Review Article

Indian J Med Res 131, January 2010, pp 17-34

Hepatitis C virus: Molecular biology & current therapeutic options
Suresh D. Sharma

Department of Biochemistry & Molecular Biology, Pennsylvania State University, Pennsylvania, USA

Received July 23, 2009 Hepatitis C virus (HCV) is a small (~55 to 65 nm), spherical, enveloped, hepatotropic RNA virus that causes acute and chronic hepatitis in humans. Persistent virus infection with HCV often leads to cirrhosis and hepatocellular carcinoma (HCC). At present there is neither a selective antiviral therapy nor a preventive vaccine. The only available treatment option is a long-acting pegylated-interferon-alpha, given in combination with nucleoside analog ribavirin, which is not very effective. Molecular studies of HCV began with the successful cloning of its genome in 1989. For many years, research to develop therapeutics was stalled by the inability to grow virus in tissue culture. A major milestone was achieved with the recent development of a robust cell culture system for HCV propagation. HCV proteins assemble and form replication complexes on modified host membranes, called as membranous webs. Even though HCV is detected and targeted by host immune mechanisms, it establishes and maintains a life-long persistent infection. HCV has evolved multiple strategies to survive and persist in hostile cellular environments; and the viral population is known to rapidly change during the course of a natural infection thereby escaping immune surveillance. Rapid mutations also help virus to survive by selecting for the variants which are resistant to antiviral drugs. Although precise mechanisms regulating HCV entry into hepatic cells via receptors remain unknown, HCV also has the capability of direct cell-to-cell transmission. The extremely complex and incompletely understood nature of the HCV lifecycle has complicated the discovery of new therapies. A complete understanding of the functional roles played by the HCV proteins during HCV lifecycle is vital for developing a successful cure. This review deals with current status of efforts in addressing these daunting tasks and challenges in developing therapeutics against chronic and rapidly changing hepatitis C virus.

Key words HCV - hepatocellular carcinoma (HCC) - novel HCV therapeutics - pegylated-Interferon - ribavirin - structural and nonstructural proteins

Introduction Hepatitis C is a complex liver disease. Its medical importance and the need to rapidly identify new therapeutic approaches has resulted in intensive study of its causative agent, hepatitis C virus (HCV). Humans are the only known natural hosts of HCV. Even after two decades since its discovery, HCV continues to be

a major cause of concern and a huge burden on public health systems worldwide. The WHO estimates that a minimum of 3 per cent of the world’s population is chronically infected with HCV1,2. HCV is a prototype member of the Hepacivirus genus (from the Greek hepar, hepatos, liver) and is further classified into at least seven major genotypes

Therefore. HCVcc (for HCV grown in cell culture) has allowed researchers for the first time to study the complete life cycle of HCV. A variety of autoimmune and immunecomplex-mediated diseases have also been associated with chronic HCV infection. The viral-RNA genome harbours a single ORF (open reading frame) which is flanked by 5’ and 3’ non translated RNA segments (NTRs). owing to the limited host range of HCV.9. in cell culture but could also generate viral particles. it can be mild (minimal inflammation of the liver) or severe and can lead to scar tissue formation (Fig. the individuals become chronic carriers. JFH1 viral genome (genotype 2a). 4. A breakthrough in the field came with the development of a complete in vitro cell culture system for HCV (JFH1) in 20057. Chronic infection eventually causes cirrhosis leading to hepatocellular carcinoma (HCC) and ultimately death. 3. Liver steatosis occurs in more than 50 per cent of the patients with chronic hepatitis C (CHC) infection. HCV circulates in infected individuals as a population of diverse but closely related variants referred to as “quasispecies”. 4.17 g/ml8. yet the virus mutates to escape surveillance5. 1). virus infection does not resolve naturally. Currently. Antibodies directed against several HCV proteins can be detected in chronic patients. Availability of injectable therapies and drugs has had a remarkable influence on HCV epidemiology. These genotypes have been further classified into sub-types (a. Neutralizing antibodies appear to be produced during the course of a natural infection.8. 2. b. within this chronically infected population the disease outcomes vary. c. although the most infectious fraction has a density of 1. Molecular Biology of HCV For many years the failure to replicate and produce authentic infectious hepatitis C virus in cell culture remained a major obstacle for innovative and costeffective therapy.18 INDIAN J MED RES. Some individuals with CHC may report non specific symptoms such as fatigue. which is approximately 9. 5. In a majority of infected people. Even among them the disease outcome can be very different (mild to severe). 6 & 7) show differences with regard to their worldwide distribution. The genomic organization of HCV is shown schematically in Fig. The cis-acting replication elements or CREs are located in both the 5' and 3' NTRs and in the NS5B coding sequence10. HCV is most commonly spread by direct contact with infected blood and blood products. RNA . autoimmune thyroiditis being one of them6. 1. averages 6-8 wk. the development of a small animal model (to study viral replication and pathogenesis) is still a big challenge in the field. transmission and disease progression3. This is the major reason why early treatment is difficult. HCV- RNA replication occurs in the cytoplasm of hepatocytes. d.6 kb (Fig. HCV virions exhibit a wide range of densities. Yet.15-1. etc). 2). cloned from a Japanese patient with fulminant hepatitis could not only replicate efficiently Fig. Chronic infection eventually causes cirrhosis leading to hepatocellular carcinoma36. The HCV genome does not enter the cell nucleus. Present inside the outer envelope. The incubation period of HCV. In fact. though ranging up to several months. making it a very difficult to detect it at an early stage. These genotypes (1. nausea and pain in the right upper quadrant. there is no vaccine to prevent hepatitis C. hepatitis C is often referred to as a “silent disease”. there is a (30-35 nm) inner core which encapsulates the single-strand viral RNA (positive-sense). 3. JANuARY 2010 that differ by about 30 per cent in their nucleotide sequence. HCV infection is often asymptomatic. What happens when infected with hepatitis C virus? Acute liver infection is followed by a chronic infection in almost 80 per cent of the infected population152. When liver fails to clear the virus. muscle aches. However.

