Using the Hepatitis C Virus RNA-Dependent RNA Polymerase as a Model to Understand Viral Polymerase Structure, Function and Dynamics

Abstract

Viral polymerases replicate and transcribe the genomes of several viruses of global health concern such as Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and Ebola virus. For this reason they are key targets for therapies to treat viral infections. Although there is little sequence similarity across the different types of viral polymerases, all of them present a right-hand shape and certain structural motifs that are highly conserved. These features allow their functional properties to be compared, with the goal of broadly applying the knowledge acquired from studying specific viral polymerases to other viral polymerases about which less is known. Here we review the structural and functional properties of the HCV RNA-dependent RNA polymerase (NS5B) in order to understand the fundamental processes underlying the replication of viral genomes. We discuss recent insights into the process by which RNA replication occurs in NS5B as well as the role that conformational changes play in this process.

Keywords: Flaviviridae; conformations; positive-strand RNA viruses.

Figure 1

(a) Right-hand structure of HCV polymerase (NS5B). Palm, fingers and thumb domains are shown in red, blue and green respectively; (b) Duplex channel in NS5B (front of the enzyme); (c) NTP channel in NS5B (back of the enzyme); (d) Motifs and functional regions of NS5B. Motif A in red, B in orange, C in yellow, D in bright green, E in pink, F in purple and G in cyan. Functional regions: I in light green, II in violet and III in tan; (e) Template channel (top view of the enzyme).

Figure 2

Schematic describing de novo initiation in Hepaciviruses and Pestiviruses. Note that Flaviruses do not anchor their C-terminus in the Endoplasmatic Reticulum (ER). This figure was generated by incorporating the descriptions provided by both Appleby et al. [35] and Choi [1]. The linker and C-terminal anchor are shown in orange as one contiguous element. The β-flap is colored red (as in Figure 3), the template strand in purple, the growing strand in green, the stabilizing GTP in blue and the Endoplasmic Reticulum (ER) in brown. The “N” and “P” indicate where the N-site (nucleotide-site) and P-site (priming-site) are. These correspond to the positions of the growing strand that bind to residues “n” and “n + 1” of the template strand respectively.

Figure 3

NS5B structure with characteristic elements highlighted. (a) Front view and (b) top view. The linker is shown in orange, the β-flap in red. The fingertips are shown in blue (the delta 1 loop) and green (the delta 2 loop).

Figure 4

Two metals ions mechanism in RdRps. The squares represent the bases that are part of the nucleotides. This figure is inspired by a similar figure from Choi et al. [1].

Figure 5

NS5B inhibitors. (a) The three allosteric sites of NS5B are highlighted with space filling representations of inhibitors that bind in these locations. Thumb site 1 (NNI-1) in yellow, thumb site 2 (NNI-2) in green, palm sites (NNI-3/4) in purple; (b) chemical structures of NIs and NNIs that are in clinical trials or have already been approved [52,53,54,55,56,57,58,59].

Figure 6

Mechanisms of inhibition for NNIs. NS5B must transition between open and closed states to perform replication (upper left). NNI-1 inhibitors have been observed to reduce enzyme stability. NNI-2 inhibitors have been shown to reduce conformational sampling, confining the enzyme in closed conformations. NNI-3 inhibitors mainly block access of the RNA template but also induce some restriction of conformational sampling. The RNA template is represented as a black rectangle and the inhibitor as an orange ellipse.