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Inhibition of Hepatitis C Virus Genotype 1a Non-Structural Proteins by Small Interference RNA in Human Hepatoma Cell Lines

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DOI: 10.4236/pp.2015.611053    3,682 Downloads   4,085 Views   Citations

ABSTRACT

Hepatitis C virus (HCV) infection and associated liver diseases are still challenging and represent a significant health care burden around the world. Although, the treatment strategies have been improved by the development of novel direct-acting antivirals, but such therapeutic options are still expensive and beyond the financial range of the most infected individuals in developing or even in resource replete countries. It demands an urgent need to search novel and improved alternate treatment strategies to treat the infection. The present study was aimed to develop an in vitro stable cell culture system, persistently expressing HCV genotype 1a non-structural genes and to characterize the inhibitory effects of synthetic siRNAs (short interference RNA) directed against the most conserved regions of nonstructural genes in an in vitro cell culture model. The continuous expression of nonstructural genes for more than 30 days post transfection was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis in stable human hepatoma cell line (Huh-7). The gene expression studies revealed significantly reduced gene expression of HCV nonstructural genes (i.e., NS2, NS4A and NS5A) both at mRNA and protein levels when treated against genome specific synthetic siRNAs in stable cell lines (51%, 47% and 54% respectively, p < 0.05). Similarly, a vivid decrease in HCV viral titer was exhibited by synthetic siRNAs in an in vitro viral replicate cell culture model (58%, 48% and 50%, respectively, p < 0.05) determined by quantitative Real-Time PCR (qPCR). Our data indicate that siRNA mediated gene silencing may be considered a promising alternate treatment strategy against HCV in combination with other effective therapeutic regimens in future.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Shahid, I. , AlMalki, W. , R. Almalki, S. , AlTurkestany, I. , AlGhamdi, H. and AlMenshawi, S. (2015) Inhibition of Hepatitis C Virus Genotype 1a Non-Structural Proteins by Small Interference RNA in Human Hepatoma Cell Lines. Pharmacology & Pharmacy, 6, 502-517. doi: 10.4236/pp.2015.611053.

References

[1] Shahid, I., AlMalki, W., Hafeez, M. and Hassan, S. (2014) Hepatitis C Virus Infection Treatment: An Era of Game Changer Direct Acting Antivirals and Novel Treatment Strategies. Critical Reviews in Microbiology, 6, 1-13.
http://dx.doi.org/10.3109/1040841X.2014.970123
[2] Shahid, I., Ahmad, W., Ijaz, B., Javed, F.T., Gull, S., Kausar, H., Sarwar, M.T., Asad, S., Sumrin, A., Khaliq, S., Jahan, S., Pervaiz, A. and Hassan, S. (2013) Stable Huh-7 Cell Line Development Expressing HCV Non-Structural Proteins of Genotype 1a. Journal of Virological Methods, 4, 4.
[3] Asad, S., Ijaz, B., Ahmad, W., Kausar, H., Sarwar, T., Gull, S., Shahid, I., Khan, M. and Hassan S. (2012) Development of Persistent HCV Genotype 3a Infection Cell Culture Model in Huh-7 Cell. Virology Journal, 9, 11.
http://dx.doi.org/10.1186/1743-422X-9-11
[4] Ansar, M., Ashfaq, U.A., Shahid, I., Sarwar, T., Javed, T., Rehman, S., Hassan, S. and Riazuddin S. (2011) Inhibition of Full Length Hepatitis C Virus Particles of 1a Genotype through Small Interference RNA. Virology Journal, 8, 203.
http://dx.doi.org/10.1186/1743-422X-8-203
[5] Ahmad, W., Ijaz, B., Javed, F.T., Jahan, S., Shahid, I., Khan, M. and Hassan S. (2010) HCV Genotype Distribution and Possible Transmission Risks in Lahore, Pakistan. World Journal of Gastroenterology, 16, 4321-4328.
http://dx.doi.org/10.3748/wjg.v16.i34.4321
[6] Ahmad, W., Shabbiri, K., Ijaz, B., Asad, S., Sarwar, M.T., Gull, S., Kausar, H., Fouzia, K., Shahid, I. and Hassan S. (2011) Claudin-1 Required for HCV Virus Entry Has High Potential for Phosphorylation and O-Glycosylation. Virology Journal, 8, 229.
