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...

Potassium channels encoded by the human ether-à-go-go-related gene (hERG) contribute to cardiac repolarization as a result of their characteristic gating properties. The hERG channel N terminus acts as a crucial determinant in gating. It is also known that the S4-S5 linker couples the voltage-sensing machinery to the channel gate. Moreover, this linker has been repeatedly proposed as an interaction site for the distal portion of the N terminus controlling channel gating, but direct evidence for such an interaction is still lacking. In this study, we used disulfide bond formation between pairs of engineered cysteines to demonstrate the close proximity between the beginning of the N terminus and the S4-S5 linker. Currents from channels with introduced cysteines were rapidly and strongly attenuated by an oxidizing agent, this effect being maximal for cysteine pairs located around amino acids 3 and 542 of the hERG sequence. The state-dependent modification of the double-mutant channels, but not the single-cysteine mutants, and the ability to readily reverse modification with the reducing agent dithiothreitol indicate that a disulfide bond is formed under oxidizing conditions, locking the channels in a non-conducting state. We conclude that physical interactions between the N-terminal-most segment of the N terminus and the S4-S5 linker constitute an essential component of the hERG gating machinery, thus providing a molecular basis for previous data and indicating an important contribution of these cytoplasmic domains in controlling its unusual gating and hence determining its physiological role in setting the electrical behavior of cardiac and other cell types.ether-à-go-go-related gene potassium channels play a key role in setting the electrical behavior of a variety of cell types (Refs.

Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking. To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations. Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms. The location of this carboxyl-terminal segment appears to interfere with the binding of doublestranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins.Recent studies have shown that members of the Norovirus genus within the Caliciviridae family are now considered one of the most common causes of outbreaks and sporadic cases of gastroenteritis in individuals of all ages worldwide (1). These pathogens have a positive-strand RNA genome and life cycle that are similar in many respects to a group of evolutionarily related viruses responsible for important human diseases such as polio, hepatitis C, dengue, yellow fever, viral encephalitis (West Nile virus and Japanese encephalitis virus), and severe acute respiratory syndrome. Many of these viruses are threats to public health, because they are highly infectious and can cause serious illnesses, in some cases leading to death. The limitations of vaccination and lack of effective antiviral chemotherapeutics for many of these diseases underline the urgency of understanding positive-stranded RNA viruses at a deeper, molecular level to allow for the development of novel treatments.Replication of the genome in all positive-strand RNA viruses is critically dependent on the activity of a virally encoded RNA-dependent RNA polymerase (RdRP) 1 (2). This enzyme is responsible for synthesizing negative-sense RNA, which is complementary to the positive-sense genomic RNA, as well as newly made positive-sense RNA genomes that can be used for the production of viral proteins or packaged into new viral particles. The three-dimensional structures of RdRPs from members of three families of positive-strand RNA viruses (poliovirus (PV) (3) from Picornaviridae, hepatitis C virus (HCV) (4 -6) from Flaviviridae, and rabbit hemorrhagic disease virus (RHDV) (7) from Caliciviridae) have previously revealed a similar overall architecture as well as a range of specific adaptations (8).Here, we report the novel structure of an RdRP from a genogroup II Norwalk virus (NV) isolate, Ast6139/01/Sp, in a metal-free form at 2.17 Å resolution, as well as in a metalbound form at 2.95 Å resolution. Although the arrangement of secondary structural elements and key catalytic motifs is similar to those see...

The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [␣-32 P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.