Phosphorylation of hepatitis C virus RNA polymerases Ser29 and Ser42 by protein kinase C-Related kinase 2 regulates viral RNA replication

Song Hee Han, Seong Jun Kim, Eun Jung Kim, Tae Eun Kim, Jae Su Moon, Geon Woo Kim, Seung Hoon Lee, Kun Cho, Jong Shin Yoo, Woo Sung Son, Jin Kyu Rhee, Seung Hyun Han, Jong-Won Oh

Research output: Contribution to journalArticle

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Abstract

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colonyforming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.

Original languageEnglish
Pages (from-to)11240-11252
Number of pages13
JournalJournal of Virology
Volume88
Issue number19
DOIs
Publication statusPublished - 2014 Jan 1

Fingerprint

Hepatitis C virus
Viral RNA
protein kinase C
DNA-Directed RNA Polymerases
DNA-directed RNA polymerase
Hepacivirus
phosphorylation
phosphotransferases (kinases)
Phosphorylation
RNA
RNA-directed RNA polymerase
Virus Replication
Proteins
RNA Replicase
Reverse Genetics
proteins
protein phosphorylation
Thumb
Phosphoamino Acids
RNA replication

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Han, Song Hee ; Kim, Seong Jun ; Kim, Eun Jung ; Kim, Tae Eun ; Moon, Jae Su ; Kim, Geon Woo ; Lee, Seung Hoon ; Cho, Kun ; Yoo, Jong Shin ; Son, Woo Sung ; Rhee, Jin Kyu ; Han, Seung Hyun ; Oh, Jong-Won. / Phosphorylation of hepatitis C virus RNA polymerases Ser29 and Ser42 by protein kinase C-Related kinase 2 regulates viral RNA replication. In: Journal of Virology. 2014 ; Vol. 88, No. 19. pp. 11240-11252.
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abstract = "Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colonyforming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.",
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Han, SH, Kim, SJ, Kim, EJ, Kim, TE, Moon, JS, Kim, GW, Lee, SH, Cho, K, Yoo, JS, Son, WS, Rhee, JK, Han, SH & Oh, J-W 2014, 'Phosphorylation of hepatitis C virus RNA polymerases Ser29 and Ser42 by protein kinase C-Related kinase 2 regulates viral RNA replication', Journal of Virology, vol. 88, no. 19, pp. 11240-11252. https://doi.org/10.1128/JVI.01826-14

Phosphorylation of hepatitis C virus RNA polymerases Ser29 and Ser42 by protein kinase C-Related kinase 2 regulates viral RNA replication. / Han, Song Hee; Kim, Seong Jun; Kim, Eun Jung; Kim, Tae Eun; Moon, Jae Su; Kim, Geon Woo; Lee, Seung Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin Kyu; Han, Seung Hyun; Oh, Jong-Won.

In: Journal of Virology, Vol. 88, No. 19, 01.01.2014, p. 11240-11252.

Research output: Contribution to journalArticle

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T1 - Phosphorylation of hepatitis C virus RNA polymerases Ser29 and Ser42 by protein kinase C-Related kinase 2 regulates viral RNA replication

AU - Han, Song Hee

AU - Kim, Seong Jun

AU - Kim, Eun Jung

AU - Kim, Tae Eun

AU - Moon, Jae Su

AU - Kim, Geon Woo

AU - Lee, Seung Hoon

AU - Cho, Kun

AU - Yoo, Jong Shin

AU - Son, Woo Sung

AU - Rhee, Jin Kyu

AU - Han, Seung Hyun

AU - Oh, Jong-Won

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