GA-Hecate antiviral properties on HCV whole cycle represent a new antiviral class and open the door for the development of broad spectrum antivirals.

In recent years, synthetic peptides have been considered promising targets for drug development that possess low side-effects, are cost-effective and are susceptible to rational design. Hecate was initially described as a potent bacterial inhibitor and subsequently as an anticancer drug with functions related to its lipid interaction property. Viruses, such as hepatitis C virus (HCV), have a lipid-dependent life cycle and could be affected by Hecate in many ways. Here, we assessed modifications on Hecate's N-terminus region and its effects on HCV and hepatotoxicity. Gallic acid-conjugated Hecate was the most efficient Hecate-derivative, presenting high potential as an antiviral and inhibiting between 50 to 99% of all major steps within the HCV infectious cycle. However, the most promising aspect was GA-Hecate's mechanism of action, which was associated with a balanced lipid interaction with the viral envelope and lipid droplets, as well as dsRNA intercalation, allowing for the possibility to affect other ssRNA viruses and those with a lipid-dependent cycle.

Heat shock proteins HSPB8 and DNAJC5B have HCV antiviral activity.

Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.

Inhibition of hepatitis C virus using siRNA targeted to the virus and Hsp90.

Hepatitis C (HCV) is a viral disease affecting millions of people worldwide, and persistent HCV infection can lead to progressive liver disease with the development of liver cirrhosis and hepatocellular carcinoma. During treatment for hepatitis C, the occurrence of viral resistance is common. To reduce the occurrence of resistance, new viral treatments should target both viral and cellular factors. Many interactions occur between viral and host proteins during the HCV replication cycle and might be used for the development of new therapies against hepatitis C. Heat shock protein 90 (Hsp90) plays a role in the folding of cellular and viral proteins and also interacts with HCV proteins. In the present study, we knocked down the expression of the Hsp90 gene and inhibited viral replication using siRNA molecules. Reducing the expression of Hsp90 successfully decreased HCV replication. All siRNA molecules specific to the viral genome showed the efficient inhibition of viral replication, particularly siRNA targeted to the 5'UTR region. The combination of siRNAs targeting the viral genome and Hsp90 mRNA also successfully reduced HCV replication and reduced the occurrence of viral resistance. Moreover, these results suggest that an approach based on the combination of cellular and viral siRNAs can be used as an effective alternative for hepatitis C viral suppression.

Evaluation of canonical siRNA and Dicer substrate RNA for inhibition of hepatitis C virus genome replication--a comparative study.

Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. The objective of this study was to test the ability of five Dicer substrate siRNAs (DsiRNA) to inhibit HCV replication and to compare these molecules to canonical 21 nt siRNA. DsiRNA molecules were designed to target five distinct regions of the HCV genome - the 5' UTR and the coding regions for NS3, NS4B, NS5A or NS5B. These molecules were transfected into Huh7.5 cells that stably harboured an HCV subgenomic replicon expressing a firefly luciferase/neoR reporter (SGR-Feo-JFH-1) and were also tested on HCVcc-infected cells. All of the DsiRNAs inhibited HCV replication in both the subgenomic system and HCVcc-infected cells. When DsiRNAs were transfected prior to infection with HCVcc, the inhibition levels reached 99.5%. When directly compared, canonical siRNA and DsiRNA exhibited similar potency of virus inhibition. Furthermore, both types of molecules exhibited similar dynamics of inhibition and frequencies of resistant mutants after 21 days of treatment. Thus, DsiRNA molecules are as potent as 21 nt siRNAs for the inhibition of HCV replication and may provide future approaches for HCV therapy if the emergence of resistant mutants can be addressed.