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

Hsu, Szu-Chun; Lo, Chieh-Wen; Pan, Ting-Chun; Lee, Kuan-Ying; Yu, Minshu

doi:10.1128/jvi.00194-17

Cited by 14 publications

(54 citation statements)

References 40 publications

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“…Our data are consistent with this (Fig 5) and we propose a model whereby S225 phosphorylation primes subsequent phosphorylation in a bidirectional fashion. Confirmation of this model would require use of additional phospho-specific antibodies, such as those used by Yu et al (33) to demonstrate that S238 phosphorylation is dependent on S235 phosphorylation. This observation is consistent with our findings and the model proposed in Fig 13.…”

Section: Discussionmentioning

confidence: 94%

“…We proceeded to use the antiserum to investigate which cellular kinases might be responsible for phosphorylation of S225. Previous studies (29, 33, 38), coupled with bioinformatic analysis (39), suggested that S225 was a potential phosphorylation site for three kinases: casein kinase-Iα (CKIα), polo-like kinase-1 (PLK1) and glycogen synthase kinase-3β (GSK3β). To investigate their potential roles in phosphorylation of S225, we treated HCV-infected Huh7 cells with three selective kinase inhibitors: D4476 (CKIα inhibitor); SBE13-HCl (PLK1 inhibitor), or GSK-3β Inhibitor VIII (GSK3β inhibitor).…”

Section: Resultsmentioning

confidence: 99%

“…These include the conservation and spacing of the serine residues, and mass spectrometric evidence for multiply-phosphorylated species corresponding to LCSI (31, 38, 43). Additionally the group of Yu (33) recently generated pS235 and pS238-specific antisera and demonstrated that pS238 was dependent on pS235, but not vice-versa, suggesting that S235 was the primary phosphorylation site leading to a hierarchical phosphorylation of other serines within LCSI. However, they did not address the potential role of S225phosphorylation, and we hypothesised that this might precede phosphorylation of S235 and initiate the hierarchical phosphorylation cascade.…”

Section: Resultsmentioning

confidence: 99%

“…In this regard LCSI is reminiscent of cellular proteins that exhibit hierarchical phosphorylation, for example β-catenin, in which serine 45 (S45) is phosphorylated by casein kinase I (CKI), priming it for phosphorylation at S41/37/33 by glycogen synthase kinase-3 (GSK-3) (32), triggering ubiquitinylation and proteosomal degradation of β-catenin. Intriguingly, CKIα has been implicated in production of the NS5A p58 as indicated by the loss of p58 following treatment of infected cells with CKIα-inhibitors (33, 34). A number of studies have used biochemical and genetic approaches to demonstrate that CKIα phosphorylates S225, S232, S235 and S238 but the relative importance of these phosphorylation events and their sequential order remains to be determined.…”

Section: Introductionmentioning

confidence: 99%

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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

Preprint

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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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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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

Preprint

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“…When analyzed with SDS-PAGE plus immunoblotting, NS5A appears as two bands at 56 and 58 kDa, which represent, respectively, hypo-and hyperphosphorylated forms of NS5A (10). Numerous studies have pinpointed a cluster of eight highly conserved serine residues (S222, S225, S228, S229, S230, S232, S235, and S238) in the LCS I region responsible for NS5A hyperphosphorylation (11)(12)(13)(14)(15)(16)(17)(18). Mutations in some of these serine residues reduce NS5A hyperphosphorylation in association with effects on viral replication and assembly (11,12).…”

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confidence: 99%

Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A

Hsu

Tsai

Lee

et al. 2018

J Virol

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The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A. The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.

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Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus

Pan

Chong

et al. 2019

J. Proteome Res.

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Protein phosphorylation is a reversible posttranslational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo-and hyperphosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo-vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo-and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

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

Cited by 14 publications

References 40 publications

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

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

Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus

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