Terminal Nucleotidyl Transferase Activity of Recombinant Flaviviridae RNA-Dependent RNA Polymerases: Implication for Viral RNA Synthesis

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.Replication of plus-strand RNA viruses requires a multisubunit enzyme, the replicase, which is composed of viral and cellular factors (6). Biochemical characterization of eukaryotic replicases is limited because of difficulty in obtaining sufficient quantities of purified replicase. Furthermore, the hepatitis C virus (HCV) replicase has not been reported to accept exogenously provided RNAs. These results have prompted studies of the recombinant HCV RNA-dependent RNA polymerase (RdRp), the subunit responsible for phosphoryl transfer (9,16,17,26,31,38). While RdRps lack many properties of replicases, they are useful for characterizing some fundamental activities, such as the recognition of the initiation site and the kinetics of nucleotide polymerization (4,18,24).The HCV RdRp has recently been demonstrated to initiate RNA synthesis preferentially from the 3Ј terminus of the template RNA (16,17,26,31). Initiation from the 3Ј terminus raises a potential problem that viruses might encounter: cellular RNases that degrade even a few 3Ј nucleotides could prevent the initiation of viral RNA replication. Several mechanisms have been proposed that might allow RNA viruses to preserve or restore the sequences at the termini of their genome. These include base-pairing-dependent and base-pairing-independent recombination (12), priming by oligonucleotides aborted during the initiation of RNA synthesis (29), telomerase-like addition of a repeated sequence (33), and nontemplated nucleotide addition (7,12). Also, terminal adenylyl transferase activity was found to be associated with poliovirus polymerase 3D pol (30), possibly causing restoration of infectivity of poliovirus RNAs lacking the wild-type poly(A) tail.Recombinant HCV RdRp was reported to possess the ability to add nontemplated nucleotides to the 3Ј end of viral RNAs (5). However, this terminal transferase (TNTase) activity was later purported to be a cellular enzyme copurifying with the HCV RdRp...

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

“…This result suggests that Cterminal region of NS5B might play a role in the initiation of RNA synthesis from 3'-end of HCV genome. To date, HCV NS5B polymerase has been shown to initiate RNA synthesis by snap-back or de novo mechanisms using a natural HCV RNA template (Behrens et al, 1996;Lohmann et al, 1997;Yamashita et al, 1998;Ishii et al, 1999;Oh et al, 2000;Sun et al, 2000;Ranjith-Kumar et al, 2001;Reigadas et al, 2001;Kashiwagi et al, 2002;Kim et al, 2002;Pellerin et al, 2002). Our results shown with several HCV RNA templates using TNTase-free NS5B are in agreement with the previous result obtained with E. coli-expressed NS5B (Oh et al, 1999), indicating that HCV NS5B proteins initiate RNA synthesis in a primer-independent manner.…”

“…We have demonstrated that NS5B proteins purified from insect cells do not have intrinsic TNTase activity, in contrast to previous reports (Behrens et al, 1996;Ranjith-Kumar et al, 2001). In addition, any 3'-terminally extended products was not detected from oligouridylic acid (U)20.…”

“…One reason for the discrepancy with respect to RNA synthesis initiation using natural HCV cis-acting element at the 3'-UTR might be due to intrinsic TNTase activity of HCV NS5B or host origin TNTase co-purified with HCV NS5B. It is still unclear whether HCV NS5B has intrinsic TNTase activity as indicated by recent data shown with the C-terminus truncated NS5B expressed in E. coli and the full-length NS5B expressed in insect cells (Ranjith-Kumar et al, 2001).…”

Arresting Initiation of Hepatitis C Virus RNA Synthesis Using Heterocyclic Derivatives

“…Heparin RdRp Assay-The RdRp assay was performed as described previously (25), with the following modifications. Briefly, after a 15-min incubation, heparin was added to the reaction at a final concentration of 10 ng/ml.…”

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

“…The newly synthesized template-sized RNA product annealed to the unlabeled template (ϩ) RNA would be resistant to RNase A treatment. However, if the template-sized product was synthesized by a putative terminal transferase activity of WNV NS5 similar to that of the hepatitis C virus NS5B polymerase reported previously (45), then the product would be digested by RNase A. To analyze the RdRP products by RNase A digestion, the RdRP reac- FIG.…”