Biochemistry of Hepatitis C PHM142 Fall 2015 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson Biochemistry of Hepatitis C Lisa Liu, Anastasia Pimenova, Brittany Nicholls

Hepatitis C liver disease caused by the Hepatitis C virus (HCV) 1 liver disease caused by the Hepatitis C virus (HCV) causes both acute and chronic infection HCV caused by blood borne virus blood-blood transmission 130-150 million people globally have chronic hepatitis ~240,000 individuals are infected in Canada current drug treatment available no vaccine available 2nd point: transmission by: inadequate sterilization of medical equipment and transfusion of unscreened blood and blood products 3rd point: epidemiology highest prevelance in central+east asia, north africa and middle east Additionally, HCV has enormous genetic diversity in infected hosts, existing in blood as a swarm of related quasispecies. This diversity is a result of the error-prone viral polymerase, and fast viral replication enables rapid adaptation to host antibody responses, cellular immune responses, and antiviral drugs current drug treatments use peginterferon alfa (act to induce antiviral response) and oral ribacirin (stop viral synthesis and mRNA capping) this combination can read to a sustained virological response (clinically seen as aviremia ; lack of virus in blood plasma) http://www.pjm.microbiology.pl/archive/vol5822009093.pdf

Prognosis 1 fibrosis: first stage of liver scarring cirrhosis- scar tissue builds up and takes over most of liver are not health /functional hepatocyte cells time and accumulation of scarring results in cirrhosis (20-25 years) http://www.epgonline.org/images/hepatitis/images/1602_03.gif

Effects of Hepatitis C 1,2 gallbladder impairment Li Y, Yamane D, Masaki T, Lemon SM. The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA. Nat Rev Microbiol. 2015;13(9):544-58. hCV virus damage liver cells -- leading to scarring and higher prevelance of hepatocytes developing into cancer cells hypothroidism: the damage and subsequent drugs used to treat Hep C can trigger autoimmune responses which in turn damages the thyroid tissue and its funciton the lack of normal blood filtering by the liver results in buildup of blood and results in blood vessel diation (as seen in portal hypertension) the lack of normal blood flow and the lack of the liver to properly filter blood also results in anemia (the inability of liver the recycle RBCs\0 as mentioned above, the lack of adequate blood flow through the liver also affect the systemic circuit in neighbouring organs such as the stocmach livers cannot produce bile as well, lack of proper digestion of fatty foods

single stranded RNA lipid and protein-enveloped virus of 9.6kb HCV structure 3 single stranded RNA lipid and protein-enveloped virus of 9.6kb http://viralzone.expasy.org/all_by_species/37.html http://www.pjm.microbiology.pl/archive/vol5822009093.pdf http://viralzone.expasy.org/all_by_species/37.html

HCV Genomic/Proteomic Structure 3 RNA codes for 3 000 amino acid polyprotein 3 structural proteins make the envelope: capsid and envelope glycoproteins E1/E2 non structural proteins for genome replication: NS2, NS3, NS4A, NS4B, NS5A, NS5B 5’ end of RNA codes for structural proteins and the rest codes for non structural http://viralzone.expasy.org/all_by_species/37.html http://viralzone.expasy.org/all_by_species/37.html

Entry into Liver Endothelium 4 HCV Capture: 2 options Via L-SIGN. Transcellular transport. Via DC-SIGN. Paracellular transport. Lyu J, Imachi H, Fukunaga K, Yoshimoto T, Zhang H, Murao K. Roles of lipoprotein receptors in the entry of hepatitis C virus. World J Hepatol. 2015;7(24):2535-42. DC SIGN: dendritic cells. Attaches to DC-SIGN L-SIGN: L stands for lectins which are a class of proteins which bind to sugar residues. In the case of HCV, L-SIGN binds to mannose found in glycoproteins in the capsid.

Entry into Hepatocyte 3 steps: Interaction with LDLR 4 3 steps: Interaction with LDLR Binding to SR-BI and CD81 Use of CLDN-1 and OCLN to result in clathrin-mediated endocytosis Roles of lipoprotein receptors in the entry of hepatitis C virus LDLR: low density lipoprotein receptor. Unknown mechanism. SR-BI: scavenger receptor class B, type 1. Mediates uptake of high density lipoprotein which is found in the E2 structural protein. CLDN-1 and OCLN: CLDN-1 interacts with CD81 which assists in virus entry. OCLN interacts with E2 structural protein.

HCV infection overview 5 after capsid uncoating, RNA is translated into protein at the rough ER proteins encoded by HCV make more HCV RNA structural proteins (capsid, E1, E2) are packaged and assembled in the Golgi capsid is assembled with RNA inside and expelled by exocytosis virus can now infect other hepatocytes http://www.ncbi.nlm.nih.gov/books/NBK1630/pdf/Bookshelf_NBK1630.pdf Tan S. Hepatitis C Viruses, Genomes and Molecular Biology. Horizon Scientific Press; 2006.

Replication of HCV: Translation 5 5’UTR has internal ribosome entry site (IRES) that facilitates hepatocytic 40s ribosome subunit binding 60s subunit recruited, can now translate RNA enters cell when capsd fuses with hepatocyte membrane http://www.ncbi.nlm.nih.gov/books/NBK1630/pdf/Bookshelf_NBK1630.pdf

Role of HCV protein segments 5 Role of HCV protein segments HCV polyprotein cleaved by hepatocyte proteases into: NS2, NS3 and NS4A: HCV proteases that cleave the polyprotein NS3: also acts as helicase, unwinds HCV RNA for replication NS5B: RNA polymerase, makes +/- HCV RNA strands NS4B: creates vesicles for viral assembly

Damage to hepatocytes HCV is not directly cytopathic to hepatocytes 6 HCV is not directly cytopathic to hepatocytes Immune mediated subvert activity of NK cells, and CD4+ and CD8+ T cells HCV-specific T cells become unresponsive and disappear Many pathways are proposed and involve the innate and adaptive immune responses http://www.ncbi.nlm.nih.gov/pubmed/17199558 Spengler U, Nattermann J. Immunopathogenesis in hepatitis C virus cirrhosis. Clin Sci. 2007;112(3):141-55. HCV and fibrosis (scarring) http://www.ncbi.nlm.nih.gov/pubmed/12655347

Damage to Hepatocytes via Cytokines 6 HCV core proteins and NS3 proteins trigger monocytes and mast cells to release cytokines IL1, IL6, IL8, TNF Due to impaired immune system, cytokines are continuously released lead to hepatocyte damage, fibrogenesis and cirrhosis via inflammation Spengler U, Nattermann J. Immunopathogenesis in hepatitis C virus cirrhosis. Clin Sci. 2007;112(3):141-55.

Summary Slide Single stranded RNA lipid and protein-enveloped virus of 9.6kb Affects liver and causes liver damage Structural proteins: E1, E2 and capsid, Non-structural proteins:NS2, NS3, NS4A, NS4B, NS5A, NS5B Entry via clathrin coated endocytosis 3 step process using LDLR, SR-BI, CD81, CLDN1, OCLN Replication via non-structural proteins Damage to liver via adaptive and innate immune responses cytokines cause inflammation and cell death

References Webster DP, Klenerman P, Dusheiko GM. Hepatitis C. Lancet. 2015;385(9973):1124-35. Li Y, Yamane D, Masaki T, Lemon SM. The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA. Nat Rev Microbiol. 2015;13(9):544-58. Budkowska A. Mechanism of cell infection with hepatitis C virus (HCV)--a new paradigm in virus-cell interaction. Pol J Microbiol. 2009;58(2):93-8. Lyu J, Imachi H, Fukunaga K, Yoshimoto T, Zhang H, Murao K. Roles of lipoprotein receptors in the entry of hepatitis C virus. World J Hepatol. 2015;7(24):2535-42. Tan S. Hepatitis C Viruses, Genomes and Molecular Biology. Horizon Scientific Press; 2006. Spengler U, Nattermann J. Immunopathogenesis in hepatitis C virus cirrhosis. Clin Sci. 2007;112(3):141-55.