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Function
HCV is most closely related to the Flaviviridae family. These viruses have a NS3 serine protease that is necessary for replication, but it is not known why. HCV also has a NS2-3 serine protease that executes cis cleavage at the 2/3 site and is autocatalytic, unlike other RNA viruses that just utilize the NS3 serine protease (2).
The function of Hepatitis C primase is to build the viral capsid. The viral capsid is built by forming alpha helices in areas with bactericide, and forming aggregates in areas that lack them. Assembly happens from interactions between dimers and endoplasmic reticulum, along with other oligomers, proteins, and lipid droplets. The viral capsid and two glycoproteins make up the genome (1).
The function of Hepatitis C helicase is to stop viral RNA from binding by stripping it of its proteins. It is necessary for viral replication (2). Helicase can process a wide range of nucleic acid sequences and unwind them (5).
Together, Hepatitis C primase and helicase functions together are still undetermined; however, it is believed that they work together to code a polyprotein and then cleave it to produce the genome and some nonstructural proteins (10).
Disease
Hepatitis C (HCV) is a viral infection that causes inflammation of the liver. As of 2022, there has been no vaccine created for Hepatitis C (6). HCV was originally thought of as a putative viral hepatitis that developed after some sort of blood transfusion of intravenous drug usage. The Hepatitis C virus causes large amounts of damage to the liver, disrupting normal liver function and damaging the quality of life for the patient, due to the normal progression of the infection. Development of hepatitis C is expected to progress to chronic liver disease, cirrhosis, and other hepatocellular carcinomas. HCV is shown to be the leading cause of chronic liver disease in both industrialized and developing countries (9). Acute HCV is clinically mild and normally unrecognized and undiagnosed due to the insignificance of the symptoms. The introductory warnings are flu-like symptoms which are not significant to HCV in particular, as flu-like symptoms are shared with many acute viral infections. More tell-tale symptoms are dark urine, anorexia, jaundice, and abdominal discomfort. The public that is more prone to become infected with HCV are healthcare workers, dialysis patients, intravenous drug users, people who received a blood product before routine screening of blood for hepatitis C (before 1990), and groups participating in high-exposure sexual activities. HCV is spread through infected blood. HCV is not particularly treatable, but spontaneous clearance of the disease is very possible in acute HCV. Studies have shown that acute HCV is cleared and does not progress to chronic illness in 15-40% of cases (9). Factors that influence clearance of the disease from happening are immune system response, manner of infection, immunosuppression by external factors, severity of the illness, HIV co-infection, and many other aspects.
The other spectrum of hepatitis C virus is Chronic HCV, which is the long-term progression of the disease. Chronic HCV is the leading cause of end-stage liver disease, hepatocellular carcinoma and liver-related deaths in the Western world (9). Chronic HCV is defined by persistent hepatic inflammation, which generally leads to the development of scarring of the liver which greatly impacts the function of the liver. Patients being unaware of the development of chronic HCV is prevalent until end-stage liver disease or failure due to the common symptoms with acute and chronic HCV. There are many factors that can aid in the advancement of chronic HCV, examples being age at infection, gender (more frequent in males), immunosuppressive therapy, genetic factors, insulin resistance, obesity, and other immunocompromising diseases. Although spontaneous clearance of the disease is common in the acute stage of HCV, it is far less common in the chronic stage.
Relevance
The relevance of Hepatitis C helicase/primase is that the helicase/protease combination in HCV is believed to play a pivotal role in the replication cycle of HCV. The helicase exists as a dimer, bearing mutations, and can be found in three different functional states (8). The three functional states include a substrate-unbound state, an ATP-bound state, and an NA-bound state. The presence of ATP transitions the protease from high NA binding affinity to low NA binding affinity. The cooperation of helicase/protease binding the DNA is affected by the length of the ss lattice, and the desired ss DNA length is around 22nt (3).
HCV is unique because it is a RNA virus that leads to chronic and/or progressive illness in 85% of affected patients. It is possible that the quick sequence changes of the envelope proteins protect the virus from being attacked by the body's immune response; nonetheless, it is still unknown why HCV works the way it does. Most RNA viruses respond incredible well to their treatment, but there is still no cure for HCV (10). Approximately 3-4 percent of the population is affected by this chronic disease, yet there is still very little known on the function and treatment of this virus. New technology and experiments, like recombinant HCV DNA created from infectious molecular clones, are being created to try and learn more about this virus (7).
Structural highlights
Method: X-Ray Diffraction.
Resolution: 2.50 Å.
Classification: Viral Protein.
Organism(s): Hepacivirus C.
Expression System: Escherichia coli.
Deposited: 2006-12-19.
