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.
Highlights
The recent introduction of hepatitis C virus (HCV) NS3, NS5B, and NS5A inhibitors, the so-called directly acting antivirals (DAAs), has greatly improved HCV treatment [1]
In order to generate novel combinatorial platforms able to interfere with HCV replication, first of all, we consulted previous literature data and online HCV resources to select 4 different short harpin RNAs (shRNAs) already known to efficiently block the viral life cycle: the internal ribosome entry site (IRES) specific HCV353 and HCV321 small interferingRNAs (siRNAs) as well as siRNAs targeting the PI4KII and CypA host factors [18,20,32]
In order to (i) allow easy quantification of the levels of HCV replication in cell culture and (ii) simultaneously normalize for well-to-well variations in cell number linked to variance in cell seeding, as well as effects on cell growth and viability due to drug treatments, transfection, and lentiviral transduction, we developed a new HCV replicon double-reporter cell line (Figure 2A)
Summary
The recent introduction of hepatitis C virus (HCV) NS3, NS5B, and NS5A inhibitors, the so-called directly acting antivirals (DAAs), has greatly improved HCV treatment [1]. Due to the biological characteristics of HCV, the emergence of drug-resistance represents an important limitation. High cost and lack of patient compliance constitute additional problems [2]. The design of innovative therapeutic strategies and the development of additional agents to fight. Silencing of specific genes can be achieved by RNA interference (RNAi) that is based on the degradation of target mRNAs by small complementary RNA molecules known as small interfering. RNAs (siRNAs) [3,4]. HCV is highly susceptible to RNAi, because its genome is a positive single-stranded RNA, which is involved both in viral transcription and cytoplasmic replication
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
