Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

Abstract

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G) 12 but not with poly(A)-oligo(U) 12 . Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses. The hepatitis C virus (HCV)1 is an RNA virus that causes acute and chronic liver disease (for reviews see Refs. 1-3). A high proportion of infected patients fail to clear the virus and contract chronic infection, which may lead to liver cirrhosis and, eventually, to hepatocellular carcinoma. Currently it is estimated that 100 -200 million people worldwide suffer from a chronic HCV infection. Thus, HCV became a focus of intensive research worldwide.Based on similarities of genome organization and virus particle structure with the flaviviruses like yellow fever virus and the animal pathogenic pestiviruses like the classical swine fever virus (CSFV), HCV has been classified as the separate genus Hepacivirus in the family Flaviviridae (4).These viruses have in common an enveloped virus particle and a single-stranded RNA genome of positive polarity encoding a polyprotein that is cleaved by host cell signalases and viral proteinases. In the case of HCV, the genome has a length of ϳ9600 nucleotides. Its single long open reading frame is flanked at the 5Ј-and 3Ј-ends by nontranslated regions of about 340 and 230 nucleotides in length, respectively. The 5Ј nontranslated region is important for efficient translation of the viral polyprotein and functions as an internal ribosome entry site (5-7). The 3Ј nontranslated region most likely required for RNA replication carries a 3Ј-terminal highly conserved sequence forming stable secondary and probably also higher order structures (8 -11).HCV polyprotein processing is accomplished by a combination of host and viral proteinase...

Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides

“…In sharp contrast, soaking the crystals with rATP, rCTP, or rUTP, even at a 10 mM concentration, does not lead to binding at the surface site, demonstrating the specificity for rGTP. Several studies have shown a specific effect of high rGTP concentrations on the efficiency of RNA synthesis by NS5B (29,30). Luo et al (30) have concluded that the rGTP effect is likely to be related to its incorporation as an initiating nucleotide, which they interpret as being rate limiting for RNA synthesis.…”

“…An additional distinctive feature of these two polymerases is their ability to initiate replication from an RNA template without the need of a primer (de novo initiation), as reported for HCV (30,34,46) and 6 (31). In addition, it has been shown that high concentrations of riboguanosine triphosphate (rGTP) stimulate RNA synthesis for both the 6 (35) and HCV (29,30) enzymes, presumably by enhancing the efficiency of the initiation process. The cocrystals of the 6 polymerase in complex with a DNA oligonucleotide and rGTP provided the first snapshot of an initiation complex formed by these enzymes (12).…”

Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: Implications for RNA–RNA recombination

“…Products longer than template length were observed with the replicases from BMV (26), dengue virus (36), turnip crinkle virus (37), and the RdRps from hepatitis C virus (38,39) and poliovirus (40). If all of the unexplained dimeric products are caused by template switch, then RNA recombination in vitro can be directly studied with RNA viruses for which RNA synthesis assays are available.…”

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

“…The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7)(8)(9)(10)(11)(12)(13)(14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided additional information about the complex of NS5B with ribonucleotides.…”