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

ABSTRACT Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have 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-β) 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.

[1]  David Pryde,et al.  Small Molecules Targeting Hepatitis C Virus-Encoded NS5A Cause Subcellular Redistribution of Their Target: Insights into Compound Modes of Action , 2011, Journal of Virology.

[2]  J. Crawford,et al.  Hepatitis C virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system. , 2007, Gastroenterology.

[3]  Ross D. Cranston,et al.  Protection of HIV Neutralizing Aptamers against Rectal and Vaginal Nucleases , 2010, The Journal of Biological Chemistry.

[4]  D. H. Burke,et al.  Broad-spectrum aptamer inhibitors of HIV reverse transcriptase closely mimic natural substrates , 2011, Nucleic acids research.

[5]  M. Houghton,et al.  Binding of hepatitis C virus to CD81. , 1998, Science.

[6]  S. Lemon,et al.  Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Darong Yang,et al.  Innate Host Response in Primary Human Hepatocytes with Hepatitis C Virus Infection , 2011, PloS one.

[8]  Charles M. Rice,et al.  Human occludin is a hepatitis C virus entry factor required for infection of mouse cells , 2009, Nature.

[9]  P. Bates,et al.  A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. , 2010, Cancer research.

[10]  Charles M. Rice,et al.  Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication , 2002, Journal of Virology.

[11]  N. Enomoto,et al.  Characterization of naturally occurring protease inhibitor-resistance mutations in genotype 1b hepatitis C virus patients , 2012, Hepatology International.

[12]  Xiao-lian Zhang,et al.  CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2 , 2009, PloS one.

[13]  R. Bartenschlager,et al.  Production of infectious hepatitis C virus in tissue culture from a cloned viral genome , 2005, Nature Medicine.

[14]  Charles M. Rice,et al.  Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry , 2007, Nature.

[15]  Xiaobing Zhang,et al.  Molecular aptamers for drug delivery. , 2011, Trends in biotechnology.

[16]  R. Cortese,et al.  The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus , 2002, The EMBO journal.

[17]  T. Baumert,et al.  Hepatitis C virus entry. , 2013, Current topics in microbiology and immunology.

[18]  Todd M. Allen,et al.  Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras. , 2011, The Journal of clinical investigation.

[19]  C. Rice,et al.  Hepatitis C Virus Entry* , 2008, Journal of Biological Chemistry.

[20]  D. Burton,et al.  Robust hepatitis C virus infection in vitro. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Huijia Dong,et al.  Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line , 2011, Journal of viral hepatitis.

[22]  D. Guyer,et al.  Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease , 2006, Nature Reviews Drug Discovery.

[23]  A. Rein,et al.  RNA Aptamers Directed to Human Immunodeficiency Virus Type 1 Gag Polyprotein Bind to the Matrix and Nucleocapsid Domains and Inhibit Virus Production , 2010, Journal of Virology.

[24]  Jin-Xiong She,et al.  Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line , 2003, Hepatology.

[25]  D. Rowlands,et al.  Formation of Higher-Order Foot-and-Mouth Disease Virus 3Dpol Complexes Is Dependent on Elongation Activity , 2011, Journal of Virology.

[26]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[27]  D. Lavanchy,et al.  The global burden of hepatitis C , 2009, Liver international : official journal of the International Association for the Study of the Liver.

[28]  Sheng-Nan Lu,et al.  Is the Cancer of the Liver Italian Program system an adequate weighting for survival of hepatocellular carcinoma? Evaluation of intrascore prognostic value among 36 subgroups , 2009, Liver international : official journal of the International Association for the Study of the Liver.

[29]  R. Bartenschlager,et al.  Replication of hepatitis C virus. , 2000, The Journal of general virology.

[30]  Toshiaki Maruyama,et al.  Complete Replication of Hepatitis C Virus in Cell Culture , 2005, Science.

[31]  J. Bukh,et al.  Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses. , 2011, Gastroenterology.

[32]  S. Locarnini,et al.  Hepatitis C virus resistance to protease inhibitors. , 2011, Journal of hepatology.

[33]  N. Sakamoto,et al.  New therapeutic approaches to hepatitis C virus , 2009, Journal of Gastroenterology.

[34]  O. Weiland,et al.  Telaprevir for retreatment of HCV infection. , 2011, The New England journal of medicine.

[35]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[36]  C. Bréchot.  Hepatitis C virus , 1996, Digestive Diseases and Sciences.

[37]  David L Bryce,et al.  Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex , 2008, Proceedings of the National Academy of Sciences.