HCV core protein was reported to interact with the 5’-NTR of plus-strand RNA13. 2. The 5′ UTR (+) is ~341 nucleotides in length and contains an internal ribosomal entry site (IRES). 3). a cis-acting replication element (CRE) was discovered18. E1 and E2 are embedded in a lipid envelope which is derived from the host8. MicroRNA. However. recent work with JHF1 viral RNA suggested that its 5’-NTR (+) does not contain RNA packaging signals14 and authors further speculate that it may reside in the RNA region encoding the replicase. HCV Structural Proteins HCV encodes a single polyprotein (NH2-C-E1E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) which is approximately 3010 amino acids (Fig. NS4B. Non coding RNA molecules or microRNAs (miR) are important in the control of gene expression and regulation. (ii) a variable region. this activation does not interfere with cap-independent translation of HCV viral proteins. II. Two membrane-associated envelope glycoproteins. 3’SLII and 3’SLIII). These host factors require further scrutiny to be considered as candidates for drug targets. Core has been implicated in the development of hepatocellular structures are known to participate and play vital roles in the multiplication of RNA viruses. Sequestration of miR-122 in liver cell lines strongly reduced HCV translation. The 5'- and the 3'-NTRs of the genome are highly conserved and contain control elements for translation of the viral polyprotein and replication. and (iii) a virtually invariant 98-nt X-tail region made up of 3 stem-loops (3’SLI. NS3. . NS5A. This region is recognized by the viral polymerase as the initiation site for plus-strand synthesis of the HCV genome16. The 3'-uTR can robustly stimulate IRESdependent translation in human hepatoma cell lines15. Hepatitis C virus particle structure: The HCV core protein interacts with viral genomic RNA to form the nucleocapsid51. However. The domain IIId of the IRES constitutes the key anchoring site for the 40S subunit11. whereas its addition stimulated translation via direct interaction of miR-122 with two sites in the 5'-uTR21. Within the 3’-end of the non-structural protein 5B (NS5B) coding sequence. The structural proteins (core. miR-122 is specifically expressed and is found to be abundant in the human liver20. E1 and E2) and the p7 protein are released from the polyprotein after cleavage by host endoplasmic reticulum (ER) signal peptidase(s). Recent studies have recognized that various stemloop structures exist in the negative strand 3’-NTR. Core: HCV core is a multifunctional protein which is highly basic in nature23. The IRES is required for cap-independent translation of viral RNA. Mutual longrange binding with both 5' and 3' sequences is suggested to stabilize the CRE at the core of a complex pseudoknot10. Three different domains can be recognized in this uTR: (i) a poly (u/uC) tract with an average length of 80 nucleotides (nt). A recent discovery showed binding of a miRNA (miR-122) to the 5’-uTR of HCV. The IRES domains III-IV have also been shown to be an activator of protein kinase R (PKR)12. The importance of long-range RNARNA interactions in the modulation of HCV lifecycle has been well documented. The HCV IRES is folded into four stem-loop motifs which are called as I. NS4A. A role for proteasome alpha-subunit PSMA7 in regulating HCV IRESmediated translation has also been demonstrated22. which is carried out by host cell ribosome. and NS5B) are cleaved by viral proteases NS23 and NS3-4A. III and IV. These studies have generated a lot of interest in the role of miR-122 in HCV multiplication and its potential as a therapeutic target. The 3'-UTR (+) is around ~200nt and is involved in RNA replication. This CRE is called as SL9266 (or 5BSL3. It forms the structural component of the virus particle. SHARMA: NEW THERAPEuTIC APPROACHES FOR HCV 19 Fig.2) and its disruption blocks RNA replication19. A recent study identified a cellular factor called Far-upstream element (FuSE) binding protein (FBP) which binds to 3'NTR by interacting with the poly(u) tract17. The non structural proteins (NS2. This proteolytic processing of the polyprotein during and after translation by host and viral proteases yield at least 10 mature viral proteins.164.

Transgenic mice expressing the core protein develop HCC.20 INDIAN J MED RES. HCV is translated as a polyprotein that is proteolytically processed by host and viral proteases59. not only with the ER but also with the outer mitochondrial membrane26. Core protein also interacts with apolipoprotein AII. a recent study suggests a potential mechanism for the development of liver cancer via the HCV-core mediated inactivation of the PML tumour suppressor pathway40. 3.34. A study with chronically HCV-infected liver also revealed a similar distribution. HCV core protein. This interaction of core with the chaperon protein resulted in an increased oxidative stress due to perturbation of normal interactions between cytochrome c oxidase and prohibitin. JANuARY 2010 Fig. A region of core protein (spanning amino acids 112 to 152) is essential for association. In infected Huh-7 cells. Lipid droplets (LDs) are intracellular organelles involved in lipid storage and also take part in intracellular vesicular trafficking30. a component of lipid droplets. the pathogeneses of liver steatosis. thus. and NS5B) are located at the carboxyl-terminal end70. as it is clear that gene expression profile in hepatocytes is dependent on the HCV-core-genotype sequence39. However. Interestingly. the core protein is associated with the surface of lipid droplets31. The HCV core protein was shown to affect the steady state levels of a subset of mitochondrial proteins. The core gene sequence data may provide useful information about HCC risk and more studies should be performed to develop it further. no genetic or functional differences were observed between genotype 3a core proteins from patients with and without HCV-induced steatosis. structural and nonstructural proteins: The viral RNA consists of a 5' untranslated region (uTR) containing the internal ribosome entry site (IRES). However. A schematic representation of HCV genome. via deposition of triglycerides in the liver30. it is important to consider the type of genotype involved. HCVsubgenomic replicon systems (which allow HCV-RNA to replicate autonomously). envelope protein 1) are located at the amino-terminal end while the nonstructural proteins (NS2. which were linked with increased HCC risk38. NS4B. HCV core protein is targeted to lipid droplets by its domain 2 (D2) and this association with lipid droplets is required for virus production. NS5A. The structural proteins (C. Core has many intriguing regulatory functions with one of the most important being recruitment of non structural proteins to the lipid droplet-associated membranes29. A study found two mutations in the core gene (36G/C and 209A). In the cytoplasm the core protein is mostly localized to the endoplasmic reticulum (ER). The HCV core protein is known to pass from the ER into mitochondria and is involved with Ca2+ regulation and apoptotic signals27. upregulation of de novo fatty acid biosynthesis by HCV core protein in Huh7 cells has been reported. E2. indicating a direct part played by the core protein in this process37. The p7 protein (ion channel or viroporin) is located at the junction of structural and nonstructural proteins59. steatosis and oncogenesis. core protein derived from genotype 3a induced higher fatty acid synthase activity than core protein derived from genotype 1b. These data are further supported by studies showing that the expression of core protein can lead to the development of steatosis in transgenic mice28. HCV makes use of lipid droplets for replication. core plays an imperative role in . suggesting a possible role of other viral proteins in the development of hepatocellular steatosis33. including prohibitin (functions as a chaperon of mitochondrial proteins)28. envelope protein 1. with core localized to both cytoplasm and the nucleus35. A recent study showed that core protein can self-assemble in HCV-like particles (HCVLPs) in ER-membranes25. The core protein is generated from a polyprotein encoded by the viral genome and is processed by cellular proteases in the endoplasmic reticulum (ER)24. followed by the genomic region for structural and nonstructural genes and the 3' uTR. circulating 'free' in non enveloped state has also been detected in HCVinfected patients36. Tumour suppressor protein promyelocytic leukaemia (PML) is known to be involved in antiviral response. have shown subcellular localization of core to be both cytoplasmic and nuclear26. core. Besides its structural and regulatory function. Interestingly. NS4A. E1. Disrupting the association of core protein with lipid droplets is deleterious for HCVcc production and this interaction is thought to contribute to steatosis. Clinical studies have reported that virus-induced steatosis is very severe with HCV genotype 3 than with other genotypes32. NS3.

