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...

The RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus, a calicivirus, is known to have a conserved GDD amino acid motif and several additional regions of sequence homology with all types of polymerases. To test whether both aspartic acid residues are in fact involved in the catalytic activity and metal ion coordination of the enzyme, several defined mutations have been made in order to replace them by glutamate, asparagine, or glycine. All six mutant enzymes were produced in Escherichia coli, and their in vitro poly(U) polymerase activity was characterized. The results demonstrated that the first aspartate residue was absolutely required for enzyme function and that some flexibility existed with respect to the second, which could be replaced by glutamate.Rabbit hemorrhagic disease virus (RHDV) has been characterized as a member of the Caliciviridae (19, 21) and recently designated as the type species of the new genus Lagovirus (24). The viral genome is a positive polarity, single-stranded, polyadenylated RNA with approximately 7.4 kb, which has a virusencoded VPg protein covalently attached to its 5Ј end (15,16,28). Viral particles also encapsidate an abundant VPg-linked polyadenylated subgenomic RNA of about 2.2 kb (16). The data obtained from the in vitro translation (28), Escherichia coli expression studies (14), and detection of viral proteins after infection of cultured hepatocytes with RHDV (8) revealed that the viral RNA is translated into a polyprotein that is subsequently cleaved to give rise to mature structural and nonstructural proteins.The extensive sequence similarities between the RNA-dependent RNA polymerase (RdRp) 3D of picornavirus and the RHDV polyprotein cleavage product p58 (8,15,28) suggested that this polypeptide could have a similar role in RHDV genome replication. Studies from our laboratory have reported the successful expression of enzymatically active RHDV RNA polymerase (3D pol ) in E. coli (13). The recombinant polypeptide exhibited rifampin-and actinomycin D-resistant poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity by using synthetic RHDV subgenomic RNA as a template in the presence or absence of an oligo(U) primer (13).The availability of a growing number of complete sequences from the genomes of positive, negative, and double-stranded RNA viruses of plant and animal origin has enabled many groups to carry out sequence alignments of the RdRps in an attempt to identify regions essential for polymerase function, which should appear as most conserved (1,7,23). Currently, eight conserved motifs have been described (23), four of which (A, B, C, and D) are now known to be located in the catalytic portion of the "palm" domain (4) of all classes of polymerases. Motif C, which formed a "␤-strand, turn, ␤-strand" structure, contained a highly conserved GDD sequence found in all RdRps (7). This structure is very similar in all classes of polymerases and positions the two aspartate residues close to the conserved aspartate of motif A (4). The location ...

All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conservation. Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus. Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates. In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate specificity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates. Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a "copy-back" mechanism that initiated at the template 3'-terminal OH.

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathioneS-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.