Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

Eyre, Nicholas S.; Hampton‐Smith, Rachel J.; Aloia, Amanda L.; Eddes, James S.; Simpson, Kaylene J.; Hoffmann, Peter; Beard, Michael R.

doi:10.1016/j.virol.2016.01.018

Cited by 30 publications

(35 citation statements)

References 52 publications

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“…A cluster of serine residues is responsible for NS5A hyperphosphorylation and functions (12); however, direct measurements of their phosphorylation levels were made possible only recently with phosphorylation-specific antibodies (21)(22)(23). In the present work, we simultaneously measured NS5A phosphorylation at S222, S235, and S238 in the HCV (J6/JFH1)-infected Huh7.5.1 cells with three high-quality antibodies (Fig.…”

Section: Discussionmentioning

confidence: 87%

“…The lipid kinase PI4KIII␣ (23,(31)(32)(33)(34) and the protein kinase CKI␣ (18,22,27,28,35) are the two most-studied kinases involved in NS5A hyperphosphorylation. To investigate their roles in S235 and S238 phosphorylation, we measured S235 and S238 phosphorylation in the T7 polymerase-expressing Huh7 cells (T7-Huh7) whose PI4KIII␣ or CKI␣ was knocked down prior to NS3-NS5B expression.…”

Section: S235 Determines Ns5a Hyperphosphorylationmentioning

confidence: 99%

“…NS5A hyperphosphorylation has been associated with viral replication and assembly (17,18). 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).…”

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confidence: 99%

“…Isoleucine mutation at S232 enhances viral replication in HCV genotype 1 (26); however, alanine mutation at the same site reduces viral replication or assembly in genotype 2 (17,18), leaving S232 a mysterious site for functions in different genotypes. Recently, S235 was shown to be a critical amino acid for HCV replication, likely via regulating replication complex formation (22,23). Thus, genetic mutations that either remove or mimic phosphorylation have been very instrumental in demonstrating the functions of the above-described serine residues.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 87%

Section: S235 Determines Ns5a Hyperphosphorylationmentioning

confidence: 99%

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confidence: 99%

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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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“…APEX2 is especially useful for EM applications in vivo because, unlike existing genetic tags (see below), APEX2 does not require irradiation with light or exogenous delivery of large molecules such as antibodies and nanoparticles. APEX2 has also been used to label viral proteins after infection of cultured mammalian cells 42,43 and to study the impact of an infectious intracellular bacterium on ER morphology 29 . It is unclear whether APEX2 can be used in plants, which contain abundant endogenous peroxidases that may create strong background staining 44 .…”

Section: Introductionmentioning

confidence: 99%

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

et al. 2017

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Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2).

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Proximity labeling approaches to study protein complexes during virus infection

Zapatero-Belinchón¹,

Carriquí-Madroñal

2021

Advances in Virus Research

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Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

Cited by 30 publications

References 52 publications

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

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

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

Proximity labeling approaches to study protein complexes during virus infection

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