HCVpp are generated by co-transfection of 3 plasmids into 293T cells: (i) gag-pol genes of HIV or MLV. Interestingly. HCVpp closely resembles the cell entry properties of genuine HCV virions and was used to identify CLDN and OCLN. In addition. infected with recombinant baculovirus expressing HCV structural proteins(Core. HDLs play a very active role in HCV entry. which include (i) frame shifting (ii) form transcriptional slippage. E1 and E2 envelope glycoproteins are required for host-cell entry via receptor binding. NS5A and NS5B are thought to be to be required for replication of the viral genome. HCVpp system does not require productive HCV replication and hence is not restricted to Huh7 or Huh7. The binding of E2 was further mapped to the major extracellular loop of CD81. HDL can inhibit HCV-neutralizing antibodies in serum of acute and chronic HCV-infected patients52. p7. E2 and C). Further analysis of HCVLPs by cryoelectron microscopy (CryoEM) revealed that they are spherical particles with smooth surfaces. In addition. and tight junction molecules claudin-1 (CLDN) and occludin (OCLN) are the most important receptors that mediate HCV entry46. A hypervariable domain near the amino terminus of E2 is the most variable part of the viral polyprotein and called as HVR-1. Both CD81 and SR-BI were identified as candidate HCV receptors based on their physical interaction with a soluble version of E247. NS4B. 2)9. Envelope glycoproteins: The HCV envelope glycoproteins. HCV-LPs for the first time allowed 3D structural analysis of HCV particles51. It is thought that E2 protein may induce thyroidal inflammation. E1 and E2 are structural components of the virion. It is now recognized that the cross-talk between structural and the non structural proteins of HCV is required for efficient virus particle production54. HCV-like particles (HCV-LPs) were isolated from insect cells. A complex interplay between HDL. can form ion channels. Insight into the mechanisms by which HCV gains entry into host cells is vital to understand primary HCV infection and re-infection post-transplantation. Since then multiple mechanisms have been proposed for the expression of ARFPs. It is interesting to note that HCV can associate with LDL and VLDL from infected patient serum. SHARMA: NEW THERAPEuTIC APPROACHES FOR HCV 21 ARFPs: Alternate reading frame proteins or ARFPs are encoded by the +1 reading frame of the viral genome which overlaps with the core protein coding sequence. ARFPs have been shown to be associated with the ER and mitochondria. These additional proteins of unknown function are also called as core+1 or F1 and were recently identified41. which display E1-E2 glycoproteins of HCV on their surface. Bases on the ability of ARFPs to bind the proteasome subunit alpha3. NS3. Amazingly. SRBI and HCV envelope glycoproteins leads to enhanced HDL-mediated HCVpp entry in cells48. it is thought that HCV may utilize glycosaminoglycans and low density receptors on hepatocytes as initial attachment factors. 43. E2 protein has been shown to bind PKR and as a consequence perturb innate immune pathways. E2 has also been shown to bind with CD81 receptors which are expressed on thyroid cell and induce a cascade of signaling pathway leading to IL-8 release. . Finally. The discovery of OCLN provides an vital advance towards efforts to develop small animal models for HCV46. HCV Non Structural Proteins The non structural proteins NS2. However. a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes45.48. They undergo post-translational modifications (N-linked addition of carbohydrate chains) while being translated in the endoplasmic reticulum (ER). They constitute the outer coat of fine spikelike projections of the HCV particle (Fig.5 that support robust replication50. The HCVpp system was subsequently used to prove that they are required for viral entry49. NS4A. HCVpp system (also called as HCV pseudoparticle) generates virus particles. which further allows studying the complex mechanisms of HCV assembly and maturation. the functional role of ARFPs in the HCV lifecycle is not clear yet. Isolated HCV-LPs were composed of all the structural proteins (E1. these proteins are labile and very short lived42. CD81 along with human scavenger receptor SR-BI. (ii) HCVgp. It has been shown to be the target for neutralizing antibodies. packaging signals and a reporter gene. it was suggested that it may regulate protein degradation in cells44. E1 and E2)51. They were found to be consistent with the native HCV virions isolated from HCV infected patients. 53. required for the production of infectious virus particles. Cell entry of HCVpp is HCV glycoprotein mediated. The non structural protein. thereby triggering thyroiditis by a bystander activation mechanism6. and (iii) a retrovirus genome with LTRs. Both. The E2 protein has the binding sites for human CD81. or (iii) from internal initiation in the +1 open reading frame (ORF) of the core protein coding sequence.

