Reverse Genetic Analysis of NS2 Mutational Diversity in Hepatitis C Virus

Amber Hassan, Riffat Mehboob, Imrana Tanvir, Husam Malibary, Bassam M. J. Addas, Almotasimbellah O. Rayes, Farhat Bano, Omar Mohammad Bahassan, Ahmad Alwazzan


Hepatitis C Virus (HCV) is a main etiological and causative agent for acute and chronic liver disease, leading to liver cancer. The NS2 protein of HCV has been recently implicated in virus particle assembly. Objective of the study is to evaluate the mutation variations in the NS2 Gene of HCV and map epitopes in the variable NS2 region After clinical evaluation, 40 serum samples from HCV patients were obtained under sterile conditions. RNA was extracted from stored serum samples. Primers were designed, and amplicons were amplified. The mean age of the patients was 47.6 years. The RNA was extracted from all 40 serum samples and was detected through agarose gel electrophoresis. A single band of 470bp with maximum concentration was obtained at 61ºC. SNPs were detected in the amplified regions. A complex network of interactions involving NS2 and other viral structural and nonstructural proteins was observed during virus assembly.


Keywords: hepatitis C virus, NS2 nonstructural proteins, single nucleotide polymorphism.


Full Text:



VESCOVO T., REFOLO G., VITAGLIANO G., FIMIA G. M., and PIACENTINI M. Molecular mechanisms of hepatitis C virus–induced hepatocellular carcinoma. Clinical Microbiology and Infection, 2016, 22(10): 853-861.

PFAENDER S., BROWN R. J., PIETSCHMANN T., and STEINMANN E. Natural reservoirs for homologs of hepatitis C virus. Emerging Microbes & Infections, 2014, 3(1): 1-9.

BORGIA S. M., HEDSKOG C., PARHY B., HYLAND R. H., STAMM L. M., BRAINARD D. M., SUBRAMANIAN M. G., MCHUTCHISON J. G., MO H., SVAROVSKAIA E., and SHAFRAN S. D. Identification of a novel hepatitis C virus genotype from Punjab, India: expanding classification of hepatitis C virus into 8 genotypes. The Journal of Infectious Diseases, 2018, 218(11): 1722-1729.

CHAHAL J. A human microRNA, miR-122, promotes Hepatitis C virus RNA accumulation through three distinct mechanisms. McGill University, Montreale, 2020.

TABATA K., NEUFELDT C. J., and BARTENSCHLAGER R. Hepatitis C virus replication. Cold Spring Harbor Perspectives in Medicine, 2019, 10(3): a037093.

WU M.-J., SHANMUGAM S., WELSCH C., and YI M. Palmitoylation of hepatitis C virus NS2 regulates its subcellular localization and NS2-NS3 autocleavage. Journal of Virology, 2019, 94(1): e00906-19.

BUKH J., MILLER R. H., and PURCELL R. H. Genetic heterogeneity of hepatitis C virus: Quasispecies and genotypes. Seminars in Liver Disease, 1995, 15(1): 41-63.

BOUKADIDA C., FRITZ M., BLUMEN B., FOGERON M.-L., PENIN F., and MARTIN A. NS2 proteases from hepatitis C virus and related hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities. PLoS Pathogens, 2018, 14(2): e1006863.

SHUNMUGAM L. In silico investigation of hepatitis c virus: a novel perspective into targeted viral inhibition of NS3 helicase, NS 3/4a protease, and NS5b RNA dependent RNA polymerase. PhD thesis, University of KwaZulu-Natal, 2019.

WALTHER T., BRUHN B., ISKEN O., and TAUTZ N. A novel NS3/4A protease dependent cleavage site within postviral NS2. Journal of General Virology, 2021, 102(10): 001666.

NEUFELDT C. J., CORTESE M., ACOSTA E. G., and BARTENSCHLAGER R. Rewiring cellular networks by members of the Flaviviridae family. Nature Reviews Microbiology, 2018, 16(3): 125-142.

LEUMI S., GUO M., LU J., WANG Z., GAN T., HAN L., NGARI J., TONG Y., XIANG X., XIE Q., WANG L., and ZHONG J. Identification of a novel replication-competent hepatitis C virus variant that confers the sofosbuvir resistance. Antiviral Research, 2022, 197: 105224.

