Dual role of the amphipathic helix of hepatitis C virus NS5A in the viral polyprotein cleavage and replicase assembly

Zhang, Yang; Zhao, Xiaomin; Zou, Jingyi; Yuan, Zhenghong; Yi, Zhigang

doi:10.1016/j.virol.2019.07.017

Cited by 3 publications

(7 citation statements)

References 38 publications

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“…We recently reported that NS5A AH regulates protein-protein interactions within the replicase components ( Zhang et al., 2019a ). Mutagenesis of NS5A AH and identification of suppressor mutations in the C terminus of NS4B indicate a genetic interaction of NS5A AH with NS4B ( Biswas et al., 2016 ).…”

Section: Resultsmentioning

confidence: 99%

“…Finally, we assessed the impact of the identified interactome on viral replication. We used the subgenomic replicon sgJFH1-sGluc expressing secreted Gaussia luciferase (sGluc) ( Zhang et al., 2019a ), introduced point mutations into sgJFH1-sGluc, and then assessed viral replication by monitoring the luciferase activity ( Figure 7 A). Several residues involved in the interactome have been reported to affect viral replication in previous studies ( Biswas et al., 2016 ; Elazar et al., 2004 ; Gouttenoire et al., 2014 ) ( Figure 7 B).…”

Section: Resultsmentioning

confidence: 99%

“…However, mutation of the NS3 docking sites (D228) and the oligomerization sites (L237) of the C-terminal α-helix H2 of NS4B abolished viral replication ( Figures 7 A and 7B), pointing to the critical role of C-terminal α-helix H2 of NS4B in replicase assembly. We recently reported that the NS5A AH regulates polyprotein processing and protein-protein interactions within the replicase ( Zhang et al., 2019a ). In this study, we identified the NS5A AH-mediated NS5A-NS4B interaction, and mutations of the residues that participate in the interaction have been reported to abolish viral replication ( Figure 7 B) ( Biswas et al., 2016 ).…”

Section: Discussionmentioning

confidence: 99%

“…NS4B S227A/D228A/R232A were introduced into plasmids phCMV-NS3-5B-4B HA and phCMV-NS3-5B-5A HA to get the plasmids phCMV-NS3-5B-4B HA -S227A/D228A/R232A and phCMV-NS3-5B-5A HA - S227A/D228A/R232A, respectively. Plasmid sgJFH1-sGluc and sgJFH1-sGluc GND were described previously ( Zhang et al., 2019a ). NS4B L2237A, NS4B S227A, NS4B D228A, NS4B R232A, NS5A W84A were introduced into sgJFH1-sGluc to get the plasmids sgJFH1-sGluc-NS4B-L237A, sgJFH1-sGluc-NS4B-S227A, sgJFH1-sGluc-NS4B-D228A, sgJFH1-sGluc-NS4B-R232A, and sgJFH1-sGluc-NS5A-W84A, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…NS5A has nonenzymatic activity and plays a central role in viral replicase assembly. NS5A has an amphipathic helix (AH) at the amino terminus, which was suggested to mediate membrane anchoring and was recently demonstrated to regulate polyprotein processing and protein-protein interactions within the replicase ( Zhang et al., 2019a ). NS5B is the viral RNA-dependent RNA polymerase (see Gu and Rice, 2013 for a review).…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Bioorthogonal dissection of the replicase assembly of hepatitis C virus

Zhang

Chen

Yuan

et al. 2021

Cell Chemical Biology

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Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bioorthogonal dissection of the replicase assembly of hepatitis C virus

Zhang

Chen

Yuan

et al. 2021

Cell Chemical Biology

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From Discovery to Cure, A Great Journey of the Hepatitis C Virus Study

Yuan

2022

Infectious Diseases &Amp; Immunity

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Hepatitis C virus (HCV) causes chronic infection in over 75% of the HCV-infected individuals, resulting in liver cancer in substantial patients. Since its discovery in 1989, HCV experiences a journey from discovery to cure, largely due to the virology studies and success of direct antiviral agent (DAA) development. We reviewed the HCV research journey, from the discovery of this virus to the development of DAAs for cure. Learning the methodology used in HCV studies and the knowledge of developing DAAs against HCV may inspire the studies of other difficult-to-culture viruses, such as hepatitis E virus and norovirus, as well as the development of DAAs for other single-stranded positive-sense RNA viruses, including the pandemic-causing SARS-CoV-2 virus, which shares the common replication strategy of forming a membrane-bound viral replicase.

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Identification of a membrane-associated element (MAE) in the C-terminal region of SARS-CoV-2 nsp6 that is essential for viral replication

Han,

Yuan,

2024

J Virol

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The coronavirus disease 2019 (COVID-19) pandemic, caused by the novel coronavirus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has rapidly spread worldwide since its emergence in late 2019. Its ongoing evolution poses challenges for antiviral drug development. Coronavirus nsp6, a multiple-spanning transmembrane protein, participates in the biogenesis of the viral replication complex, which accommodates the viral replication-transcription complex. The roles of its structural domains in viral replication are not well studied. Herein, we predicted the structure of the SARS-CoV-2 nsp6 protein using AlphaFold2 and identified a highly folded C-terminal region (nsp6C) downstream of the transmembrane helices. The enhanced green fluorescent protein (EGFP)-fused nsp6C was found to cluster in the cytoplasm and associate with membranes. Functional mapping identified a minimal membrane-associated element (MAE) as the region from amino acids 237 to 276 (LGV–KLL), which is mainly composed of the α-helix H1 and the α-helix H2; the latter exhibits characteristics of an amphipathic helix (AH). Mutagenesis studies and membrane flotation experiments demonstrate that AH-like H2 is required for MAE-mediated membrane association. This MAE was functionally conserved across MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-HKU1, and HCoV-NL63, all capable of mediating membrane association. In a SARS-CoV-2 replicon system, mutagenesis studies of H2 and replacements of H1 and H2 with their homologous counterparts demonstrated requirements of residues on both sides of the H2 and properly paired H1–H2 for MAE-mediated membrane association and viral replication. Notably, mutations I266A and K274A significantly attenuated viral replication without dramatically affecting membrane association, suggesting a dual role of the MAE in viral replication: mediating membrane association as well as participating in protein-protein interactions. IMPORTANCE Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) assembles a double-membrane vesicle (DMV) by the viral non-structural proteins for viral replication. Understanding the mechanisms of the DMV assembly is of paramount importance for antiviral development. Nsp6, a multiple-spanning transmembrane protein, plays an important role in the DMV biogenesis. Herein, we predicted the nsp6 structure of SARS-CoV-2 and other human coronaviruses using AlphaFold2 and identified a putative membrane-associated element (MAE) in the highly conserved C-terminal regions of nsp6. Experimentally, we verified a functionally conserved minimal MAE composed of two α-helices, the H1, and the amphipathic helix-like H2. Mutagenesis studies confirmed the requirement of H2 for MAE-mediated membrane association and viral replication and demonstrated a dual role of the MAE in viral replication, by mediating membrane association and participating in residue-specific interactions. This functionally conserved MAE may serve as a novel anti-viral target.

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Dual role of the amphipathic helix of hepatitis C virus NS5A in the viral polyprotein cleavage and replicase assembly

Cited by 3 publications

References 38 publications

Bioorthogonal dissection of the replicase assembly of hepatitis C virus

Bioorthogonal dissection of the replicase assembly of hepatitis C virus

From Discovery to Cure, A Great Journey of the Hepatitis C Virus Study

Identification of a membrane-associated element (MAE) in the C-terminal region of SARS-CoV-2 nsp6 that is essential for viral replication

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