PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Advances in understanding Hepatitis C Virus - Is an antiviral/vaccine on the Horizon? by Suresh D. Sharma, Ph.D.  Suresh D. Sharma, Ph.D. Department of Biochemistry and Molecular Biology Pennsylvania State University, University Park, Pennsylvania 16802, USA. INTRODUCTION Hepatitis C virus (HCV) is a small-enveloped RNA virus, known to cause hepatitis C, a life threatening disease. An estimate by WHO suggests that a minimum of 2-3% of the world's population is chronically infected with HCV [1-3]. HCV is spread by direct contact with infected blood and blood products. Availability of injectable therapies and illicit uses of injectable drugs have had a tremendous influence on HCV epidemiology in developed nations. In contrast, in the developing countries, lack of proper cleaning, disinfection of tools and equipments used in hospital and dental clinics remain a major source of virus transmission.  Figure-1: What happens after an infection with HCV? HCV infection leads to the establishment of an acute phase. Only in a very small percentage (~20%), HCV infection can resolve naturally [4]. An amazing fact about this virus is that it can establish a chronic infection in the majority (~80%) who get infected, despite the fact that it is detected and targeted by innate, cellular and humoral immune mechanisms [5]. Hepatitis C virus seems to have evolved multiple strategies to combat antiviral attacks of the host. HCV infection is known to follow a slow progressive clinical course. Individuals with chronic hepatitis C virus infection frequently exhibit no symptoms. Some may report non-specific symptoms such as fatigue, muscle aches, nausea and anorexia. In chronic patients, antibodies directed against several HCV proteins can be detected. A variety of autoimmune or immune complex-mediated diseases have also been associated with chronic HCV infection [6]. In a significant fraction of chronically infected patients, liver disease slowly progresses from fibrosis to cirrhosis and (over a period of 20-30 years) eventually leads to hepatocellular carcinoma (HCC) or liver cancer. Some of the factors that accelerate progression of hepatitis C disease include: alcohol intake, age at infection and co-infections with other viruses (e.g., HBV and HIV). HCC is the leading cause for liver transplantation in the US [7]. HCV is responsible for almost 75% of all cases of HCC in Japan [8]. There is no therapeutic or prophylactic vaccine against HCV yet [9]. HCV is classified into at least six major genotypes that differ by ~30% in their nucleotide sequence. These genotypes show differences with regard to their worldwide distribution, transmission and disease progression, and have been further classified into multiple sub-types. Administration of pegylated interferon-alpha along with ribavirin (a nucleoside analogue) leads to an increase in the sustained virologic response (SVR) rate. This combination is indeed the only treatment of choice for hepatitis C. However, both these compounds are toxic and their administration causes adverse effects that are severe and difficult to tolerate (headache, fever, severe depression, myalgia, arthralgia and hemolytic anemia) [10]. Moreover, only 50% of the patients achieve sustained virologic responses with genotype-1 infections (most prevalent world wide). This combination therapy achieves a better SVR (75%–80%) in patients infected with genotypes-2 and 3. A recent estimate projects the current treatment market for HCV to be around US $3 billion per year. This market is expected to grow rapidly and reach around US $8 billion by 2010 [11]. MOLECULAR BIOLOGY OF HCV HCV is a prototype member of the Hepacivirus genus and replicates predominantly in hepatocytes or liver cells (108 -1011 copies of HCV RNA per gram of tissue). However, recent reports provide good evidence that HCV can also infect cells of extrahepatic origin, such as T and B lymphocytes, dendritic cells (myeloid and plasmacytoid lineages), gut epithelium and the central nervous system [12, 13]. The HCV genome consists of a single-stranded, positive-sense RNA of approximately 9.6 kb (Figure 2).  Figure 2: HCV RNA genome The RNA genome harbors a single ORF that is flanked by 5' and 3'-nontranslated RNA segments (NTR's). The 5'- and the 3'-NTR's sequences of the viral genome are highly conserved. The 5' UTR contains an internal ribosomal entry site (IRES) for cap-independent translation of viral RNA. The 3'UTR has a poly (U/UC) tract. Translation of the single, long ORF yields a polyprotein of ~3010 amino acids (Figure-3).  