http://dx.doi.org/10.1186/1743-422X-8-229
[7] Sarwar, M.T., Kausar, H., Ijaz, B., Ahmad, W., Ansar, M., Sumrin, A., Ashfaq, U.A., Asad, S., Gull, S., Shahid, I. and Hassan S. (2011) NS4A Protein as a Marker of HCV History Suggests that Different HCV Genotypes Originally Evolved from Genotype 1b. Virology Journal, 8, 317.
http://dx.doi.org/10.1186/1743-422X-8-317
[8] Lazaro, A., Chang, M., Tang, W., Campbell, J., Sullivan, G., Gretch, R., Corey, L., Coombs, W. and Fausto N. (2007) Hepatitis C Virus Replication in Transfected and Serum-Infected Cultured Human Fetal Hepatocytes. American Journal of Pathology, 170, 478-489.
http://dx.doi.org/10.2353/ajpath.2007.060789
[9] Butt, S., Idrees, M., Rehman, U., Ali, L., Hussain, A., Ali, M., Ahmed, N., Saleem, S. and Fayyaz M. (2011) Establishment of Stable Huh-7 Cell Lines Expressing Various Hepatitis C Virus Genotype 3a Protein: An In-Vitro Testing System for Novel Anti-HCV Drugs. Genetics Vaccines Therapy, 9, 12-17.
http://dx.doi.org/10.1186/1479-0556-9-12
[10] Buck, M. (2008) Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures. PLoS One, 3, e2660.
http://dx.doi.org/10.1371/journal.pone.0002660
[11] Molina, S., Castet, V., Pichard-Garcia, L., Wychowski, C., Meurs, E., Pascussi, M., Sureau, C., Fabre, M., Sacunha, A. and Larrey, D. (2008) Serum-Derived Hepatitis C Virus Infection of Primary Human Hepatocytes Is Tetraspanin CD81 Dependent. Journal of Virology, 82, 569-574.
http://dx.doi.org/10.1128/JVI.01443-07
[12] Lisowski, L., Elazar, M., Chu, K., Glenn, J. and Kay, M. (2013) The Anti-Genomic (Negative) Strand of Hepatitis C Virus Is Not Targetable by shRNA. Nucleic Acids Research, 41, 3688-3698.
http://dx.doi.org/10.1093/nar/gkt068
[13] Zheng, D., Giljohann, A. and Chen, L. (2012) Topical Delivery of siRNA-Based Spherical Nucleic Acid Nanoparticle Conjugates for Gene Regulation. Proceedings of the National Academy of Sciences of the United States of America, 109, 11975-11980.
http://dx.doi.org/10.1073/pnas.1118425109
[14] Gao, K., and Huang, L. (2009) Nonviral Methods for siRNA Delivery. Molecular Pharmacology, 6, 651-658.
http://dx.doi.org/10.1021/mp800134q
[15] Zintchenko, A., Philipp, A., Dehshahri, A. and Wagner, E. (2008) Simple Modifications of Branched PEI Lead to Highly Efficient siRNA Carriers with Low Toxicity. Bioconjugation Chemistry, 19, 1448-1455.
http://dx.doi.org/10.1021/bc800065f
[16] Caplen, J., Parrish, S., Imani, F., Fire, A. and Morgan, A. (2001) Specific Inhibition of Gene Expression by Small Double-Stranded RNAs in Invertebrate and Vertebrate Systems. Proceedings of the National Academy of Sciences of the United States of America, 98, 9742-9747.
http://dx.doi.org/10.1073/pnas.171251798
[17] Coburn, A. and Cullen, R. (2002) Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference. Journal of Virology, 76, 9225-9231.
http://dx.doi.org/10.1128/JVI.76.18.9225-9231.2002
[18] Brummelkamp, R., Bernards, R. and Agami, R. (2002) Stable Suppression of Tumorigenicity by Virus-Mediated RNA Interference. Cancer Cell, 2, 243-247.
http://dx.doi.org/10.1016/S1535-6108(02)00122-8
[19] Hannon, J. (2002) RNA Interference. Nature, 418, 244-251.
http://dx.doi.org/10.1038/418244a
[20] Korf, M., Meyer, A., Jarczak, D., Beger, C., Manns, P. and Kruger, M. (2007) Inhibition of HCV Subgenomic Replicons by siRNAs Derived from Plasmids with Opposing U6 and H1 Promoters. Journal of Viral Hepatology, 14, 122-132.