Released: 2007-07-31.
Deposition Author(s): Prongay, A.J., Guo, Z., Yao, N., Fischmann, T., Strickland, C., Myers Jr., J., Weber, P.C., Malcolm, B., Beyer, B.M., Ingram, R., Pichardo, J., Hong, Z., Prosise, W.W., Ramanathan, L., Taremi, S.S., Yarosh-Tomaine, T., Zhang, R., Senior, M., Yang, R., Arasappan, A., Bennett, F., Bogen, S.F., Chen, K., Jao, E., Liu, Y., Love, R.G., Saksena, A.K., Venkatraman, S., Girijavallabhan, V., Njoroge, F.G., Madison, V.
Primary Structure: 180 amino acids.
Secondary Structure: Three alpha helices with 2 connecting loops and 9 anti-parallel beta sheets (11).
Tertiary Structure: 2 domains. Beta clam motif and helix-loop-helix motif (1).
Method: X-Ray Diffraction.
Resolution: 2.30 Å.
Classification: Helicase.
Organism(s): Hepacivirus C.
Expression System: Escherichia coli BL21(DE3).
Deposited: 1998-03-13.
Released: 1999-04-20.
Deposition Author(s): Cho, H.S., Ha, N.C., Kang, L.W., Oh, B.H.
Primary Structure: 620 amino acids.
Secondary Structure: beta sheets sandwiched between Alpha helices (12).
Tertiary Structure: Domains: 3 domains; domain 2 is linked to domains 1 and 3 by flexible linkers. Motifs present: 6 motifs; the Walker A motif, the Phe loop, and the Arg-clamp motif (12).
References
[1] Gawlik, K.; Gallay, P. A. HCV Core Protein and Virus Assembly: What We Know without Structures. Immunologic research 2014, 60 (1), 1–10.
[2] Kolykhalov, A. A.; Mihalik, K.; Feinstone, S. M.; Rice, C. M. Hepatitis c Virus-Encoded Enzymatic Activities and Conserved RNA Elements in the 3′ Nontranslated Region Are Essential for Virus Replication in Vivo. Journal of Virology 2000, 74 (4), 2046–2051.
[3] Donmez, I.; Rajagopal, V.; Jeong, Y.-J.; Patel, S. S. Nucleic Acid Unwinding by Hepatitis c Virus and Bacteriophage T7 Helicases Is Sensitive to Base Pair Stability. Journal of Biological Chemistry 2007, 282 (29), 21116–21123.
[4] Turkington, C. Hepatitis C; McGraw-Hill/Contemporary, 1998.
[5] Rajagopal, V.; Gurjar, M.; Levin, M. K.; Patel, S. S. The Protease Domain Increases the Translocation Stepping Efficiency of the Hepatitis c Virus NS3-4A Helicase. Journal of Biological Chemistry 2010, 285 (23), 17821–17832.
[6] Locatelli, G. A.; Spadari, S.; Maga, G. Hepatitis c Virus NS3 ATPase/Helicase: An ATP Switch Regulates the Cooperativity among the Different Substrate Binding Sites†. Biochemistry 2002, 41 (32), 10332–10342.
[7] Bartenschlager, R. Hepatitis C virus : from molecular virology to antiviral therapy. Springer: London, 2013.
[8] Centers for Disease Control and Prevention (U.S.), National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (U.S.). Division of Viral Hepatitis. Hepatitis C : information about testing. Department of Health and Human Services: Atlanta, 2010.
[9] Rachel H. Westbrook, Geoffrey Dusheiko. Natural History of Hepatitis C. Journal of Hepatology. Volume 61, Issue 1. 2014. Pgs S58-S68. ISSN 0168-8278. https://doi.org/10.1016/j.jhep.2014.07.012. (https://www.sciencedirect.com/science/article/pii/S0168827814004814)
[10] Peter Simmonds. Variability of Hepatitis C Virus. Hepatology. Volume 21, Issue 2. February 1995. Pgs 570-583. ISSN 0270-9139. https://doi.org/10.1016/0270-9139(95)90121-3.
(https://www.sciencedirect.com/science/article/pii/0270913995901213)
[11] Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.; Mathieu, M.; De Francesco, R.; Rey, F. A. Crystal Structure of the RNA-Dependent RNA Polymerase of Hepatitis C Virus. Proceedings of the National Academy of Sciences 1999, 96 (23), 13034–13039. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23895/)
[12] Frick, D. N.; Tan, S. L. In Hepatitis C viruses: Genomes and molecular biology; Horizon Bioscience: Norfolk U.K., 2006. (https://www.ncbi.nlm.nih.gov/books/NBK1614/)