Figure-3: HCV Polyprotein Proteolytic processing of the polyprotein during and after translation by host and viral proteases yields at least 11 mature viral proteins (core, "F" protein, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). HCV structural proteins are located in the amino-terminal end of the polyprotein and include Core, E1, E2 and p7. Core is a highly basic protein. It encapsulates the HCV-genome. HCV encodes two envelope glycoproteins, E1 and E2. The interaction of the E1- E2 glycoproteins with cell surface receptors is thought to mediate HCV entry (via receptor mediated endocytosis). HCV-E2 glycoprotein has been shown to bind human CD81 [14]. To evaluate the early steps of HCV infection, pseudotype virus systems based on the vesicular stomatitis and retroviruses have been recently developed. HCVpp system generates pseudotype virus particles, which display E1-E2 glycoproteins on their surface. HCVpp closely resembles the cell entry properties of genuine HCV virions and several candidates for HCV receptor have been identified by utilizing this system [15, 16]. The cellular receptors implicated in HCV entry include CD81, scavenger receptor BI (SR-BI), the low-density lipoprotein receptor (LDLR) and the tight-junction Claudin-1. Out of these, SR-BI binds to HDL, LDL and VLDL. It is interesting to note that HCV is also found associated with LDL and VLDL from infected patient serum. HDL has been shown to inhibit HCV-neutralizing antibodies (in sera of acute and chronic HCV-infected patients) by stimulating cell entry via activation of the Scavenger receptor-BI [17]. HCV non-structural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A and NS5B are required for viral genome replication. NS2 auto-protease cleaves at the NS2-NS3 junction. NS3 is a tri-functional protein with a serine-protease, RNA-helicase and NTPase activities. The serine protease activity resides in the N-terminal (one-third) portion while the C-terminal portion possesses the NTPase and helicase activity. NS4A is a cofactor for NS3. The N-terminal portion of 4A is responsible for membrane association of the NS3-4A complex. The NS3 serine protease is responsible for cleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions. NS3 is indispensable for virus replication and therefore represents a very promising target for anti-HCV therapy [18]. Several inhibitors of NS3-4A have been designed and are currently in clinical trials (e.g., ketoamide telaprevir (VX-950 PhaseIII clinical trail), boceprevir (SCH503034-PhaseIII) [19, 20], MK- 7009 and TMC435350 [21]. NS5A is a Zinc metalloprotein and can exist in basally phosphorylated (56 kDa) and hyperphosphorylated (58 kDa) forms. Rigorous biochemical studies have shown that NS5A is an RNA binding protein [22]. It has an amphipathic alpha-helix at its amino terminus with which it is anchored to the ER-derived membranes [23, 24]. Existence of an interferon (IFN) sensitivity determining region (ISDR), is still highly debated and controversial [25]. A significant advance came in the field (1999) with the development of sub-genomic replicons capable of replicating stably in human hepatoma cell lines (Huh-7). The first of its kind, subgenomic replicon utilized a HCV genotype 1b clone called a Con1 in which the HCV nonstructural genes were replaced by a neomycin resistance gene [26]. Mutations that enhance the capacity of sub-genomic HCV RNA to replicate in cell culture (human hepatoma cells Huh-7) were mapped to the NS5A-coding sequence. Following transfection of in vitro transcribed RNA into Huh-7 cells, antibiotic G418-resistant cells could be obtained in which the sub-genomic RNA replicated autonomously [27]. However, the failure to produce authentic infectious hepatitis C virus in cell culture remained a major blockade for many years. A major breakthrough in the field came with the development of a complete in vitro cell culture system for HCV in the year 2005. HCVcc (for HCV grown in cell culture), allowed researchers for the first time to study the complete life cycle of HCV [28, 29]. NS5B is a RNA dependent RNA polymerase (RDRP). It initiates synthesis of complementary negative-strand RNA using the HCV genome (positive polarity) as a template. Subsequently, it generates positive-strand RNA from this negative-strand RNA template [30]. NS5B lacks a proofreading function. Due to high