Interference of hepatitis C virus RNA replication by short interfering RNAs

Abstract

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN␣ in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.H epatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (1, 2). Viral clearance during acute HCV infection is usually associated with a multispecific CD4 ϩ and CD8 ϩ T cell response, which is weak or undetectable in subjects who do not control the infection (3-5). Importantly, most chronically infected patients fail to resolve HCV infection after combination therapy with .The HCV genome is a positive-stranded Ϸ9.6-kb RNA molecule consisting of a single ORF, which is flanked by 5Ј and 3Ј UTR. The HCV 5Ј UTR contains a highly structured internal ribosome entry site (8-13). The HCV ORF encodes a single polyprotein that is 3,008-3,037 aa in length and is posttranslationally modified to produce at least ten different proteins: core, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1,13,14). Despite considerable advances in the understanding of the function of these proteins, the basic mechanism(s) of HCV replication still remain unclear because of the absence of a tissue culture system that can sustain productive virus infection (1). The recent development of subgenomic and full-length HCV replicons that replicate and express HCV proteins in stably transfected human hepatoma cell-derived Huh-7 cells has facilitated the analysis of the role of cellular pathways required in HCV replication and the efficacy of antiviral drugs (15-19). For example, by using HCV replicons, the antiviral effects of IFN␣ and IFN␥ have been clearly demonstrated (20-23). Although IFN treatment can efficiently inhibit HCV replication in cultured Huh-7 cells, Ͼ60% of patients treated with IFN do not eliminate the virus (24)(25)(26)(27)(28). This suggests that HCV may be able to induce a state of IFN resistance in the infected liver. In keeping with this notion, HCV E2 and NS5A proteins have been demonstrated to interfere with the IFN-induced signaling pathway by interacting with protein kinase R (PKR) and inhibiting its kinase activity (29-32). Thus, alternative approaches to the treatment of chronic HCV infection seem to be warranted. Double-s...

Prostate cancer invasion is influenced more by expression of a CD44 isoform including variant 9 than by Muc18

“…Others have documented that the RNAi effect takes up to 4 days after transfection to become maximal and may persist up to 6 days post-transfection. 36 We also noted no change in protein expression by Western blot after 24-48 h; therefore, we waited 90 h before performing the Matrigel invasion assay.…”

“…Transfection in serum-free medium proceeded for 6 h. Others have found a 3-4 day interval of further incubation optimal in order for depletion of previously synthesized protein. 36 We tried post-transfection incubations ranging from 1-6 days, and optimized the interval for abrogation of protein expression by Western blot at 90 h, in serum-containing medium.…”

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

“…Total RNA was isolated as described above and RPA was performed as described in ref. 45. The sequences for each primer are as follows: ATP citrate lyase (sense) 5Ј-CTCCGATGAGACCATCTACATT-3Ј and (antisense) 5Ј-ATCAAGCTTGTACTCTTGCCAGTTCAT-TGAGA-3Ј; acetyl-CoA synthetase (sense) 5Ј-AAGGAT-GCCCGGCTATGAT TGGT-3Ј and (antisense) 5Ј-AT-CA AGCT TCGAGTGGTGTGATGCCGAGGAC-3Ј; FAS (sense) 5Ј-TGTCGTTGGTGCTCATCGTC-3Ј and (antisense) 5Ј-ATCA AGCT T TGGTCT TGAGAGATGGCT-TGA-3Ј; hydroxymethylglutaryl (HMG)-CoA synthase (sense) 5Ј-ATCA AGCT T TATGCCCTGGTAGT TGCAGGAG-3Ј and (antisense) 5Ј-T TGCCTCT T TCTGCCACTGG-3Ј; HMG-CoA reductase (sense) 5Ј-ATCAAGCTTTACCA-TGTCAGGGGTACGTC-3Ј and (antisense) 5Ј-CAAGCCT-AGAGACATAATCATC-3Ј; squalene synthase (sense) 5Ј-A A A A ACTCTGCCATCCCA ATGC-3Ј and (antisense) 5Ј-ATCAAGCTTAAGAAGGTCCCGCTGTTACACAA-3Ј; L32 (sense) 5Ј-GCGATCTCGGCACAGTAAGATTT-3Ј and (antisense) 5Ј-ATCAAGCTTCTTGATGCCCAACATTG-GTTATG-3Ј.…”

Inhibition of influenza virus production in virus-infected mice by RNA interference

“…Because siRNAs confer transient interference of gene expression in a sequence-specific manner, they represent a new class of molecules that are likely to have significant medical applications (14,15). siRNAs have been shown to inhibit virus infection if they are transfected into cultured cells by electroporation or by using cationic lipids (16)(17)(18)(19)(20). Similarly, we have previously shown that siRNAs specific for conserved regions of the influenza A virus genes can inhibit virus production profoundly in cultured cells and embryonated chicken eggs (21).…”