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

Abstract

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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“…In line with the present data, previous studies showed alanine mutations in serine 225 led to NS5A hyper-phosphorylation reduction and replication of HCV genotype 2 [4,58,59]. In addition, Goonawardane et al [60] focused on serine 225 (S225) and determined the contribution of S225 phosphorylation in the regulation of genome replication, interactions of NS5A with several host proteins, and the sub-cellular localization of NS5A. Mutations can affect the phosphorylation sites and our data suggested mutation in amino acid 131 could omit this phosphorylation site in 1a genotype.…”

“…There is some evidence showed NS5A induces liver pathogenesis, steatosis, and hepatocellular carcinoma that NS5A phosphorylation can affect these important functions directly or indirectly [62]. Furthermore, there are some reports suggested S225 phosphorylation disruption reduced the HCV replication [60] and virus assembly [16,47,59]. NS5A is a target for DAAs such as daclatasvir (DCV) that S225 can be one of the targets for such effective drugs.…”

Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories

“…In mammalian cells, hierarchical phosphorylation has been described for β-catenin, in which Ser45 phosphorylated by CK1α primes it for hyperphosphorylation by glycogen synthase kinase 3 (GSK-3) (38, 39), which triggers its ubiquitination and proteasomal degradation (40). A similar phosphorylation mechanism has recently been described for nonstructural protein NS5A of the hepatitis C virus (HCV), with the hyperphosphorylation cascade primed by the initial phosphorylation of serine 225 by CK1α and the subsequent phosphorylation of neighboring residues involving other kinases (41, 42). Moreover, NS5A phosphorylation has been shown to play a key role in controlling the establishment of replication complexes during HCV infection (43).…”

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“…Proteins were separated by electrophoresis on a 7.5 % SDS-PAGE gel, and following electrophoresis, proteins were transferred onto PVDF membrane and blocked with 50 % (v/v) Odyssey blocking buffer (LiCor) in Tris-buffered saline (TBS). The membrane was then incubated with primary antibody as labelled, sheep anti-NS5A [15], rabbit anti-pS225 [16] or rabbit anti-pS232 [17] at 4 °C overnight. After washing with TBS, membranes were incubated with fluorescently labelled anti-sheep (800 nm) and anti-rabbit (680 nm) secondary antibodies for 1 h at room temperature.…”

“…Interestingly, DBN3a NS5A did exhibit the expected two bands corresponding to basally and hyper-phosphorylated species, however, compared to JFH-1, the ratio of hyper : basally phosphorylated species was much lower. To investigate this further, we used antisera specific for NS5A phosphorylated at either S225 [16] or S232 [17]. These antisera had been shown to detect hyper-phosphorylated NS5A only, consistent with hierarchical phosphorylation of serine residues within LCSI.…”