STEINMANN E., & PIETSCHMANN T. Cell culture systems for hepatitis C virus. In: BARTENSCHLAGER R. (Ed.) Hepatitis C Virus: From Molecular Virology to Antiviral Therapy. Springer, Berlin, 2013: 17-48.

BARTENSCHLAGER R., FRESE M., and PIETSCHMANN T. Novel insights into hepatitis C virus replication and persistence. Advances in Virus Research, 2004, 63: 72-181.

PIETSCHMANN T., & BROWN R. J. Hepatitis C virus. Trends in Microbiology, 2019, 27(4): 379-380.

ZHENG F., LI N., XU Y., ZHOU Y., and LI Y.-P. Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum. Journal of Biological Chemistry, 2021, 296: 100018.

JIRASKO V., MONTSERRAT R., APPEL N., JANVIER A., EUSTACHI L., BROHM C., STEINMANN E., PIETSCHMANN T., PENIN F., and BARTENSCHLAGER R. Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. The Journal of Biological Chemistry, 2008, 283(42): 28546-28562.

MORADPOUR D., & BLUM H. E. A primer on the molecular virology of hepatitis C. Liver International, 2004, 24(6): 519-525.

KHAN M. U., SADIA H., IRSHAD A., BAIG A. A., ASHIQ S., ZAHID B., SHEIKH R., ROSHAN S., ALI A., SHAMAS S., BHINDER M. A., and AHMAD R. Detection, quantification, and genotype distribution of HCV patients in Lahore, Pakistan by real-time PCR. African Health Sciences, 2020, 20(3): 1143-1152.

WASITTHANKASEM R., VICHAIWATTANA P., SIRIPON N., POSUWAN N., AUPHIMAI C., KLINFUENG S., THANETKONGTONG N., VUTHITANACHOT V., SAIYATHA S., THONGMAI C., SOCHOO S., PONGSUWAN N., POOVORAWAN K., TANGKIJVANICH P., and POOVORAWAN Y. Liver disease burden and required treatment expenditures for hepatitis C virus (HCV) infection in Thailand: Implications for HCV elimination in the new therapeutic era, a population-based study. PLoS One, 2018, 13(4): e0196301.

HAYES C. N., IMAMURA M., and CHAYAMA K. Management of HCV patients in cases of direct-acting antiviral failure. Expert Review of Gastroenterology & Hepatology, 2019, 13(9): 839-848.

UTRILLA-TRIGO S., JIMÉNEZ-CABELLO L., CALVO-PINILLA E., MARÍN-LÓPEZ A., LORENZO G., SÁNCHEZ-CORDÓN P., MORENO S., BENAVIDES J., GILBERT S., and NOGALES A. The Combined Expression of the Non-structural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge. Journal of Virology, 2021, 96(3).

SU Y., LIN T., LIU C., CHENG C., HAN X., and JIANG X. MicroRNAs, the Link Between Dengue Virus and the Host Genome. Frontiers in Microbiology, 2021: 2240.

BARNARD T. R., ABRAM Q. H., LIN Q. F., WANG A. B., and SAGAN S. M. Molecular Determinants of Flavivirus Virion Assembly. Trends in Biochemical Sciences, 2021, 46(5): 378-390.

SINIGAGLIA L., GRACIAS S., DÉCEMBRE E., FRITZ M., BRUNI D., SMITH N., HERBEUVAL J.-P., MARTIN A., DREUX M., TANGY F., and JOUVENET N. Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons. Scientific Reports, 2018, 8(1): 1-15.

JIRASKO V., MONTSERRAT R., LEE J. Y., GOUTTENOIRE J., MORADPOUR D., PENIN F., and BARTENSCHLAGER R. Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly. PLoS Pathogens, 2010, 6(12): e1001233.

ZHOU H., QIAN Q., SHU T., XU J., KONG J., MU J., QIU Y., and ZHOU X. Hepatitis C virus NS2 protein suppresses RNA interference in cells. Virologica Sinica, 2020, 35(4): 436-444.


  • There are currently no refbacks.