Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein

Abstract

c Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated tohave strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-␤) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients. Hepatitis C virus (HCV) infects 3% of the world's population, and persistent virus infection causes chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma (1). There is no vaccine available, and alpha interferon (IFN-␣)-based therapy is the current treatment for patients with chronic hepatitis C (2). Many patients do not response to the therapy. There is an urgent need to develop well-tolerated and effective therapeutic drugs against HCV infection (3).HCV is an enveloped, single positive-strand RNA virus of the Flaviviridae family. The 9.6-kb viral genome encodes one polyprotein that is processed by viral and cellular proteases to produce structural proteins including core protein and envelope proteins E1 and E2 as well as the nonstructural proteins consisting of the p7 ion channel, NS2-3 protease, NS3 serine protease, RNA helicase, NS4A polypeptide, NS4B, NS5A proteins, and NS5B RNA-dependent RNA polymerase. E1 and E2 are type I membrane glycoproteins and form a noncovalent complex, which is believed to be the building block for the viral envelope (4). E2 is thought to be primarily responsible for receptor binding (5). Infection of the host cells by HCV is initiated through the interactions between the E1E2 protein and several previously identified HCV entry receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1 (CLDN1), and occludin (OCLN) (6-9). The essential role of E1E2 i...

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

“…The selected aptamers have higher affinity to genotype 1a, 1b and 2a than others, and strongly prevented HCV viral infection in Huh7 5.1 cells [81]. Afterwards, Yang et al described the potential antiviral action of DNA aptamers selected against E1E2 protein by HCV infection suppression in HuH7.5 cells without innate immune response action [117]. In the case of core, an essential protein for HCV viral assembly, Shi et al have applied for therapy the above-mentioned aptamers in diagnostics.…”

Application of Nucleic Acid Aptamers to Viral Detection and Inhibition

“…Aptamer ZE2 showed the highest affinity and specificity to E2 and was able to detect HCV particles and block HCV infection on human cultured hepatocytes by CD81 binding inhibition [89]. A similar inhibition mechanism was observed on the DNA aptamer E1E2-6, which inhibited viral infection by blocking host cell binding [90]. A new system developed to quantify immobilized infectious HCV particles in microplates (so-called enzyme linked apto-sorbent assay or ELASA) used aptamers against E2 instead of antibodies and resulted in an effective and easy-to-use tool to quantify infectious units of HCV and to monitor anti-HCV drug efficacies [91].…”

Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

“…Interestingly, Sc5-c3 interaction with HPV-16 VLPs appears more stable than the described HPV-33 interaction with the cell surface (K d = 100 pM) which is mainly governed by electrostatic interactions and probably conserved for high-risk HPVs [14,41]. This provides an insight to the potential Sc5-c3 applications as a competitor to block HPV infection, as previously shown for aptamers against human influenza virus [42,43], hepatitis C virus [44], and cytomegalovirus [36] infections.…”