Crystal Structures of Active and Inactive Conformations of a Caliciviral RNA-dependent RNA Polymerase

Abstract

The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-Å resolution. The overall structure resembles a "right hand," as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either "closed" or "open" conformations differing by a rigid body rotation of ϳ8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes. Rabbit hemorrhagic disease virus (RHDV)1 belongs to the Caliciviridae family of positive-stranded RNA viruses and causes a highly contagious and lethal disease in rabbits (1, 2). First described in China in the 1980s, RHDV has spread at an alarming rate in the rabbit population throughout Asia, Europe, and Australia. Caliciviruses cause a number of severe diseases in other mammals; in humans, Norwalk viruses are responsible for ϳ95% of cases of non-bacterial gastroenteritis and represent a growing public health problem in need of new, more effective treatments (3). Caliciviruses are also closely related to the picornaviruses (e.g. polio virus, rhinovirus, and foot-and-mouth disease virus) and to the flaviviruses (e.g. hepatitis C, dengue, and yellow fever viruses), which cause many serious diseases in humans and other mammals.A virally encoded RNA-dependent RNA polymerase (RdRP) is the central enzyme responsible for replicating the genomic RNA of caliciviruses and other positive-stranded RNA viruses (4). The RHDV RdRP has been produced in Escherichia coli and has been shown to efficiently synthesize RNA from RNA primer-template duplexes in the presence of divalent cations and ribonucleoside triphosphates (5-7). Sequence analysis and sitedirected mutagenesis studies of RdRPs from a wide spectrum of positive-stranded RNA viruses (4), as well as the crystal structures of RdRPs from polio virus (PV) (8) and hepatitis C virus (HCV) (9 -11), suggest that the members of this large family of enzymes share a common architecture and enzymatic mechanism. Structural and enzymological studies on a wide range of RdRPs are beginning to reveal the essential features of RdRP function, and it is hoped that this body of molecular information will facilitate the design of more effective treatments for viral diseases.Although the previously determined crystal structures of the PV and HCV RdRPs have shed light on the structural basis of polymerase function, many important questions remai...

Characterization of the Metal Ion Binding Properties of the Hepatitis C Virus RNA Polymerase

“…The conformations adopted by the rabbit hemorrhagic disease virus polymerase can vary dramatically depending on the ions present in the crystallization solution. It was suggested that these structural changes may be important for the enzymatic activity of the protein (38). As reported previously for DNA polymerases (39,40), these conformational changes are probably required for the catalytic activity of rabbit hemorrhagic disease virus and HCV polymerases.…”

“…In fact, comparisons with closely related polymerases strongly suggest that conformational changes are required for NS5B to efficiently initiate polymerization (28). However, analysis of the crystal structure the RNA polymerase of the rabbit hemorrhagic disease virus, a virus closely related to HCV, revealed the presence of both active and inactive conformations within the same crystal form (38). The conformations adopted by the rabbit hemorrhagic disease virus polymerase can vary dramatically depending on the ions present in the crystallization solution.…”

“…These two metal ions are in contact with both the phosphate of the nucleotide and several acidic amino acids residues (41). Two metal ions are also bound in the active site of the rabbit hemorrhagic disease virus (38) and HCV polymerases (28). A catalytic mechanism has been proposed in which one metal ion is involved both in positioning the substrate and in the activation of an incoming nucleophile (42).…”

Structures of EV71 RNA-dependent RNA polymerase in complex with substrate and analogue provide a drug target against the hand-foot-and-mouth disease pandemic in China

“…To date, representative RdRps from five families of RNA viruses have known three-dimensional structures: (1) Picornaviridae, poliovirus (Hansen et al, 1997;Thompson and Peersen, 2004;Thompson et al, 2007), human rhinovirus (Love et al, 2004), coxsackievirus (Campagnola et al, 2008), foot-and-mouth-disease virus (FMDV) (Ferrer-Orta et al, 2004); (2) Caliciviridae, rabbit hemorrhagic disease virus (Ng et al, 2002), Norwalk virus (Zamyatkin et al, 2008); (3) Flaviviridae, hepatitis C virus (Bressanelli et al, 1999;Lesburg et al, 1999), bovine viral diarrhea virus (Choi et al, 2006), Dengue virus (Yap et al, 2007), West Nile virus ; (4) Cystoviridae: phage f6 (Butcher et al, 2001;Salgado et al, 2004); and (5) Reoviridae, reovirus (Tao et al, 2002). The RdRp enzymes share a similar overall structure Figure 1.…”

3D structure of the influenza virus polymerase complex: Localization of subunit domains

“…The atomic structures of several viral RNA (vRNA)-dependent RNA polymerases from positive-and double-stranded RNA viruses are available (1)(2)(3)(4)(5)(6). These proteins show little sequence homology with other DNA-dependent polymerases but contain specific sequence motifs shared with other polymerases (7).…”