The membrane association of NS2 is p7-independent and occurs co-translationally67. It can oligomerize following its inclusion into a lipid membrane creating ion channels. Another study also reported similar findings. NS2 also appears to be involved in a particle assembly step which happens post core. JANuARY 2010 The p7 ion channel: The HCV p7 is a small 63 amino acid protein. HCV NS2: The non structural protein 2 (NS2) is a 23-kDa transmembrane hydrophobic protein. It appears to be dispensable for viral RNA replication. The p7 protein is highly hydrophobic in nature. The ion channel blocker.68. The cleavage between NS2 and NS3 is absolutely required for persistent viral infection in a chimpanzee72. 62. was thought to interfere with the ion channel activity of p7 based on studies with artificial lipid bilayer system. This phosphorylation event appears to regulate the stability of NS2 protein (at least from genotype 1a). It is thought that this precursor may have a role in the regulation of HCV lifecycle. It has two amphipathic. the precise role and existence of these precursors in a natural HCV infection is not known. The search for molecules or inhibitors targeting NS2. Interestingly. all the active site residues (H952. are located entirely in NS273. This requirement of NS3 remained intriguing until the recent discovery showing that the zinc binding domain of NS3 could in fact stimulate the protease activity of NS2. The NS3 enzyme has a chymotrypsin-like serine protease activity76. This hexameric (42 kDa) protein complex was found to depict flower-shaped architecture with protruding petals oriented toward the ER lumen59. NS2 followed by only 2 amino acids of NS3 produces a basal proteolytic activity in vitro. amantadine. dose-dependent reductions of virus titres were achieved with iminosugars64. the necessity for a timely release of the individual proteins at the appropriate time in the viral lifecycle. the N-terminal 180 aa of NS3 are required besides NS2 for a robust protease activity. However when co-expressed with p53 (tumour suppressor). The p7 protein belongs to a family of viral proteins called as viroporins that form ion channels. This process is undoubtedly vital for virus multiplication. NS4A/4B.66. Nonetheless. should without a doubt. NS5A. until recently the only known function for NS2 was its autocleaving activity at the NS2/3 junction. The 3dimensional structure of p7-complex was determined by using single-particle electron microscopy. E972 and C993). Along with its cofactor NS4A is responsible for cleavage at the NS3/4A. NS4B/5A and NS5A/5B junctions. These studies implicate a role of NS2 in promoting steatosis. However. However. the simplest being. that p7 ion channel function is not affected by amantadine64. an RNA helicase and NTPase activities (Fig. it became clear after clinical trials. Many models have been proposed to explain the potential significance of these precursor forms. However. amantadine65. The cleavages mediated by signal peptidases between p7 and NS2 occurs slowly and are partial at the E2/p7 and p7/NS2 sites55. This results in the formation of an E2p7NS2 precursor56. NS2 protein can be phosphorylated by casein Kinase II on Ser residue at position 168.70. The N- and C-termini are exposed to the extracellular environment58. NS2 interacts with itself forming homo-dimers. It is localized in the ER-membranes when encoded by a replication-competent genome57. The absolute dependence of HCV on- ion channel function of p7 protein for infectivity makes it an attractive candidate target for antiviral intervention. as replicons lacking the p7 gene replicate or make viral RNA efficiently61. where HCV strains were found resistant to the ion channel blocker. The p7 ion channel protein serves an essential role in the production of infectious virus particles during HCV lifecycle60. 4). HCV NS3: The non structural protein 3 (NS3) is a member of the superfamily 2 DExH/D-box helicases and its crystal structure has been determined. advance the development of new therapeutics against HCV. NS2 is a membraneassociated cysteine protease69. needed for the catalytic activity of the NS2/3 cysteine protease.22 INDIAN J MED RES. It is a 67 kDa tri-functional protein with a serine protease. positioned at the junction of the structural and non structural proteins. it is . The p7 protein could form amantadine-sensitive ion channels in this artificial system63. Expression of NS2 in Huh7 cells resulted in upregulation of transcriptional factor sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase. and NS3 assembly75. TM1 and TM2 (spanning amino acids 19-32 and 36-58) which are embedded in the ER-membrane. The functional sub-domains in NS3 essentially function as its regulatory cofactor73 again highlighting tight regulation of the proteolytic processing. Moreover NS2 has also been shown to interact with all the other HCV non structural proteins74. transmembrane regions. NS3/4A are localized in ER cisternae surrounding mitochondria77. required for HCVcc infectivity71.

