Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line

Hossein M Elbadawy, Mohi I Mohammed Abdul, Naif Aljuhani, Adriana Vitiello, Francesco Ciccarese, Mohamed A Shaker, Heba M Eltahir, Giorgio Palù, Veronica Di Antonio, Hanieh Ghassabian, Claudia Del Vecchio, Cristiano Salata, Elisa Franchin, Eleonora Ponterio, Saleh Bahashwan, Khaled Thabet, Mekky M Abouzied, Ahmed M Shehata, Cristina Parolin, Arianna CalistriGualtiero Alvisi

Research output: Contribution to journalArticlepeer-review

Abstract

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

Original languageEnglish
Article number1044
Number of pages21
JournalViruses
Volume12
Issue number9
DOIs
Publication statusPublished - Sep 18 2020

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