Screening, Design And Functional Mechanism Of Anti-HCV Peptides

Hepatitis C virus (HCV) is a positive single strand RNA virus and classified in the Hepacivirus genus within the Flaviviridae family. HCV infection is a major cause of chronic liver disease, such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). More than200million people are infected with HCV all over the world. The current standard of care for patients with chronic hepatitis C depends on combination therapy with pegylated interferon a (IFNa) and ribavirin. The most recently developed therapy contains protease plus the above dual therapy. However, these therapies are unsatisfactory in clinical because of the side effects, drug resistance and low sustained virological respons for many patients. Besides, there is no effective vaccine for the preventation of HCV infection. Therefore, development of potential novel antiviral agents is urgently needed.The venom gland cDNA libraries of the scorpion C. tricostatus and C. tryznai were constructed and characterized by our group. After sequence analysis,13peptides were classified as antimicrobial peptides and their mature peptides were chemically synthesized and screened for the ability of inhibiting HCV (JFH-1) infection in Huh7.5.1cells. In vitro studies revealed that one peptide named Ctry2459displayed poten inhibitory effect on HCV infection via inactivating infectious viral particles. The50%effective contration (EC50) of Ctry2459peptide wasl.84μg/ml, its50%cytotoxic concentration (CC50) and50%hemolytic concentration (HC50) were79.8μg/ml and137.9μg/ml, the selective index was43.4, revealing admirable pharmacological properties. Although Ctry2459peptide exhibited inhibitory effect on HCV infection, but it cannot suppress established infection of HCV because of the poor cellular uptake and restriction of endosomes. The bioavailability of Ctry2459peptide can be improved by regulating the cellular uptake and endosomal escape pathway. Based on the molecular template of the Ctry2459peptide, two histidine-rich peptides (Ctry2459-H2and Ctry2459-H3) were designed. The derived peptides Ctry2459-H2and Ctry2459-H3adopted the same a helix comformation with Ctry2459peptide and maintained the similar hibitory effect on HCV initiate infection. The two peptides inhibited HCV infection efficiently with EC50of1.08μg/ml and0.85μg/ml. They also can inhibit established HCV infection by breaking through the barriers of cellular uptake and endosome. When treated48hour postinfection, Ctry2459-H2and Ctry2459-H3peptides can inhibit more than40%and70%intracellular HCV RNA content. In distinct contrast to Ctry2459, Ctry2459-H2and Ctry2459-H3peptides demonstrated lower cytotoxic and hemolytic activities, with CC50of more than500μg/ml, and HC50of203.3μg/ml and416.4μg/ml, revealing much more admirable pharmacological properties.Based on the structure and function characteristic of HCV p7ion channel, we designed four peptides from the transmembrane domain of p7ion channel which may disrupt the normal channel assembly. The four peptides were chemical synthesized and in vitro study revealed that one peptide named p7avp3potently inhibited established HCV infection with EC50of2.19±0.16μM. Moreover, the CC50of p7avp3peptide against Huh7.5.1cell was83.64±6.95μM, the selective index was38.19. Although p7avp3peptide can suppress established HCV infection, but it has a low selective index. On the basis of p7avp3, we designed10new peptides with modification of the polarity and net charge. In vitro study exhibited that one peptide named p7avp5exhibited stronger inhibitory effect on HCV proliferation in vitro, with EC50of1.09±0.13μM, CC50was115.99±16.68μM, the selective index was106.41. Moreover, both p7avp3and p7avp5peptides exhibited low hemolytic activities, and displayed high serum stability.The anti-HCV mechanism of p7avp5peptide was further determined. A time of peptide addition experiment revealed that p7avp5peptide displayed multiple inhibitory effect of HCV infection, which contains viral and cellular inactivation, viral entry inhibition and postentry inhibiton. The most efficient antiviral effect occurs when p7avp5was used to pretreat viral supernatant, revealing a potent and rapid viral inactivation effect, but without any effect on the viral RNA. Confocal microscopy showed that p7avp5peptide rapidly interacted with cell membrane and was absorbed on the membrane surface, which significantly inhibit the initiate HCV infection but without any influence of subsequent viral replication. p7avp5peptide gradually entered cells after about24hour incubation and resulted the inhibition of intracellular viral infectivity. Thus, p7avp5peptide is likely to have three antiviral modes, containing freely viral and cellular inactivation and intracellular viral inactivation. Moreover, electrophysiological experiment was used to detect the influence of p7avp5peptide on normal p7ion channel. P7avp5peptide was added either after transfection of p7and incubated throughout the whole experiment before the channel current detection, or added as a blocking agent in the patch clamp experiment. Both the two experiment determined that p7avp5peptide has no effect on the assembly of p7ion channel or block the channel current, corresponding with the effect on viral release.In conclusion, this work identified a new natural anti-HCV peptide Ctry2459from the scorpion peptide libraries, obtained derived peptides with enhanced bioavailability and potent anti-HCV activities by rational design. This work also designed a new anti-HCV peptide p7avp5based on the structure and function characteristic of HCV p7ion channel and identified its antiviral activities and mechanism. This work designed new anti-HCV peptides with potential application value, enriched the exsisting development strategies of antiviral peptides, provided theoretical basis for the discovery and application of natural animal toxins and the molecular design of new antiviral peptides.