RNA unwinding activity of NS3 helicase is modulated by this interaction with NS5B polymerase78. A direct interaction between NS3 and NS5B occurs through the protease domain of NS3. 4. NS3 unwinds both RNA and DNA substrates. regulates helicase activity). SHARMA: NEW THERAPEuTIC APPROACHES FOR HCV (Helicase domain) Protease domain Domain I 1192 1214 1353 23 Domain II 1507 Domain III 1658 1027 Fig. This activity may be necessary but does not appear to be sufficient for long-term viral persistence since there is (cytotoxic) T cellmediated clearance of NS3/4A-expressing hepatocytes in vivo82. Retinoic acid inducible gene or RIG-I is a cytoplasmic RNA helicase79. Palmitoylation of 4B protein facilitates oligomerization which appears to be essential for HCV replication87. In addition to its role in viral polyprotein processing and HCV multiplication. HCV NS3/4A protease has remarkably evolved to target and cleave ISP-1 (or MAVS at Cys-508). NS3 therefore represents a very promising target for anti-HCV therapy.β promoter stimulator-1 or IPS-1(IPS-1 is also known VISA. This interaction of RIG-I with IPS-1 can signal downstream activation of IRFs and NFkB to trigger alpha/beta-INFs. It couples unwinding of RNA strands (with extensive secondary structures) to ATP hydrolysis. It is an essential pathogen recognition receptor (PRR) for HCV. thereby halting alpha/beta interferon expression81. Thus. in addition to its essential role in HCV replication it is also involved in viral pathogenesis by affecting cellular functions. The multifunctional roles played by NS3 in the lifecycle of HCV. Cardif or MAVS)80. cytoplasmic changes were seen in hepatocytes of infected chimpanzees with the help of electron microscope. NS3 undergoes other posttranslational modifications. NS4A is localized not only on the ER. where viral replication complex is assembled. The immunoelectron microscopy of Huh7 cells supporting replication of sub-genomic localized along with p53 in the nucleus. such as phosphorylation and N-terminal acetylation83. This function of MAVS is abolished if the mitochondrial-targeting domain of MAVS is deleted81. another important function of NS3 involves antagonizing host innate-immune pathways. Innate immune defense mechanisms activated by alpha/beta INFs represent an essential first line of protection against viral infections. Further. A recent study utilizing sub-genomic replicon demonstrated that besides methylation (which . A characteristic feature of Plus-strand RNA viruses is their ability to induce alterations in cellular membranes and then utilize it to replicate their own genomes. other HCV proteins are most likely responsible for interfering with the adaptive immunity. consisting of vesicles in a membranous matrix. RIG-I upon binding viral RNA undergoes changes in conformation and can then interact with IFN. although there is no DNA intermediate involved in HCV lifecycle. HCV NS4B: The HCV non structural protein 4B (NS4B) is a 27-KDa polypeptide. Therefore. The Nterminal portion of 4A is responsible for membrane association of the NS3-4A complex.88. a study reported altered intracellular distribution of mitochondria and its damage due to NS4A expression in Huh7 cells85.164. It contains four transmembrane domains and is palmitoylated at two C-terminal cysteine residues. makes it indispensable for virus multiplication. It is a highly hydrophobic integral ER-membrane protein86. Egger et al92 showed that NS4B induced a distinct membrane alteration when expressed in cultured human osteosarcoma (u-2 OS) cells. a protein which had no known function until then was shown to induce a tight structure. It has an amphipatic helix at the N-terminal and a C-terminal domain which are both associated with membranes89. Recently. Thus. The role played by this small polypeptide during HCV replication is very significant. Interestingly. The induction of type I interferon (IFN) genes (Type I IFNs include several IFN-α subtypes and a single IFN-β subtype) is regulated at the step of transcription and is best understood for the IFN-β promoter. NS4A also associates with other HCV proteins on the ER-derived membranous webs. Sponge-like inclusion composed of a dense matrix and irregularly arranged membranes were reported91. designated membranous web. Back in the 1980s. but also on mitochondria84. HCV NS4A: Non structural protein 4A (NS4A) is a cofactor for NS3 serine protease activity. Allelic variation in the NS4B sequence between closely related HCV isolates was found to drastically impact HCV replication in cell culture90. The localization of MAVS to the mitochondria is critical for its ability to induce IFNs. A diagrammatic representation of HCV NS3 protein: The protease and the helicase domain organization is shown with boundaries defining these domains (amino acids 1027 to 1658 in the polyprotein)163.

HCV NS5A: The non structural protein 5A (NS5A) has RNA-binding activity96. Cleavage by caspase 3 and 6 leads to genearation of N-terminally truncated NS5A fragments which get localized within the nucleus. It is an important protein for HCV lifecycle. NS5A specifically binds to the G/U rich sequences in the 3’ ends of HCV genomic RNA96. A role for NS5A-DIII in virus assembly was shown via phosphorylation of a serine residue at position 2433 by CKII101. This clone was engineered such that HCV-structural genes were replaced by a neomycin resistance gene. The crystal structure of the conserved domain I (DI) was solved recently and revealed a unique fold. OSBP is co-localized to the golgi with NS5A and this functional interaction is suggested to play a role in HCV particle release105. The replication of HCV is a very intricate process. PKR. The C-terminal domain of NS5A can associate with c-Raf kinase103. NS5A has an amphipathic alpha-helix at its amino terminus with which it is anchored to the ER membrane. A significant association between variations in sequences between 2209-2248 nucleotides of NS5Agene. Genotype 2 HCV isolates have a 19 residue insertion near the C terminus of DIII. Phosphorylation status of NS5A is also known to affect the interaction of NS5A with VAP-A99. Mutations that enhance the capacity of sub-genomic HCV RNA to replicate in cell culture (Huh7 cells) were mapped to the NS5A-coding sequence and were called as adaptive mutations. NS5A is further divided into three-domains which are separated by linker regions. In vitro expression studies of NS4B from all major genotypes have demonstrated the importance of N terminus of NS4B94. Further. JANuARY 2010 replicon revealed a loss of the organization and other morphological alterations of the ER showing convoluted cisternae and paracrystalline structures77. Disrupting the amphipathic-helix in the N terminus of NS4B abolishes the ability of NS4B to rearrange membranes. The first of its kind. NMR studies have shown DII of NS5A to be flexible and disordered. Crystal structures for both DII and DIII are not available yet. Full length HCV NS5A has a cytoplasmic localization. Domains II (DII) and III (DIII) are more variable among HCV genotypes. negatively impacts HCV replication104. the mechanism of inhibition remains unknown. NS5A protein can be found in basally phosphorylated (56 kDa) and hyper- phosphorylated (58 kDa) forms in cells. Following transfection of in vitro transcribed RNA into Huh-7 cells. This interaction appears to be essential for HCV replication in Huh7 cells. Oxysterol binding protein (OSBP) can interact with the N-terminal region of DI. Interestingly. NS5A inhibitor (BMS-824) and the NS3 protease inhibitor block hyperphosphorylation of NS5A. These membranous webs are made up of small vesicles (80-180 nm in diameter) embedded in a membranous matrix and are found closely associated with the rough endoplasmic reticulum. Even before an important role in formation of membranous web like structures was discovered for this interesting protein. P58 form of NS5A is hyperphosphorylated at additional sites that remain unmodified in the p56 form by casein kinase I-alpha97. NS4B colocalized within this web together with other structural and NS proteins92. antibiotic G418-resistant cells could be obtained in which the sub-genomic RNA replicated autonomously.24 INDIAN J MED RES. called as Con1. This region of NS5A (amino acids 237−276) is called as ISDR or interferon sensitivity determining region. The ISDR was also found to associate with the antiviral molecule. a role for NS4B in malignant transformation of NIH3T3 cell in association with the Ha-ras oncogene had been proposed95. upon its deletion. replicate in Huh7 cells. a recent study with JFH1 virus showed that DIII of NS5A could influence RNA replication. Patients with a mutant-type . the involvement of a protease inhibitor in inhibiting p58 is quite intriguing100. However. Loss of hyperphosphorylation (or NS5A-p58 form) stimulates RNA replication of the HCV genotype 1b replcion in Huh-7 cells. NS5A-DII contains the PKR and the HCV-NS5B binding domains. 98. HCV-replication-complex was first identified in Huh7 cells supporting sub-genomic replicon in 200393. Human vesicle-associated membrane protein (hVAP) subtype A is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. as sequestration of c-Raf by truncated NS5A into the nucleus. NS4B alone could induce the formation of membranous web. DI has a zinc binding motif and also forms a nucleic acid-binding domain. the sub-genomic replicon utilized a HCV genotype-1b clone. The adaptive mutations alter the phosphorylation state of the NS5A protein. NS5A domain III is not required for RNA replication as sub-genomic replicon lacking DIII. authors observed a delay in both RNA replication and particle assembly102 thereby suggesting a role in viral multiplication. and response to interferon treatment has been proposed. however. occurring through protein–RNA and protein–protein interactions.

