Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

Ross‐Thriepland, Douglas; Mankouri, Jamel; Harris, Mark

doi:10.1128/jvi.02995-14

Cited by 45 publications

(59 citation statements)

References 26 publications

(54 reference statements)

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“…Several host kinases, including PI4K-IIIα and casein kinase Iα (CKIα), and multiple viral nonstructural proteins, including NS2 and NS4A, have been implicated in the generation of basal (56 kDa) and hyperphosphorylated NS5A species 37, 111 . Mechanistic details are lacking, but hyperphosphorylation appears to have a negative effect on the capacity of NS5A to support replicase formation and RNA synthesis, while enhancing its movement to lipid droplets and promoting virion assembly 112, 113 . Hyperphosphorylation of NS5A is thus likely to deplete the intracellular (+)RNA pool.…”

Section: The Yin Of Hepatitis C: Hcv Rna Synthesismentioning

confidence: 99%

The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA

Yamane

Masaki

et al. 2015

Nat Rev Microbiol

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PREFACE Hepatitis C virus (HCV) is an unusual RNA virus that possesses a striking capacity to persist for life in the majority of infected individuals. In order to persist, HCV must balance viral RNA synthesis and decay in infected cells. In this Review, we focus on interactions between the positive-sense RNA genome of HCV and host RNA-binding proteins and microRNAs (miRNAs) that influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. We discuss how these processes impact HCV pathogenesis and therapeutic strategies against the virus.

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Section: The Yin Of Hepatitis C: Hcv Rna Synthesismentioning

confidence: 99%

The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA

Yamane

Masaki

et al. 2015

Nat Rev Microbiol

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“…Our previous studies had pointed to a key role for S225 phosphorylation – the S225A phosphoablatant mutant had a dramatic phenotype: a loss of p58, a 10-fold reduction in JFH-1 genome replication, a lack of interactions between NS5A and a range of host proteins, and a perinuclear restricted localisation of NS5A and other replication complex components (23, 37). This phenotype prompted us to investigate the role of S225 phosphorylation more directly, therefore to complement the analysis of S225 mutants, we raised an antiserum to pS225-NS5A and used this unique reagent to probe the functions of S225-phosphorylated wildtype NS5A.…”

Section: Introductionmentioning

confidence: 99%

A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

Goonawardane

Harris

2018

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241, importance: 150, main text (excluding figure legends): 4984 Abstract 18NS5A is a multi-functional phosphoprotein that plays a key role in both viral replication 19 and assembly. The identity of the kinases that phosphorylate NS5A, and the 20 consequences for HCV biology, remain largely undefined. We previously identified 21 serine 225 (S225) within low complexity sequence (LCS) I as a major phosphorylation 22 site and used a phosphoablatant mutant (S225A) to define a role for S225 23 phosphorylation in the regulation of genome replication, interactions of NS5A with 24 several host proteins and the sub-cellular localisation of NS5A. To investigate this 25 further, we raised an antiserum to S225 phosphorylated NS5A (pS225). Western blot 26 analysis revealed that pS225 was exclusively found in the hyper-phosphorylated 27 NS5A species. Furthermore, using kinase inhibitors we demonstrated that S225 was 28 phosphorylated by casein kinase 1α (CK1α) and polo-like kinase 1 (PLK1). Using a 29 panel of phosphoablatant mutants of other phosphorylation sites in LCSI we obtained 30 the first direct evidence of bidirectional hierarchical phosphorylation initiated by 31 phosphorylation at S225. 32Using super-resolution microscopy (Airyscan and Expansion), we revealed a unique 33 architecture of NS5A-positive clusters in HCV-infected cells -pS225 was concentrated 34 on the surface of these clusters, close to lipid droplets. Pharmacological inhibition of 35 S225 phosphorylation resulted in the condensation of NS5A-positive clusters into 36 larger structures, recapitulating the S225A phenotype.Although S225 37 phosphorylation was not specifically affected by daclatasvir treatment, the latter also 38 resulted in a similar condensation. These data are consistent with a key role for S225 39 phosphorylation in the regulation of NS5A function. Importance 41 NS5A has obligatory roles in the hepatitis C virus lifecycle, and is proposed to be 42 regulated by phosphorylation. As NS5A is a target for highly effective direct-acting 43 antivirals (DAAs) such as daclatasvir (DCV) it is vital to understand how 44 phosphorylation occurs and regulates NS5A function. We previously identified serine 45 225 (S225) as a major phosphorylation site. Here we used an antiserum specific for 46 NS5A phosphorylated at S225 (pS225-NS5A) to identify which kinases phosphorylate 47 this residue. Using super-resolution microscopy we showed that pS225 was present 48 in foci on the surface of larger NS5A-positive clusters likely representing genome 49 replication complexes. This location would enable pS225-NS5A to interact with cellular 50 proteins and regulate the function and distribution of these complexes. Both loss of 51 pS225 and DCV treatment resulted in similar changes to the structure of these 52 complexes, suggesting that DAA treatment might target a function of NS5A that is also 53 regulated by phosphorylation. 54 56 Hepatitis C virus (HCV) infects an estimated 73 million people and frequently results 57 in chronic liver disease -cirrhosis and he...

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“…Many studies, largely based on genetic mutations, have pinpointed several serine residues in the LCS I region responsible for NS5A hyperphosphorylation and functions (17)(18)(19)(20)(21)(22)(23). For example, S225 was reported to participate in the formation of viral replication protein complex (24) as well as viral assembly (18). S229 seems an irreplaceable amino acid critical to the viral life cycle (17,18), as both alanine (phosphorylation-ablated) and aspartate (phosphorylationmimicking) mutations abolished viral replication (17,18).…”

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Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication

Hsu

Pan

et al. 2017

J Virol

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The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo-and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase I␣, which requires a priming phosphorylation at S235. IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypoand hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.KEYWORDS NS5A, antibody, hepatitis C virus, protein phosphorylation, proteomics H epatitis C virus (HCV) is an enveloped virus with a 9.6-kb single-stranded positivesense RNA genome. It infects about 185 million people worldwide and is a leading cause of liver diseases and related complications (1, 2). The HCV genome encodes a long polyprotein composed of 10 proteins from NH 2 to the COOH terminus: core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. A complete HCV life cycle requires 3

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Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

Cited by 45 publications

References 26 publications

The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA

The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA

A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication

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