lanes 2 to 7) assays performed with BacFL using template LE19. (E) RNA synthesis (lanes 1 and 2) and TNTase (lanes 3 and 4) assays performed with Δ51 using template LE19. The sizes of the RdRp products in this and other experiments were assigned by comparison with a series of BVDV RdRp products that varied by one nucleotide (M. Kim and C. Kao, data not shown).
FIG. 2
FIG. 2
BVDV NS5B has TNTase activity that cofractionates with RdRp activity. (A) RNA synthesis and TNTase assays performed on RNA LE19. The nucleotide substrates used are shown above the autoradiogram. The asterisk corresponds to the radiolabeled nucleotide. r3 denotes the presence of GTP, ATP, and UTP. Sizes of the reaction products (in nt) are given on the side of the autoradiogram. Lanes 1 and 2 contain results from RNA synthesis and TNTase assays, respectively, performed using HΔ21, while lanes 3 to 8 were performed with BΔ23. (B) Silver-stained SDS-PAGE profile depicting the various SP-Sepharose column fractions. Fraction numbers are at the bottom of the gel. (C) Quantification of relative RNA synthesis and TNTase activity of the BΔ23 eluted from an SP-Sepharose column. RNA synthesis and TNTase activities in fraction 22 were set at 100%, and other fractions were normalized to these values.
FIG. 3
FIG. 3
Template requirements for TNTase activity. RdRp and TNTase assays performed with HΔ21. The nucleotide substrates used are shown above the autoradiogram. Asterisk (∗) identifies the radiolabeled nucleotide. r3 denotes the presence of GTP, ATP, and UTP in the reactions. The lengths of the reaction products (in nucleotides) are indicated on the side of the autoradiogram. (A) Reactions performed with RNA LE19 are in lanes 1 and 2, while those performed with the puromycin-modified LE19P are in lanes 3 to 6. (B) Comparison of TNTase activity performed with HΔ21, [α-32P]rCTP, and either an RNA or DNA version of LE19. (C) Autoradiogram of the products from TNTase (lanes 1 and 2) and RNA synthesis (lanes 3 and 4) reactions, untreated (U) or treated with RNase T1 (T1).
FIG. 4
FIG. 4
Effect of Mn2+ and KCl on TNTase activity. (A) RdRp and TNTase assays using HΔ21. Both assays were performed with enzyme HΔ21 and RNA LE19. The nucleotides used in the reactions are listed above the autoradiogram. Asterisk (∗) identifies the radiolabeled nucleotide. r3 denotes the presence of GTP, ATP, and UTP in the reactions. Lengths of the reaction products (in nucleotides) are given between the two autoradiograms. Lanes + and −, reactions performed in the presence and absence of Mn2+, respectively. (B) RdRp and TNTase assays for BΔ23. The layout of the figure is identical to that in panel A. (C) Effects of increasing KCl concentration on RNA synthesis and TNTase activity of HΔ21. (D) Effects of increasing KCl concentration on RNA synthesis and TNTase activity of BΔ23.
FIG. 5
FIG. 5
TNTase activity does not require the initiation nucleotide. (A) RNA synthesis (lanes 1 and 2) and TNTase (lanes 3 to 12) assays performed with HΔ21 using LE19+1G. The nucleotide substrates used in each reaction are listed above the autoradiogram. Asterisk (∗) identifies the radiolabeled nucleotide. r3 denotes the presence of GTP, ATP, and UTP in the reaction. The length of the reaction products (in nucleotides) is on the side of the autoradiogram. T1, reactions treated with RNase T1, U, untreated control. (B) LE19+1G retains interaction with HΔ21. This assay measures the synthesis from template SLD3 as affected by the presence of a second RNA in the reaction. SLD3 has the sequence 5′-GGGCUUGCAUAGCAAGUCUGAGACC-3′ (17). RNAs 14–16 and 14–17 have sequences 5′-AAAUCCUCUGAUAU-3′ and 5′-AAAUCCUCUGAUAA-3′, respectively.
FIG. 6
FIG. 6
TNTase activity can restore de novo initiation on an initiation-incompetent RNA. (A) Schematic of the treatments used in the experiments shown in panels B and C. APase, alkaline phosphatase. (B) RNA synthesis and TNTase assays performed with HΔ21, RNA LE19, and the nucleotide used in the TNTase assay (T-NTPs) indicated above the autoradiogram. The asterisk (∗) denotes that the nucleotide is radiolabeled. RNA synthesis reaction in lanes 3 to 6 used [α-32P]CTP and unlabeled ATP, GTP, and UTP. Lengths of the reaction products (in nucleotides) are to the right of the autoradiogram. Lanes + and −, reactions performed in the presence and absence of 75 mM KCl, respectively. (C) TNTase and RdRp reactions performed using 14-1, 14–16, and 14–17 as the templates. Nucleotides used in the TNTase reactions are identified in the column labeled T-NTP. The absence and presence of 75 mM KCl is indicated (− and +, respectively). RNA synthesis reactions contained [α-32P]CTP and unlabeled ATP, GTP, and UTP.
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