rate of error-prone replication, complex mutant swarms are generated [31], and poses a huge hurdle for the development of antiviral/vaccine. Currently several NS5B inhibitors are in clinical trials (e.g., valopicitabine, BILB1941, IDX184-Phase I, ANA598, PF-866554, GSK625433-Phase I, A-782759 and A-878837 [21]. HCV has a remarkable ability to establish persistent infection. Research so far indicates that HCV probably employs multiple strategies to antagonize host-immune responses. RIGI and IPS-1 signaling molecules initiate innate defenses to HCV infection. After binding to HCV-RNA, RIGI undergoes changes in conformation and can then interact with IPS-1 (IPS-1 is also known as VISA, MAVR.) This interaction of RIGI with IPS-1 can signal downstream activation of IRFs and NFkB to trigger production of alpha/beta INF's. NS3/4A protease cleaves ISP-1 thereby halting interferon expression. Thus NS3/4A protein can antagonize innate immune control mechanisms. PKR is considered a component of the cellular antiviral response and regulates translation through phosphorylation of eIF2-alpha. HCV NS5A directly binds and prevents PKR activation. Nevertheless, there are reports indicating progress with therapeutic vaccines for HCV. IC41, a synthetic peptide vaccine, contains HCV T-cell epitopes. IC41 can induce HCV-specific INF-gamma-secreting CD4+ and CD8+ T cells in healthy individuals. It was shown to induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV non-responder patients [32]. There is also evidence that broadly neutralizing antibodies against antigenic regions of E2 can protect Alb-uPA/SCID mouse (human liver-chimeric mouse model), against HCV challenge [33]. Attempts are also underway at Tacere Therapeutics, Inc., to inhibit virus replication using RNAi technology. TT-033 contains three separate RNAi molecules designed to shut down replication of all strains of the Hepatitis C virus. This strategy sounds promising and could potentially stop the generation of viral escape mutants [34].  The HCV virus life cycle CONCLUSIONS Despite current advances in our understanding of HCV, many questions remain unanswered. The clinical outcome of HCV infection varies; some individuals can eliminate the virus, whereas others remain chronically infected. We don't know yet why and how this occurs, or what factors are involved. This insight can help in developing more effective treatments. At present, the viral genotype is the only available pre-treatment predictor of treatment response. Newer and effective strategies are urgently needed to enhance our ability to predict treatment responsiveness. Why do we observe differences in treatment response with different genotypes; this is still an open question. There is also an urgent need to develop more effective and less toxic derivatives of interferon. Every step in the viral life cycle can be a potential antiviral target. The search for "specific targeted antiviral therapy for HCV" (STAT-C) has been in progress for a long time now. Numerous pharmaceutical labs are trying to develop antivirals for the targets, which include IRES, protease, helicase and RNA dependent RNA polymerase. Various resistant loci have been identified with the help of replicon-systems and more importantly in clinical studies. A major hurdle apparent from these studies is the selection of resistant genotypes to antivirals. However, there is some hope that various antivirals, targeting multiple steps in viral life cycle, along with existing combination therapy may eradicate the virus completely. However, there are many issues (such as toxicity/side-effects) that need to be investigated before these antivirals can reach the market. HCV envelope glycoprotein-2 (hypervariable regions: HVR1, HVR2 and HVR3) and the C-terminal region of the NS5A protein (V3 domain) contain mutational hotspots and evolve rapidly. As old epitopes are recognized by the immune system the new epitopes generated as a result of mutations evade the adaptive immune response. Thus as HCV replicates, genetically distinct viruses called "quasispecies" are generated even within a single infected host. HCV can therefore pass under the radar of the immune system and avoid detection. Very rapid genetic variability presents a huge challenge in the development of a prophylactic (preventive) vaccine against HCV. An ideal goal would be to stop chronicity and disease by inducing sterilizing immunity. REFERENCES
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