Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Mandal, Amrita; Ganta, Krishna Kumar; Chaubey, Binay

doi:10.1155/2016/9671031

Cited by 10 publications

(21 citation statements)

References 67 publications

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“…The purified transcript was transfected in overnight grown 70-80% confluent naive Huh-7.5 cells in a 60-mm tissue culture dish using a Lipofectamine-2000 transfection reagent (Invitrogen), as described before [26]. After 6 h the cells were incubated in fresh media.…”

Section: Hcv Production and Quantificationmentioning

confidence: 99%

“…The forward and reverse primer sequences were: HCV 5′UTR: forward, 5′-CCTAATAGGGGCGACACTCCG-3′; reverse, 5′-CCACAAGGCCTTTCGCAACC-3′. Assays were performed using a Sybr green real-time PCR master mix reagent (Invitrogen) on a Mastercycler ep RealPlex2 (Eppendorf, Germany) as described earlier [26].…”

Section: Hcv Production and Quantificationmentioning

confidence: 99%

“…Therefore, a multiple siRNA-based approach could be a likely answer [13,25]. Recent studies have also shown synergistic anti-HCV effects using a combinatorial RNAi approach [13,14,[25][26][27][28][29]. Earlier, we reported a synergistic effect on HCV inhibition by using combination of siRNAs designed against La autoantigen along with HCV NS5B or hVAP-A genes [26].…”

Section: Introductionmentioning

confidence: 97%

“…Recent studies have also shown synergistic anti-HCV effects using a combinatorial RNAi approach [13,14,[25][26][27][28][29]. Earlier, we reported a synergistic effect on HCV inhibition by using combination of siRNAs designed against La autoantigen along with HCV NS5B or hVAP-A genes [26]. In order to minimize the risk of escape mutants and to increase viral inhibition, simultaneous expression of multiple shRNAs could yield better therapeutic results.…”

Section: Introductionmentioning

confidence: 99%

“…In continuation of our previous study [26], we constructed a third-generation bicistronic SIN lentiviral vector expressing shRNA against La autoantigen and NS5B under U6 and H1 promoters, respectively. Expression of shRNA through a single round of transduction with lentiviral vectors in HCV-infected Huh7.5 cells demonstrated a sustained inhibition of HCV replication.…”

Section: Introductionmentioning

confidence: 99%

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Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Mandal

Chaubey

2018

Intervirology

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Background: The RNAi-based transient therapeutic approach has been well explored for its potential against the hepatitis V virus (HCV). However, to achieve a sustained virological response, a consistent presence of siRNA is needed and it can be achieved by constitutively expressing shRNAs. In this context, the lentiviral vector has emerged as an attractive tool for shRNA delivery against HCV. Methods: We monitored HCV inhibition after single and multiple rounds of siRNA treatments against La autoantigen and HCV-NS5B in Huh-7.5 cells infected with the FL-J6/JFH chimeric HCV strain. A bicistronic self-inactivating third-generation lentiviral vector expressing shRNA under U6 and H1 promoters was constructed. To ascertain the long-term HCV inhibition, cells were transduced with lentiviral vectors and HCV inhibition was monitored by RT-PCR and Western blotting at regular intervals. Results: We observed transient antiviral activity after a single round of siRNA treatment, and consecutive rounds of treatments with siRNA demonstrated a sustained HCV inhibition. Delivery of duplex shRNA expressing lentiviral vectors provided constant expression of shRNA leading to synergistic and sustained HCV inhibition. Conclusion: A lentiviral vector-based delivery system is a “single-shot” therapeutic strategy. It can express duplex shRNA for long-term synergistic inhibition of HCV and qualify as a promising therapeutic approach for sustained inhibition of HCV replication.

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Section: Hcv Production and Quantificationmentioning

confidence: 99%

Section: Hcv Production and Quantificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Mandal

Chaubey

2018

Intervirology

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Anti-HCV Activity from Semi-purified Methanolic Root Extracts ofValeriana wallichii

et al. 2017

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Hepatitis C virus (HCV) is a serious global health problem affecting approximately 130-150 million individuals. Presently available direct-acting anti-HCV drugs have higher barriers to resistance and also improved success rate; however, cost concerns limit their utilization, especially in developing countries like India. Therefore, development of additional agents to combat HCV infection is needed. In the present study, we have evaluated anti-HCV potential of water, chloroform, and methanol extracts from roots of Valeriana wallichii, a traditional Indian medicinal plant. Huh-7.5 cells infected with J6/JFH chimeric HCV strain were treated with water, chloroform, and methanol extracts at different concentrations. Semi-quantitative reverse transcription polymerase chain reaction result demonstrated that methanolic extract showed reduction in HCV replication. The methanolic extract was fractionated by thin layer chromatography, and the purified fractions (F1, F2, F3, and F4) were checked for anti-HCV activity. Significant viral inhibition was noted only in F4 fraction. Further, intrinsic fluorescence assay of purified HCV RNA-dependent RNA polymerase NS5B in the presence of F4 resulted in sharp quenching of intrinsic fluorescence with increasing amount of plant extract. Our results indicated that methanolic extract of V. wallichii and its fraction (F4) inhibited HCV by binding with HCV NS5B protein. The findings would be further investigated to identify the active principle/lead molecule towards development of complementary and alternative therapeutics against HCV. Copyright © 2017 John Wiley & Sons, Ltd.

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In silico prediction and experimental validation of siRNAs targeting ORF1ab of MERS-CoV in Vero cell line

Sohrab

El‐Kafrawy

Mirza

et al. 2021

Saudi Journal of Biological Sciences

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The Middle East Respiratory Syndrome Coronavirus is well known to cause respiratory syndrome and this virus was identified and isolated for the first time from Jeddah, Saudi Arabia in 2012 from infected patient. In this report, we have conducted the in-silico prediction, designing and evaluation of siRNAs targeting Middle East Respiratory Syndrome Coronavirus orf1ab gene to inhibit the virus replication. By using bioinformatics software, total twenty-one functional, off-target reduced siRNA were selected from four hundred and sixty-two siRNAs based on their greater potency and specificity. We have evaluated only seven siRNAs to analyze their performance and efficacy as antivirals by reverse transfection approach in Vero cells. There was no cytotoxicity of siRNAs at various concentrations was observed in Vero cells. Based on the real-time PCR results, better inhibition of viral replication was observed in the siRNA-1 and 4 as compared to other siRNAs. The results generated from this work provided suitable information about the efficacy of siRNAs which encouraged us to further evaluate the remaining siRNAs to determine their inhibitory effect on the virus replication. We concluded that the insilico prediction and designing resulted in the screening of potential siRNAs with better efficiency, and strength. This can be used to develop oligonucleotide-based antiviral therapeutics against MERS-CoV in the near future.

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Combinations of siRNAs against La Autoantigen with NS5B or hVAP-A Have Additive Effect on Inhibition of HCV Replication

Cited by 10 publications

References 67 publications

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Expression of Duplex shRNAs through a Lentiviral Vector against Cellular and Viral Genes Inflicts Sustained Inhibition of Hepatitis C Virus Replication

Anti-HCV Activity from Semi-purified Methanolic Root Extracts ofValeriana wallichii

In silico prediction and experimental validation of siRNAs targeting ORF1ab of MERS-CoV in Vero cell line

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