The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "␤-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the ␤-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA. Hepatitis C virus (HCV)7 is a member of the Flaviviridae family that induces severe liver disease in humans (1). The viral genome is a single-stranded RNA of positive polarity containing a single open reading frame (ORF) flanked by two untranslated regions (UTRs), the 5Ј-UTR and 3Ј-UTR. The single ORF is translated into a large (ϳ3000 residues) polyprotein that is processed into some 10 mature proteins. Thus, the RNA-dependent RNA polymerase (RdRp) NS5b is cleaved from the C terminus of the polyprotein. In vivo the 591-residue NS5b is the central player in the synthesis of new genomic RNAs, in association with other viral and cellular proteins. This viral replication complex is associated with membranes (2) with the highly hydrophobic C-terminal 21 residues of NS5b forming a transmembrane helix (3). In vitro, NS5b has been shown to be capable of template-directed RNA synthesis on its own, requiring only divalent metals (magnesium or manganese) as cofactors. Indeed, NS5b can catalyze both de novo synthesis from a singlestranded template (4) and primer extension from the subsequent RNA duplex or from a preannealed template/primer duplex. The NS5b C-terminal transmembrane helix is dispensable for these activities, and C-terminal deletions of 21 residues (NS5b_⌬21) or more (NS5b_⌬47 to NS5b_⌬60) have been used in most activity and all crystallographic studies. The latter (5-7) has shown that the catalytic core of NS5b comprises residues 1-530 (Fig. 1E). They have also brought a puzzle to light; the 40-residue stretch (termed "linker" throughout this manuscript) between the catalytic core (fingers, palm, and thumb, Fig. 1) and the C-terminal membrane anchor occludes the catalytic cleft (5) in the crystal structures in which it is present (i.e. _⌬21 forms). The only reported exception is the case of the consensus subty...

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR–PXR and potentially several other nuclear receptor heterodimers.