Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line
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 doub...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-09-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/12/9/1044 |
| id |
doaj-cfffe25b4ddd4231ac75d53254bc1142 |
|---|---|
| record_format |
Article |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Hossein M. Elbadawy Mohi I. Mohammed Abdul Naif Aljuhani Adriana Vitiello Francesco Ciccarese Mohamed A. Shaker Heba M. Eltahir Giorgio Palù Veronica Di Di Antonio Hanieh Ghassabian Claudia Del Del Vecchio Cristiano Salata Elisa Franchin Eleonora Ponterio Saleh Bahashwan Khaled Thabet Mekky M. Abouzied Ahmed M. Shehata Cristina Parolin Arianna Calistri Gualtiero Alvisi |
| spellingShingle |
Hossein M. Elbadawy Mohi I. Mohammed Abdul Naif Aljuhani Adriana Vitiello Francesco Ciccarese Mohamed A. Shaker Heba M. Eltahir Giorgio Palù Veronica Di Di Antonio Hanieh Ghassabian Claudia Del Del Vecchio Cristiano Salata Elisa Franchin Eleonora Ponterio Saleh Bahashwan Khaled Thabet Mekky M. Abouzied Ahmed M. Shehata Cristina Parolin Arianna Calistri Gualtiero Alvisi Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line Viruses hepatitis C virus reporter cell line antivirals gene therapy siRNA |
| author_facet |
Hossein M. Elbadawy Mohi I. Mohammed Abdul Naif Aljuhani Adriana Vitiello Francesco Ciccarese Mohamed A. Shaker Heba M. Eltahir Giorgio Palù Veronica Di Di Antonio Hanieh Ghassabian Claudia Del Del Vecchio Cristiano Salata Elisa Franchin Eleonora Ponterio Saleh Bahashwan Khaled Thabet Mekky M. Abouzied Ahmed M. Shehata Cristina Parolin Arianna Calistri Gualtiero Alvisi |
| author_sort |
Hossein M. Elbadawy |
| title |
Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line |
| title_short |
Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line |
| title_full |
Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line |
| title_fullStr |
Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line |
| title_full_unstemmed |
Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line |
| title_sort |
generation of combinatorial lentiviral vectors expressing multiple anti-hepatitis c virus shrnas and their validation on a novel hcv replicon double reporter cell line |
| publisher |
MDPI AG |
| series |
Viruses |
| issn |
1999-4915 |
| publishDate |
2020-09-01 |
| description |
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. |
| topic |
hepatitis C virus reporter cell line antivirals gene therapy siRNA |
| url |
https://www.mdpi.com/1999-4915/12/9/1044 |
| work_keys_str_mv |
AT hosseinmelbadawy generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT mohiimohammedabdul generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT naifaljuhani generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT adrianavitiello generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT francescociccarese generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT mohamedashaker generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT hebameltahir generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT giorgiopalu generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT veronicadidiantonio generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT haniehghassabian generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT claudiadeldelvecchio generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT cristianosalata generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT elisafranchin generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT eleonoraponterio generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT salehbahashwan generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT khaledthabet generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT mekkymabouzied generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT ahmedmshehata generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT cristinaparolin generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT ariannacalistri generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline AT gualtieroalvisi generationofcombinatoriallentiviralvectorsexpressingmultipleantihepatitiscvirusshrnasandtheirvalidationonanovelhcvreplicondoublereportercellline |
| _version_ |
1724588065082572800 |
| spelling |
doaj-cfffe25b4ddd4231ac75d53254bc11422020-11-25T03:27:37ZengMDPI AGViruses1999-49152020-09-01121044104410.3390/v12091044Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell LineHossein M. Elbadawy0Mohi I. Mohammed Abdul1Naif Aljuhani2Adriana Vitiello3Francesco Ciccarese4Mohamed A. Shaker5Heba M. Eltahir6Giorgio Palù7Veronica Di Di Antonio8Hanieh Ghassabian9Claudia Del Del Vecchio10Cristiano Salata11Elisa Franchin12Eleonora Ponterio13Saleh Bahashwan14Khaled Thabet15Mekky M. Abouzied16Ahmed M. Shehata17Cristina Parolin18Arianna Calistri19Gualtiero Alvisi20Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyPharmaceutics and Pharmaceutical Technology Department, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61519, EgyptDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Almadinah Almunawwarah 41477, Saudi ArabiaDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDepartment of Molecular Medicine, University of Padua, 35121 Padua, ItalyDespite 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.https://www.mdpi.com/1999-4915/12/9/1044hepatitis C virusreporter cell lineantiviralsgene therapysiRNA |