http://dx.doi.org/10.1111/j.1365-2893.2006.00793.x
[21] Seo, Y., Abrignani, S., Houghton, M. and Han, H. (2003) Small Interfering RNA-Mediated Inhibition of Hepatitis C Virus Replication in the Human Hepatoma Cell Line Huh-7. Journal of Virology, 77, 810-812.
http://dx.doi.org/10.1128/JVI.77.1.810-812.2003
[22] Khaliq, S., Jahan, S., Ijaz, B., Ahmad, W., Asad, S. and Hassan, S. (2011) Inhibition of Hepatitis C Virus Genotype 3a by siRNAs Targeting Envelope Genes. Archives of Virology, 156, 433-442.
http://dx.doi.org/10.1007/s00705-010-0887-6
[23] Zekri, R., Bahnassy, A., El-Din, M. and Salama, M. (2009) Consensus siRNA for Inhibition of HCV Genotype-4 Replication. Virology Journal, 6, 13.
http://dx.doi.org/10.1186/1743-422X-6-13
[24] Shin, D., Lee, H., Kim, I., Yoon, Y. and Kim M. (2009) Optimization of Linear Double-Stranded RNA for the Production of Multiple siRNAs Targeting Hepatitis C Virus. RNA, 15, 898-910.
http://dx.doi.org/10.1261/rna.1268209
[25] Khaliq, S., Jahan, S., Ijaz, B., Ahmad, W., Asad, S., Pervaiz, A., Samreen, B., Khan, M. and Hassan, S. (2010) Inhibition of Core Gene of HCV 3a Genotype Using Synthetic and Vector Derived siRNAs. Virology Journal, 7, 318.
http://dx.doi.org/10.1186/1743-422X-7-318
[26] Ahmad, W., Ijaz, B., Javed, T., Kausar, H., Sarwar, M.T., Gull, S., Asad, S., Shahid, I. and Hassan, S. (2011) HCV Genotype-Specific Correlation with Serum Markers: Higher Predictability for Genotype 4a. Virology Journal, 8, 293.
http://dx.doi.org/10.1186/1743-422X-8-293
[27] Grimm, S. (2004) The Art and Design of Genetic Screens: Mammalian Culture Cells. Nature Reviews in Genetics, 5, 179-189.
http://dx.doi.org/10.1038/nrg1291
[28] Wurm, M. (2004) Production of Recombinant Protein Therapeutics in Cultivated Mammalian Cells. Nature Biotechnology, 22, 1393-1398.
http://dx.doi.org/10.1038/nbt1026
[29] Jarczak, D., Korf, M., Beger, C., Manns, P. and Kruger, M. (2005) Hairpin Ribozymes in Combination with siRNAs against Highly Conserved Hepatitis C Virus Sequence Inhibit RNA Replication and Protein Translation from Hepatitis C Virus Subgenomic Replicons. FEBS Journal, 272, 5910-5922.
http://dx.doi.org/10.1111/j.1742-4658.2005.04986.x
[30] Kim, M., Shin, D., Kim, S. and Park, M. (2006) Inhibition of Hepatitis C Virus Gene Expression by Small Interfering RNAs Using a Tri-Cistronic Full-Length Viral Replicon and a Transient Mouse Model. Virus Research, 122, 1-10.
http://dx.doi.org/10.1016/j.virusres.2006.05.003
[31] Liu, M., Ding, H., Zhou, P., Qin, L., Gao, J., Cao, M., Luan, J., Wu, B. and Qi, T. (2006) RNA Interference Effectively Inhibits mRNA Accumulation and Protein Expression of Hepatitis C Virus Core and E2 Genes in Human Cells. Bioscience Biotechnology and Biochemistry, 70, 2049-2055.
http://dx.doi.org/10.1271/bbb.60001
[32] Grakoui, A., McCourt, W., Wychowski, C., Feinstone, M. and Rice, M. (1993) Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites. Journal of Virology, 67, 2832-2843.
[33] Bartenschlager, R., Lohmann, V., Wilkinson, T. and Koch, O. (1995) Complex Formation between the NS3 Serine-Type Proteinase of the Hepatitis C Virus and NS4A and Its Importance for Polyprotein Maturation. Journal of Virology, 69, 7519-7528.
