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Title: Structural basis for RNA replication by the hepatitis C virus polymerase

Authors:
; ; ; ; ; ; ; ; ; ; ; ;  [1];  [2]
  1. (Gilead)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
INDUSTRY
OSTI Identifier:
1184239
Resource Type:
Journal Article
Resource Relation:
Journal Name: Science; Journal Volume: 347; Journal Issue: 02, 2015
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Appleby, Todd C., Perry, Jason K., Murakami, Eisuke, Barauskas, Ona, Feng, Joy, Cho, Aesop, Fox III, David, Wetmore, Diana R., McGrath, Mary E., Ray, Adrian S., Sofia, Michael J., Swaminathan, S., Edwards, Thomas E., and Beryllium). Structural basis for RNA replication by the hepatitis C virus polymerase. United States: N. p., 2016. Web. doi:10.1126/science.1259210.
Appleby, Todd C., Perry, Jason K., Murakami, Eisuke, Barauskas, Ona, Feng, Joy, Cho, Aesop, Fox III, David, Wetmore, Diana R., McGrath, Mary E., Ray, Adrian S., Sofia, Michael J., Swaminathan, S., Edwards, Thomas E., & Beryllium). Structural basis for RNA replication by the hepatitis C virus polymerase. United States. doi:10.1126/science.1259210.
Appleby, Todd C., Perry, Jason K., Murakami, Eisuke, Barauskas, Ona, Feng, Joy, Cho, Aesop, Fox III, David, Wetmore, Diana R., McGrath, Mary E., Ray, Adrian S., Sofia, Michael J., Swaminathan, S., Edwards, Thomas E., and Beryllium). 2016. "Structural basis for RNA replication by the hepatitis C virus polymerase". United States. doi:10.1126/science.1259210.
@article{osti_1184239,
title = {Structural basis for RNA replication by the hepatitis C virus polymerase},
author = {Appleby, Todd C. and Perry, Jason K. and Murakami, Eisuke and Barauskas, Ona and Feng, Joy and Cho, Aesop and Fox III, David and Wetmore, Diana R. and McGrath, Mary E. and Ray, Adrian S. and Sofia, Michael J. and Swaminathan, S. and Edwards, Thomas E. and Beryllium)},
abstractNote = {},
doi = {10.1126/science.1259210},
journal = {Science},
number = 02, 2015,
volume = 347,
place = {United States},
year = 2016,
month = 6
}
  • Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP andmore » dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol's high error rates on pyrimidines and determines the incorporation specificity.« less
  • 7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase {eta} (Pol{eta}). We show that the open active site cleft of Pol{eta} can accommodate an 8-oxoG lesion in the anti conformation with only minimal changesmore » to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Pol to prevent the mutagenic replication of 8-oxoG lesions in cells.« less
  • Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptidemore » at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.« less
  • Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using amore » customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.« less
  • Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins wasmore » confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.« less