title: Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.
abstract: Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA. todd.appleby@gilead.com tedwards@be4.com.
authors: Todd C
authors: TC
authors: Appleby
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Jason K
authors: JK
authors: Perry
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Eisuke
authors: E
authors: Murakami
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Ona
authors: O
authors: Barauskas
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Joy
authors: J
authors: Feng
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Aesop
authors: A
authors: Cho
authors: Beryllium, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors: David
authors: D
authors: Fox
authors: Beryllium, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA.
authors: Diana R
authors: DR
authors: Wetmore
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Mary E
authors: ME
authors: McGrath
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Adrian S
authors: AS
authors: Ray
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: Michael J
authors: MJ
authors: Sofia
authors: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404, USA.
authors: S
authors: S
authors: Swaminathan
authors: Beryllium, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA. todd.appleby@gilead.com tedwards@be4.com.
authors: Thomas E
authors: TE
authors: Edwards
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry*; D000069474:Sofosbuvir; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D012265:Ribonucleotides; D017361:Viral Nonstructural Proteins; D014542:Uridine Monophosphate; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D000069474:Sofosbuvir