5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl)methyl]-L-proline Inhibits Hepatitis C Virus Entry

Nguyen, Lap P.; Park, Chorong; Luong, Trang T. D.; Park, Eun-Mee; Choi, Dongkyu; Han, Kang Min; Han, Nannan; Nguyen, Huu C; Lim, Yun-Sook; Hwang, Soon B.

doi:10.1038/s41598-019-43783-6

Cited by 6 publications

(7 citation statements)

References 32 publications

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“…Cells seeded on a 24-well plate were transfected with the indicated siRNAs. At the indicated time points, cell viability was measured by using water-soluble tetrazolium salt (WST) (Dail Lab) as we reported previously (5,55).…”

Section: Methodsmentioning

confidence: 99%

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PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

Nguyen

Tran

Suetsugu

et al. 2020

J Virol

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Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly. IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.

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Section: Methodsmentioning

confidence: 99%

“…DAAs are still too expensive for the majority of HCV patients (2,3). In addition, resistance-associated variants will likely increase with broader use of DAAs (4,5). Lastly, protective immunity is often insufficient for protection against reinfection after viral clearance with DAAs (6).…”

mentioning

confidence: 99%

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

Nguyen

Tran

Suetsugu

et al. 2020

J Virol

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“…Pseudoparticle production and entry assay. HCV pseudoparticle (HCVpp) with E1 and E2 glycoproteins derived from genotype 1a (H77) or genotype 2a (JFH1) and vesicular stomatitis virus pseudoparticles (VSVpp) were generated and the pseudoparticle entry assay was performed as we reported previously (61,62).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoblot analysis. Immunoblot assays were performed using an ECL kit (Abfrontier) as we reported previously (16,62).…”

Section: Methodsmentioning

confidence: 99%

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Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly

Nguyen

et al. 2020

J Virol

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Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. siRNA-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in assembly step without affecting viral entry, HCV IRES-mediated translation, and replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs) and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating phosphorylation of cortactin at tyrosine residue 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration. IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in virus life cycle has not yet been fully understood. The most significant finding is that cortactin strongly interacted with both HCV core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the LDs with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosine 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

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Synergistic Herb-Drug Interactions Against Viral Diseases

Hemaiswarya¹,

Prabhakar²,

Doble³

2022

Herb-Drug Combinations

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5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl)methyl]-L-proline Inhibits Hepatitis C Virus Entry

Cited by 6 publications

References 32 publications

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly

Synergistic Herb-Drug Interactions Against Viral Diseases

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