[34] Tanji, Y., Hijikata, M., Satoh, S., Kaneko, T. and Shimotohno, K. (1995) Hepatitis C Virus Encoded Non-Structural Protein NS4A Has Versatile Functions in Viral Protein Processing. Journal of Virology, 69, 1575-1581.
[35] Macdonald, A., Crowder, K., Street, A., McCormick, C. and Harris, M. (2004) The Hepatitis C Virus NS5A Protein Binds to Members of the Src Family of Tyrosine Kinases and Regulates Kinase Activity. Journal of General Virology, 85, 721-729.
http://dx.doi.org/10.1099/vir.0.19691-0
[36] Reed, E., Xu, J. and Rice, M. (1997) Phosphorylation of the Hepatitis C Virus NS5A Protein in Vitro and in Vivo: Properties of the NS5A-Associated Kinase. Journal of Virology, 71, 7187-7197.
[37] Tan, L. and Katze, G. 92001) How Hepatitis C Virus Counteracts the Interferon Response: The Jury Is Still Out on NS5A. Virology, 284, 1-12.
http://dx.doi.org/10.1006/viro.2001.0885
[38] Gale, J., Korth, M.J., Tang, M., Tan, L., Hopkins, A., Dever, E., Polyak, J., Gretch, R. and Katze, G. (1997) Evidence That Hepatitis C Virus Resistance to Interferon Is Mediated through Repression of the PKR Protein Kinase by the Non-Structural 5A Protein. Virology, 230, 217-227.
http://dx.doi.org/10.1006/viro.1997.8493
[39] El-Awardy, K., Tabll, A., El-Abd, S., Bahgat, M., Shoeb, A., Youssef, S., El-Din, B., Redwan, M., El-Demellawy, M., Omran, H., El-Garf, T. and Goueli, A. (2006) HepG2 Cells Support Viral Replication and Gene Expression of Hepatitis C Virus Genotype 4 in Vitro. World Journal of Gastroenterology, 12, 4836-4842.
[40] Lazaro, A., Chang, M., Tang, W., Campbell, J., Sullivan, G., Gretch, D., Corey, L., Coombs, W. and Fausto, N. (2007) Hepatitis C Virus Replication in Transfected and Serum-Infected Cultured Human Fetal Hepatocytes. American Journal of Pathology, 170, 478-489.
http://dx.doi.org/10.2353/ajpath.2007.060789
[41] Guha, C., Lee, W., Chowdhury, R. and Chowdhury, J. (2005) Cell Culture Models and Animal Models of Viral Hepatitis. Part II: Hepatitis C. Laboratory Animals, 34, 39-47.
http://dx.doi.org/10.1038/laban0205-39
[42] Kanda, T., Steele, R., Ray, R. and Ray, B. (2007) Small Interfering RNA Targeted to Hepatitis C Virus 5’ Nontranslated Region Exerts a Potent Antiviral Effect. Journal of Virology, 81, 669-676.
http://dx.doi.org/10.1128/JVI.01496-06
[43] Kapadia, B., Brideau-Andersen, A. and Chisari, V. (2003) Interference of Hepatitis C Virus RNA Replication by Short Interfering RNAs. Proceedings of the National Academy of Sciences of the United States of America, 100, 2014-2018.
http://dx.doi.org/10.1073/pnas.252783999
[44] McCaffrey, P., Mause, L., Pham, T., Conklin, S., Hannon, J. and Kay, A. (2002) RNA Interference in Adult Mice. Nature, 418, 38-39.
http://dx.doi.org/10.1038/418038a
[45] Pan, Q., Henry, D., Metselaar, J., Scholte, B., Kwekkeboom, J., Tilanus, W., Janssen, L. and van der Laan, J. (2009) Combined Antiviral Activity of Interferon-Alpha and RNA Interference Directed against Hepatitis C without Affecting Vector Delivery and Gene Silencing. Journal of Molecular Medicine, 87, 713-722.
http://dx.doi.org/10.1007/s00109-009-0470-3
[46] Smith, M., Smolic, R., Volarevic, M. and Wu, Y. (2007) Positional Effects and Strand Preference of RNA Interference against Hepatitis C Virus Target Sequences. Journal of Viral Hepatitis, 14, 194-212.
http://dx.doi.org/10.1111/j.1365-2893.2006.00794.x

  
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