Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François‐Loïc

doi:10.1053/j.gastro.2016.11.047

Cited by 29 publications

(40 citation statements)

References 48 publications

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“…5C. Consistent with previous observations (6,23,40), the core proteins of wild-type JFH1 were largely coating LD (75% on LD, 5% on the ER, and 20% on LD and the ER), whereas the majority of the cells infected with the M21T mutant had core proteins either on the ER or on both the ER and LD (5% on LD, 30% on the ER, and 65% on LD and the ER), which was similar to the case with Jc1 (3% on LD, 71% on the ER, and 26% on LD and the ER). These results demonstrated that the M21T mutation altered the subcellular localization of core from LD to the ER for successive virion assembly processes.…”

Section: Resultssupporting

confidence: 92%

“…The nascent HCV RNA genomes are encapsidated by core proteins on lipid droplets (LD), and the resulting nucleocapsids are then translocated to the ER for budding. Previous work showed that HCV virion assembly rate was linked to the subcellular localization of the core (6,23,40). The Jc1 strain, whose core proteins are located on the ER, has more efficient virion assembly than the JFH1 strain, whose core proteins are mostly located on LD.…”

Section: Resultsmentioning

confidence: 99%

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A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

Boson

et al. 2017

J Virol

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The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic ␣ helix (designated helix ␣ 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix ␣ 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix ␣ 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix ␣ 0 and aid understanding of the role of NS3 in HCV virion morphogenesis.IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix ␣ 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix ␣ 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.KEYWORDS HCV, NS3, core, helix ␣ 0 , assembly, hepatitis C virus

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

Boson

et al. 2017

J Virol

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“…Wildtype NS5A positive punctae were on average smaller (peak 0.09-0.12 μm 2 ) than those in infected cells, however the S225A mutant exhibited significantly condensed and larger punctae (peak 0.18-0.21 μm 2 ). It has been reported that treatment of HCV-infected cells with the NS5A inhibitor DCV resulted in a perinuclear accumulation of NS5A (13), reminiscent of that observed for the S225A mutant. We therefore treated wildtype SGR harbouring cells with DCV and analysed the distribution of total NS5A and pS225-NS5A by ExM (Fig 10C).…”

Section: Resultsmentioning

confidence: 83%

“…DAAs target NS3/4A protease, NS5A, and NS5B RNA-dependent RNA polymerase, revolutionising HCV therapy (11). Daclatasvir (DCV) exemplifies a class of DAAs proposed to be potent inhibitors of NS5A (12, 13). However, the mode(s) of action for these compounds remains obscure as it is not clear which of the myriad functions of NS5A are inhibited.…”

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

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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“…By using an HCV replicase surrogate system, Berger et al demonstrated that DCV blocks the early biogenesis of the viral membranous replication factories (19). In addition, DCV blocks transfer of viral genome to the assembly sites to affect virion assembly (20).…”

Section: Introductionmentioning

confidence: 99%

Hepatitis C virus NS5A inhibitor daclatasvir allosterically impairs NS4B-involved protein-protein interactions within the viral replicase and disrupts the replicase quaternary structure in a replicase assembly surrogate system

Zhang

Zou

Zhao

et al. 2018

Preprint

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22Daclatasvir (DCV) is a highly potent direct-acting antiviral that targets the non-structural 23 protein 5A (NS5A) of hepatitis C virus (HCV) and has achieved great clinical successes. 24 Previous studies demonstrate its impact on the viral replication complex assembly. However 25 the precise mechanism by which DCV impairs the replication complex assembly remains 26 elusive. In this study, by using HCV subgenomic replicons and a viral replicase assembly 27 surrogate system that expresses the HCV NS3-5B polyprotein to mimic the viral replicase 28 assembly, we dissected the impacts of DCV on aggregation and tertiary structure of NS5A, 29 the protein-protein interactions within the viral replicase and the quaternary structure of the 30 viral replicase. We found that DCV didn't affect aggregation and tertiary structure of NS5A. 31 DCV induced a quaternary structural change of the viral replicase, evidenced by selectively 32 increasing of the NS4B's sensitivity to proteinase K digestion. Mechanically, DCV impaired 33 the NS4B-involved protein-protein interactions within the viral replicase. The DCV-resistant 34 mutant Y93H was refractory to the DCV-induced reduction of the NS4B-invoved protein 35 interactions and the quaternary structural change of the viral replicase. In addition, Y93H 36 reduced NS4B-involed protein-protein interactions within the viral replicase and attenuated 37 viral replication. We propose that DCV may induce a position change of NS5A, which 38 allosterically affects the protein interactions within the replicase components and disrupts the 39 replicase assembly.40 41 quaternary structure; protein-protein interaction 42 Importance: 43 3 The development of the direct-acting antivirals (DAA) has resulted in great clinical 44 achievements for Hepatitis C Virus (HCV) treatment. Daclatasvir (DCV) is an inhibitor 45 targeting the non-enzymatic NS5A, with the 50% effective concentration values in the 46 picomolar range. Accumulated data suggest that DCV blocks the biogenesis of the HCV 47 109 6 suggesting the reduction of NS5A hyper-phosphorylation is an early event of DCV action and 110 maybe used as an indicator of the action of DCV. DCV treatment with levels greater than 10 111 nM didn't further reduce the NS5A hyper-phosphorylation (Fig. 1E and F). 112 DCV blocks biogenesis of the HCV replication complex in a replicase assembly 113 surrogate system (19). We took a similar strategy and expressed the HCV NS3-5B (JFH1) 114 polyprotein in HEK293T cells. We selected HEK293T cell for certain reasons. First, it 115 supports replication of the JFH1 subgenomic replicon (24), making it physiologically relevant 116 to study the replicase assembly in these cells. Second, the expression of HCV NS3-5B 117 polyprotein in HEK293T cells induces membranous web structures in the perinuclear regions 118 (Fig. 1G), similarly as in Huh7 cells (4). Third, the high transfection efficiency in these cells 119 facilitates the expression of the viral polyprotein to a similar level as found in the replicon 120 cells (Dat...

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Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites

Cited by 29 publications

References 48 publications

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

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

Hepatitis C virus NS5A inhibitor daclatasvir allosterically impairs NS4B-involved protein-protein interactions within the viral replicase and disrupts the replicase quaternary structure in a replicase assembly surrogate system

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Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites

Cited by 29 publications

References 48 publications

A Point Mutation in the N-Terminal Amphipathic Helix α 0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

A Point Mutation in the N-Terminal Amphipathic Helix α 0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

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

Hepatitis C virus NS5A inhibitor daclatasvir allosterically impairs NS4B-involved protein-protein interactions within the viral replicase and disrupts the replicase quaternary structure in a replicase assembly surrogate system

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A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding