HCV Life Cycle
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HCV Life Cycle
Hepatitis-C Virus (HCV) belongs to the Flaviviridae family and is the leading cause of chronic liver disease globally. It is estimated to infect about 170 million people around the world (WHO, 1997). Chronic HCV infection frequently leads to liver Fibrosis and Cirrhosis, and is associated with the occurrence of Hepatocellular Carcinoma. Acute infection occurs in only a few patients. In most cases the virus results in chronic infection taking 10-20 years before the emergence of liver disease, which is often accompanied by only mild or vague symptoms. Despite the seemingly benign onset of the disease, a significant number of patients with chronic hepatitis develop cirrhosis and its complications.

HCV infects only humans and chimpanzees; there are no small-animal models. The liver is its primary target organ, and the hepatocyte is its primary host cell but replication has also been described for PBMCs (Peripheral Blood Mononuclear Cells) as well as several B and T-Cell lines. HCV is primarily transmitted percutaneously. The HCV genome is a 9.6-kilobase uncapped linear ssRNA (single-stranded RNA) molecule with positive polarity (Ref.1). It contains 5 and 3 UTRs (Untranslated Regions) including control elements required for translation and replication. The UTRs flank an uninterrupted ORF (Open Reading Frame) encoding a single polyprotein of 3,010 or 3,011 amino acids, which is processed into structural (capsid protein C, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) subunits by host and viral proteases (Ref.2). Recent studies also show the existence of an alternative ORF within the core-coding region that encodes a novel HCV protein of unknown function (Ref.3 & 4). The 5 terminus of the HCV genome possesses a complex secondary structure that functions as IRES (Internal Ribosome-Entry Site) to mediate viral protein translation in a cap-independent manner.

The HCV life cycle is entirely cytoplasmic; the viral RNA does not need to enter the cell’s nucleus. Both cellular proteins and viral proteins facilitate the progression of HCV through its replication cycle. The entry of HCV into the target begins with receptor-mediated endocytosis, in which a cell internalizes a ligand-bound surface receptor. The nature of the receptors enabling HCV entry into cells has been the subject of debate. On one hand, CD81, a member of the Tetraspanin superfamily of proteins, has been proposed as a direct ligand for the E2 glycoprotein but it fails to explain virus tropism. On the other, virus present in infected blood is found associated with LDL (Low-Density Lipoprotein) and the LDLR (LDL Receptor) has been suggested as a receptor for the virus (Ref.5). Other studies also suggest GAGs (Glycosaminoglycans) as probable receptors for HCV. The virus is probably engulfed by receptor-mediated endocytosis and uncoating releases the viral genome from its capsid shell into the cells cytoplasm in order to begin replication. Once released inside the cell, HCV RNA is used as a blueprint for the production of viral proteins. The HCV RNA uses the host cellular components (in particular, ribosomes) for translation into proteins. The primary product of HCV translation is a single polyprotein which contains all ten HCV structural and nonstructural proteins required for HCV replication. This polyprotein is cleaved, or sliced up to produce structural proteins (capsid protein C, E1, E2 and p7 ), and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), in order for the viral proteins to function properly. Cleavage of the HCV polyprotein uses both viral and cellular enzymes called proteases which catalyze reactions that separate the individual proteins. The core protein C interacts with viral RNA and forms the nucleocapsid. E1 and E2 are heavily glycosylated viral-envelope proteins that can interact with plasma membranes of hepatocytes and other cells. E1 and E2 can form heteromeric complexes and bind to cell membranes. It might also have a role in the subcellular localization of virion components and assembly of virus particles. The nonstructural proteins encode enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the HCV nonstructural polypeptide is catalyzed by two virally encoded proteases. The NS2-NS3 zinc-dependent metalloproteinase undergoes autocatalytic cleavage to produce NS2 and NS3. The released NS3 serine protease catalyzes the cleavage of the remaining nonstructural polyprotein to yield NS4A, NS4B, NS5A and NS5B,which, together with NS3 and possibly cellular proteins, form the replication complex or ‘Replicase’ of HCV. The carboxy-terminal segment of the NS3 protein also has NTPase (Nucleoside Triphosphatase) and RNA Helicase activity. The NS4A protein has at least two functions: to form a stable complex with NS3 to facilitate the membrane localization of the NS3-NS4A complex in the ER, and to act as a cofactor for NS3 protease activity. NS4B might have a role in the modulation of NS5A hyperphosphorylation. The NS5A phosphoprotein is implicated in mediating HCV resistance to IFN therapy and also function to regulate viral replication through its interaction with the NS5B protein, which is the RdRp (RNA-dependent RNA polymerase) that catalyzes the replication of HCV RNA (Ref.6). The replicase is assembled at the ER membrane, where it directs the synthesis of intermediate negative-strand RNA, which is subsequently used as a template for the generation of positive-strand RNAs. This new positive-sense HCV RNA forms the genome of a new Hepatitis-C virion. Because the positive-sense and negative-sense strands are complementary, each can serve as a template for synthesizing the other. The new positive-sense HCV RNA is encapsidated with the structural proteins and is known as the nucleocapsid. The nucleocapsid is presumably enveloped by budding into the lumen of the ER. Finally, infectious virions are then transported through the Golgi compartment to the plasma membrane and released to infect new cells (Ref.6). The HCV RdRp has no proofreading mechanism to correct errors during strand synthesis, so mistakes made by the HCV RdRp get incorporated into new HCV RNA as mutations. This propensity for error during genomic replication results in a quasispecies population of closely related but genetically distinct HCV variants (Ref.7).

