Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation

Abstract

Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the ␤-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information.Approximately 3% of the world's population is infected with the hepatitis C virus (HCV), 2 including four-five million people in the United States, and chronic HCV infections lead to fibrosis, cirrhosis, and hepatocellular carcinoma (1, 2). The hepatitis C viral genome is replicated by NS5B, a virally encoded RNAdependent RNA polymerase requiring de novo initiation of RNA synthesis. Antivirals acting directly against viral proteins including NS5B have been developed recently that have dramatically improved the prognosis for treatment (3-5). However, despite these advances, the biochemical mechanisms of action of drugs currently on the market are poorly understood, and very little is known about the kinetics and mechanism of initiation and elongation governing RNA polymerization. In this and the companion paper (6), we begin to address these deficiencies.Replication proceeds through three distinct phases: 1) initiation, in which the 3Ј-end of the RNA acts as a template to direct the formation of the initial dinucleotide followed by the addition of two-three more nucleotides; 2) transition, in which the polymerase dramatically changes structure to switch from the initiation to elongation mode; 3) elongation, in which the polymerase catalyzes rapid and highly processive synthesis (7). A ␤-loop structure projecting from the "thumb" domain is thought to facilitate the initiation reaction but a...

Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase

“…The order Lomibuvir Ͼ GS-9669 Ͼ Filibuvir, observed in ranking the magnitude of the effects on ⌬% D, correlates with the potency of inhibition seen in kinetic studies (11).…”

“…A companion paper (11) showed that NNI2s inhibit RNA replication by slowing the transition from initiation to elongation, implying long range structural effects of NNI2s on enzyme dynamics. To gain insights into the underlying molecular basis for this allosteric inhibition, we used HDX kinetics to quantify changes in enzyme dynamics in the absence and presence of three different NNI2s.…”

“…Peptic fragments displaying significant ⌬% D at any incubation time point for all three NNI2 were mapped on NS5B (3FQL.pdb) using a surface rendering and colored dark blue as in A. Dinucleotide primed template (orange and yellow spheres) and representative inhibitor (magenta sticks) were positioned through alignment with appropriate structure, 4WTK.pdb for the dinucleotide primed template and 3FRZ.pdb for the inhibitor (Filibuvir). (8,11), HDX studies using a reaction mixture used in kinetic studies would be uninterruptable. Moreover, there are multiple states (initiation, transition, and elongation) that one would like to study individually.…”

“…In our recent work we showed that NNI2s inhibit replication by blocking the transition from initiation to the elongation mode (11), which is thought to proceed with a substantial rearrangement of the enzyme involving, at the very least, the movement of the ␤-loop out of the active site. Here we complement these studies using hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS).…”

“…This process is very slow and inefficient in vitro and is followed by several rounds of additional nucleotide incorporation with significant accumulation of abortive intermediates. After incorporation of ϳ4 -6 nucleotides the polymerase undergoes a transition to rapid processive elongation (8,11). NNIs might in principle impact any one or more of these various stages of RNA synthesis (namely, initiation, transition, or elongation), and it is important to understand which steps are inhibited and how an allosteric effector binding to the surface of the enzyme can attenuate reactions at the active site.…”