Through the years HCV treatment has improved with the development of modified pegylatedinterferon (PEG-INF). VCH-759. GS-9190) act by allosteric mechanism. Current Therapy The very first treatment shown to be effective against HCV involved systematic administration of INF-α. In vitro NS5B cannot distinguish between natural and synthetic templates. It was further discovered that inhibitors of NS5B phosphorylation. However. HCV must also maintain highly conserved genomic segments and a balance between conserved and variable viral elements is above all important to avoid "error catastrophe". NS5B lacks a “proofreading” function. PSI6130/R7128. GSK625433. recent studies have questioned the existence of an ISDR in NS5A. it would be interesting to see if resistant mutants emerge over a period of time.fold-higher specific activity when compared to J6 NS5B113. It could either be due to the stimulation of its catalytic activity or due to a conformational change induced by NS5A binding112. The treatment of liver cell lines (stably replicating HCV sub-genomic replicon) with these inhibitors cleared viral RNA suggesting that PRK2 inhibitors act by suppressing HCV replication via inhibition of NS5B activity118. NS5A can repress the PKR pathway. A SVR is defined as negative HCV RNA. Indeed. NS5A stimulates NS5B during elongation. The termination of RNA synthesis in vitro is not understood well. Nevertheless. very high rates of genetic variation ensure the survival of hepatitis C viruses under every changing hostile cellular environment. HCV NS5B: The HCV non structural protein 5B (NS5B) is a RNA dependent RNA polymerase (RDRP) containing the GDD motif in its active site108. Due to a high rate of error-prone replication.115. NS5A can directly bind to NS5B and modulate its polymerase (poly A template/poly u primer) activity111. A study where patients infected with genotype 1a were treated with ribavirin. NS5A also has the ability to inhibit IFN-gamma production106. has longer biological half-life. six months after treatment. Non-nucleoside inhibitors (NNIs) or analogs (ABT-333. palm and thumb sub-domains. More critical would be the normal functions affected which are regulated by PRK2. A remarkable study with NS5A transgenic mice suggested that NS5A protein could impair both the innate and adaptive hepatic immune response107. Subsequently it generates positive-strand RNA from this negativestrand RNA template. However. There is also evidence for recombination events leading to genetic variation in HCV120. It belongs to a class of integral membrane proteins termed tail-anchored proteins. HA1077 (fasudil) and Y27632 suppressed the activation of PRK2. They bind to allosteric sites on NS5B and thereby inhibit RNA synthesis114. An important lead was provided by a study which established that NS5B associates with cyclophilin A (CypA) via its enzymatic pocket and exploits the isomerase/chaperone activity of CypA to replicate in cells116. complex mutant swarms are generated. The NS5B crystal structure shows the typical fingers. SHARMA: NEW THERAPEuTIC APPROACHES FOR HCV 25 NS5A-ISDR had a higher rate of sustained virologic response (SVR) than those with the wild-type NS5AISDR. NS5B initiates synthesis of complementary negative-strand RNA using the HCV genome (positive polarity) as a template. However. NS5A (at substoichometric levels) stimulates replication by NS5B on templates derived from the 3’ end of the positive strand112. INF is known to activate several direct and indirect antiviral . There appears to be a difference in polymerase specific activity in vitro. however the mechanism of action is not clear. IDX184. NS5B nucleoside inhibitors (NIs) or analogs (NM-107/NM-283. Thus. INF was actually discovered in 1957. depending on the type of HCV genotype. NS5B is phosphorylated by the protein kinase Crelated kinase 2 (PRK2) in cell culture117. The JFH1 NS5B enzyme shows a 10. PF868554. NS5B is anchored to the ER-derived “membranous webs" via its C-terminal 21 amino-acid residues109. during studies with influenza virus121. Existence of an ISDR is an ongoing debate and controversial. INFs are produced naturally by host’s immune system in response to a viral infection. MK-0608) compete with cellular ribonucleoside triphospahtes act as functional chain terminators114. Studies with sub-genomic replicon revealed that the membrane association is indispensable for viral RNA replication110. NS5A has attracted tremendous attention in the HCV field and represents a very promising target for anti-HCV therapy. which is a stabilized version. led to the emergence of a Phe-to-Tyr (F415Y) mutation in the viral polymerase119. Over the past few years numerous nucleoside and non nucleoside inhibitors of the polymerase have been discovered and demonstrated clinical efficiency and advanced to clinical trials. The continuous generation and selection of resistant variants allows HCV to escape control by these inhibitors/antiviral drugs.