HCV does not kill the cells it infects, but triggers an immune mediated inflammatory response (Hepatitis) that either rapidly clears the infection or slowly destroys the liver, causing the development of hepatocellular carcinoma. HCV evades the immune system with two distinct strategies; in the first strategy, HCV blocks the immune response by inhibiting IFNs (Interferons) (Ref.10). The HCV protease, NS3/4A can block the phosphorylation and effectors action of IRF3 (IFN Regulatory Factor-3) by inactivating signaling by RIG-I (Retinoic Acid-Inducible Gene-I), a cytoplasmic dsRNA binding protein that activates cellular kinases and stimulate IRF3. IRF3 coordinately regulates the expression of Type-I IFNs such as IFN-Beta. Thus a blocking of IRF3 inhibits some of the immune responses against HCV. In the second strategy, mutations allow HCV to evade the immune system. Mutational inactivation of B- and T-Cell epitopes is common in HCV infection. B-Cell epitopes are concentrated in the hyper variable region of the E2 protein, probably allowing the virus to persist in the presence of antibody that is neutralizing to the non-mutated viral ancestors. The T-Cell epitope mutations span the viral polyprotein, often in residues that bind to MHC (Major Histocompatibility Complex) molecules or are otherwise involved in antigen presentation (Ref.8). These evasive strategies of HCV have made it rather difficult for researchers to develop vaccines and drugs against HCV.

HCV infection is infrequently diagnosed during the acute phase of infection since the interval between infection and the development of cirrhosis can exceed 30 years Clinical manifestations can occur, usually within 7 to 8 weeks (range, 2 to 26) after exposure to HCV, but the majority of persons have either no symptoms or only mild symptoms. In cases in which symptoms of acute hepatitis have been documented, they usually consisted of jaundice, malaise, and nausea. The infection becomes chronic in most cases, and chronic infection is typically characterized by a prolonged period in which there are no symptoms. An estimated 74 to 86 percent of persons will have persistent viremia, and this range may prove to be low as more sensitive tests become available to detect viremia. The factors most strongly associated with HCV infection are injection-drug use, blood-borne transmission, transmission via blood products and sexual or congenital transmission (Ref.9). Other factors that accelerate clinical progression include alcohol intake, which has a pronounced effect on the course of the disease; coinfection with HIV1 or HBV; male sex; and an older age at infection. These sporadic cases represent more than 20% of infected individuals. In industrialized countries, HCV is responsible for 20% of acute hepatitis, 70% of chronic hepatitis, 40% of decompensated cirrhosis, 60% of hepatocellular carcinoma and 30% of liver transplants.

In addition to hepatic disease, there are important extra hepatic manifestations of HCV infection. Most of these syndromes are associated with autoimmune or lymphoproliferative states and may be related to the possibility that HCV is able to replicate in lymphoid cells. Only a small fraction of affected persons (10 to 15 percent) have symptomatic disease. These symptoms are often related to vasculitis and consist of weakness, arthralgias, and purpura. The most severe cases are associated with membranoproliferative glomerulonephritis, as well as involvement of the nerves and brain. HCV is the chief cause of essential mixed Cryoglobulinemia (Type-II Cryoglobulinemia), with up to 90 percent of affected persons having HCV viremia. A higher incidence of non-Hodgkins lymphoma has also been observed in HCV infection, both with and without mixed cryoglobulinemia. Other diseases, including lichen planus, sicca syndrome, and porphyria cutanea tarda, have been linked to HCV infection (Ref.9). There is currently no vaccine available for HCV and progress in this area is hampered because there are considerable barriers to the development of anti-HCV therapeutics, which include the persistence of the virus, the genetic diversity during replication in the host, the development of drug resistant virus mutants and the lack of reproducible infectious culture systems and small-animal models for HCV replication and pathogenesis. Moreover, given the mild clinical course of HCV infection in most cases and the ever-complex biology of the liver, careful consideration must be given to antiviral drugs, which are likely to have marked side effects. IFN-Alpha (Interferon-Alpha) is currently the recommended treatment for HCV and is believed to act indirectly by binding to specific cellular receptors that induce an intracellular antiviral response (Ref.10).