In (+) RNA coronaviruses, replication and transcription of the giant approximately 30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5'-(G/U)CC-3' trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-Å resolution of active coxsackievirus B3 RdRp (also named 3D pol ) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D pol at this site excludes its uridylation by the carrier 3D pol . We suggest that VPg at this position is either uridylated by another 3D pol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D pol /VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2-C-methyl CTP (2-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2-modified nucleotide analogues. 2-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2-Omethyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5-to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.Hepatitis C virus (HCV) infection is the most common blood-borne infection and a major cause of chronic liver disease in developed countries. More than 170 million individuals in the world are infected with HCV, making these individuals at risk of developing liver cirrhosis and hepatocellular carcinoma. Persistent HCV infection can be controlled by antiviral therapy (30, 40) or even eradicated from infected patients (43).Current antiviral therapies rely on the combination of pegylated alpha interferon and the nucleoside analogue ribavirin (16,18,29). Less than 50% of treated patients respond when they are infected with the most prevalent HCV genotype, i.e., genotype 1 (23). The HCV RNA-dependent RNA polymerase (RdRp), the NS5B protein, plays an essential role in the replication of the viral genome and is thus an attractive target for the development of new antiviral drugs (36). Intensive drugscreening programs led to the discovery of two classes of HCV NS5B inhibitors, namely, nonnucleoside inhibitors (NNIs) and nucleoside inhibitors (NIs). NNIs have been described to bind to one of the three allosteric sites present at the NS5B surface (for a review, see reference 11). All of the NNIs are noncompetitive inhibitors relative to nucleoside triphosphate (NTP) incorporation and target the alloenzyme free of substrate. They are inactive when the enzyme has entered into the processive elongation phase (3,17,31,45,46) of in vitro RdRp reactions or when NS5B is c...

The dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a messenger RNA cap 1 structure (7MeGpppA2'OMe) at the 5' end of the viral genome, and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for the full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, small-angle X-ray scattering (SAXS) experiments were carried out on the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms wherein the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.