In addition molecules targeting host factors which play a role in HCV replication. in the presence of alpha-glucosidase inhibitors. responses to treatment vary widely depending on the genotype of HCV. 127. A clear understanding of the function of genes involved in the IFN-α pathway could possibly explain some of these leading questions. The mechanisms or pathways involved. Therefore. fever. JANuARY 2010 mechanisms (such as viral RNA degradation. This suggests that targeting the glycans of viral envelope proteins holds potential in the development of antiviral therapies. Thus. NS5A and NS5B. direct inhibition of the viral RNA polymerase (by ribavirin triphosphate) and lethal mutagenesis of HCV-RNA genomes caused via incorporation of ribavirin triphosphate by NS5B126. ribavirin can be a substrate for the HCV RNA polymerase although surprisingly it is not very effective in monotherapy128. there is no approved (HCV specific) antiviral therapy. However. dministration of ribavirin A in combination with PEG-INF-alfa-2a (40kDa) to HCV infected patient’s results in an increased SVR rate124. Moreover.132. Ribavirin was first used in treating children with respiratory syncytial virus infection122. pegylated interferon (PEG-INF) and ribavirin combination has become the current “gold standard” regimen or treatment of choice for hepatitis C. unfortunately. search for “specific targeted antiviral therapy for HCV” (STAT-C) has been ongoing for a long time. this novel class of inhibitors can target host-directed glycosylation and may prevent the appearance of drug . It binds to envelope glycoproteins and inhibits the interaction between E2 and CD81130. only when administered along with interferon in combination therapy. leading to anti-HCV effects are not fully understood yet. despite which SVR is often not achieved. the antiviral mechanism of IFN in vivo remains unknown. This was further confirmed by using HCV-LPs. viral glycoproteins were misfolded. (ii) HCVpp. The latest development being. retained in the ER and also impaired in their interaction with calnexin134. modulation of the host immune responses. These initial events in virus lifecycle offer an attractive target for antiviral development. HCV enters into hepatocytes by interacting with cell surface receptors. the ability to generate HCV particles that are infectious both in cell culture and in vivo with the Con1 wild type genome54. Current State of Antivirals At present. the cost of therapy is very high. We are approaching a very productive as well as challenging phase in the field of HCV virology. Genotypes 1 and 4 are the most difficult-to-treat.26 INDIAN J MED RES. myalgia. 4triazole-3-carboxamide) is a purine-analog with broadspectrum antiviral activity123. Another interesting class of inhibitors such as celgosivir. Age is also an important factor as older patients exhibit lower response to therapy than younger patients. These inhibitors target HCV receptors. NS3/4A. The lectin cyanovirin-N (CV-N) was originally discovered against HIV and subsequently also shown to be a potent inhibitor of HCV. Alpha-glucosidase enzyme plays a critical role in HCV maturation by initiating the processing of the N-linked oligosaccharides of viral envelope glycoproteins133. halt viral translation). Thus their inhibitors can affect HCV morphogenesis. inhibition of host inosine monophosphate dehydrogenase (by ribavirin monophosphate). (iv) HCV-LPs to study HCV have further improved our understanding of HCV lifecycle and potential therapeutic targets. Thus. arthralgia and haemolytic anaemia. current available treatment options for non responders are very few. (iii) HCVcc-JHF1. the existing treatment is not very effective and causes significant side effects. 2. severe depression. both these compounds are toxic and their administration causes side effects such as headache. HCV-IRES.125. Some of the proposed mechanisms include: modulation of interferon-stimulated gene expression. The factors determining the triumph or failure of the antiviral therapy are not well understood. are also being pursued. Several months of therapy is required for eradication of chronic HCV infection. n-butyl deoxynojirimycin and N-(n-nonyl) deoxynojirimycin target host alpha-glucosidase enzyme and may offer alternative treatment options131. Moreover. In summary. This insight and further research exploiting a relationship between therapy response and host genetic polymorphisms in the IFN-α pathway may lead to the development of new therapeutic strategies129. Only 40-50 per cent of the patients achieve SVR with genotype 1 infections. Ribavirin (1-beta-D-ribofuranosyl-1. However. Wide varieties of inhibitors or directly acting antiviral agents are at different phases of development and some are currently in clinical trials. Advances in the model systems such as: (i) sub-genomic and genomic replicons. Development of better and improved treatment is required urgently and represents a major challenge for virologists In addition. It was discovered more than 30 yr ago and is effective against HCV.

Zinc mesoporphyrin holds immense potential as a novel drug to treat HCV infection143. verbenalin) and amphipathic DNA polymers (APs) are novel antiviral molecules which inhibit HCV entry. the major drawback of ribavirin is side effects. A recent study showed that CD81 has no role in the cell-to-cell spread of HCVcc. 4-propynyl1-beta-D-ribofuranosylpyrazole-3-carboxamide. Further research is required to advance. Celgosivir (rapidly converted to castanospermine) is currently undergoing clinical trials. telaprevir (VX-950) was put to clinical test recently with some success. this mode of transmission appears to be shielded from neutralizing antibodies. required to process viral polyprotein. which happens to be the most difficult to treat140. HCV polymerase is an attractive target owing to its essential role in viral RNA replication. In addition. Inhibitors that disrupt phosphorylation with a concomitant decrease in virus replication are indeed very attractive and should be developed further. NNIs on the other hand . showed significantly improved SVR rates in patients with genotype 1. Currently only Roche-1728 NI is under active development and clinical trials144. PI4KIII-α and β are very important host factors for HCV replication. PI4K-III-α also plays an essential role in membrane alterations leading to the formation of HCV replication complexes. there were higher rates of discontinuation in the treatment because of the adverse effects such as pruritus. Other recent studies have demonstrated that iridoids (such as aglycones of shanzhiside methyl ester: loganin. APs inhibit HCV internalization owing to their amphipathic nature. However. A specific inhibitor of NS3. refine and develop non-toxic versions of these inhibitors. Another study which involved administration of interferon with telaprevir highlighted the importance of retaining ribavirin during the treatment. Another NI from Merck. A study where phosphatidylinositol 4-kinase type III-alpha (PI4KIII-α) was knocked down prevented infection by HCVpp or by HCVcc (JFH-1). thereby perturbing the interferon response. Idenix NI IDX-184 is under phase I trial in patients infected with genotype 1. The current thought in the field being that a combination therapy along with inhibitors which simultaneously inhibit multiple steps in the virus lifecycle (protease and replicase functions) may yield better SVR. Inhibitors loganin and verbenalin were shown to have anti-HCV entry and anti-infectivity activities137. rash. Another study showed that PI4K-III-α is involved in events vital for membrane alterations. Indeed. since NS3-4A protease also functions to inhibit host innate response by cleaving IFN-β promoter stimulator-1 (IPS-1) and TRIF139. In addition. and 4-phenylethynyl-1-beta-D-ribofuranosylpyrazole-3carboxamide) are being evaluated and could offer an alternative and better therapeutic option142. As response rates were lowest when ribavirin was not present in the treatment141. which are essential for the formation of HCV replication complexes136. It is beyond any doubt the most validated target for the development of antiviral therapy. However. Targeting NS3-4A has additional advantage. SHARMA: NEW THERAPEuTIC APPROACHES FOR HCV 27 resistance when given in combination with the current standard of care132. Valopicitabine (NM-283) was the first NS5B analogue to demonstrate proof of concept for 5B nucleoside inhibitors in the clinic. The NIs can be utilized by the polymerase once converted to the 5’-triphosphate form. BMS-824 is a small molecule which targets NS5A and inhibits hyperphosphorylation or p58 formation100. This study highlights the urgent need to develop strategies aimed at disrupting direct cell-tocell HCV transmission as targeting cell-free virus alone may not be enough to halt a chronic infection and a dual approach is required138. These kinases are potential therapeutic targets. NS5A plays a vital role in HCV replication and is emerging as an important target for the therapy of HCV infection. There is some progress in this direction and several ribavirin analogues (such as 4-iodo-1-beta- Dribofuranosylpyrazole-3-carboxamide. the susceptibility to HCVpp infection and replication in cells was dependent on the presence of phosphatidylinositol 4-kinase type III-beta (PI4KIIIβ)135. There is supporting evidence of a decreased SVR and relapse when ribavirin was prematurely discontinued or ribavirin doses were frequently missed. The significance of targeting NS3-4A protease is clearly evident from its vital function. Another molecule. recent clinical trials where PEG-IFN and ribavirin were administered along with telaprevir. and anaemia. MK-0608 is in preclinical trials. zinc mesoporphyrin (a synthetic heme analogue with a central zinc of the mesoporphyin macrocycle) causes a dramatic downregulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent degradation. Thus APs target the cell entry step of HCV. The latter activity of NS3 affects signaling of the RIG-I and Toll-like-receptor-3 pathways.

There is also evidence that broadly neutralizing antibodies against antigenic regions of E2 can protect Alb-uPA/SCID mouse (human liver-chimeric). preferentially in the initiation phase. This drug has also been used to further enhance the antiviral effects of INF148.148. MicroRNAs (miR) are (~22 nucleotide) naturally occurring noncoding RNAs. PF-00868554 (Pfizer) is currently in phase II clinical trials where it is given along with PEG-INF and ribavirin. mevastatin was found to prevent selection of replicons resistant to the non nucleoside inhibitor HCV-796149. However. which contains three separate RNAi molecules to shut down replication of all strains of the hepatitis C virus. The RNA helicase DDX3 is a component of P bodies and is required for HCV RNA replication154. a drug that interferes with lipid metabolism. LDL and geranylgeraniol. These belong to a family of small RNA molecules that perform and regulate critical cellular functions. IC41 can induce HCV-specific INFgamma-secreting CD4+ and CD8+ T cells in healthy individuals. and SCY-635) effectively block HCV replication in vitro116. A link between HCV and P bodies has also been observed153. GS-9190 (Gilead) is under development and currently a study with VCH-759 (ViroChem Pharma) is ongoing. It was shown to induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV non-responder patients156. a better understanding of the role for miR-122 binding to the HCV is required for effective design of antiviral therapies. A further advance came with the concept of administering inhibitors of both. However. These P bodies are important for many viruses in their lifecycles. The significance of core-DDX3 helicase interaction in HCV pathogenesis is an active area of research. IC41. are currently in clinical trials146. These are also implicated in apoptosis and oncogenesis151. An interesting study utilizing replicon system showed that statins (mevastatin or simvastatin) in combination with polymerase or protease inhibitors could effectively clear the cultures from HCV replicons. An interesting study established the role of miR-29a and P bodies in regulating HIV-1 production and infectivity152. . HCV core protein binds and colocalizes with DDX3 in P bodies155. This strategy sounds promising and could potentially stop the generation of viral escape mutants165. Remarkable work is being done in this regard with a specific host factor cyclophilin (Cyp). NIM811. MicroRNAs serve as gene regulators controlling the cell cycle and tissue differentiation150. thereby inhibiting cholesterol biosynthesis and also causing a decrease in prenylated proteins147. A NNI. It is required for HCV replication. These cause a reduction in intracellular mevulonate. Cyclosporine A derivatives such as Debio 025 that lack the immunosuppressive function. GSK-625433 (GlaxoSmithKline) and ABT-333 (Abbott) have reached phase I clinical trials. a combination of specific viral inhibitors (viral protease or polymerase inhibitors) with NIM811 prevented the development of resistance very effectively. It has now been established that cyclosporine A and other non immunosuppressive Cyp inhibitors (such as Debio 025. These cell culture studies suggest that a combination of specific viral inhibitors with statins may delay or prevent the development of resistance to viral inhibitors. this remains to be tested in clinical trials. Attempts are underway utilizing RNAi technology and one such attempt is the designing of TT-033. Statins are inhibitors of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase. Recent studies have further highlighted the importance of miRNAs and P bodies in modulating host cell interactions with viruses152. Statins have been approved clinically for the treatment of hypercholesterolaemia. Cyclosporine A inhibits HCV replication by binding to cyclophilin. a synthetic peptide vaccine. In addition. which belongs to a family of cellular peptidyl-prolyl isomerases. JANuARY 2010 are non-competitive inhibitors which target the viral polymerase. The miRNA (miR-122) is required for HCV replication and development of antiviral therapies targeting on specific miRNA has great potential for exploration. against HCV challenge157. viral and host proteins. Interestingly. HCV has developed complex mechanisms to use the variety and intricacy of the host lipidome. A recent study investigated the effects of combining NIM-811 (host inhibitor) with specific inhibitors of viral protein using replicon system145.28 INDIAN J MED RES. Another promising area for exploring therapeutic options are targeted at processing bodies (P bodies). This seems a very intelligent choice for a rapidly mutating virus like HCV and might be vital to success in forthcoming therapies. There are also reports indicating progress with therapeutic vaccines for HCV. A promising candidate for anti-HCV therapy emerged with the recent development of statins. Several NNIs such as VCH-916 (ViroChem Pharma). containing HCV T-cell epitopes has been recently shown to be safe and is currently in clinical trial.

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