Diversity:
Heterogeneity is characteristic of the hepatitis C virus. Like other RNA viruses, HCV has a high mutation rate, due to an error-prone polymerase that is lacking in proofreading ability. Of all the viral proteins, the highest mutation rates have been observed in the envelope proteins (1). The genetic diversity of HCV is attributed to differences in genotypes as well quasispecies. The term genotype refers to genetic heterogeneity amongst HCV isolates world-wide, and reflects the accumulation of mutations during the long-term evolution of these viruses. The term quasispecies refers to genetic heterogeneity within an infected individual, due to de novo infection with a heterogeneous virus population, or accumulation of mutations during the course of the infection. 

A variety of HCV isolates or 'strains' exist around the world (see Figure 1). The most diverse of these isolates belong to major genotypes and are identified with arabic numerals (1, 2, 3, etc.). More closely related isolates, within these major genotypes, are considered subtypes, and are identified with lower case letters (a, b, c, etc.). In addition, genotypes are grouped together to form clades (see Table 1). 
 

Worldwide, 6 major clades, and more than one hundred different subtypes of the virus have been identified by nucleotide sequencing. The genotypic diversity of HCV, due to the mutation rate of the virus, interferes with effective humoral immunity against it. Although neutralizing antibodies to HCV have been detected in the serum of infected patients, these are, at best, short-lived (2). Moreover, HCV infection has not been shown to induce lasting immunity against re-infection with different virus isolates, or even the same isolate. Thus, neither homologous nor heterologous immunity appears to develop after acute HCV infection. 

Some HCV genotypes are distributed worldwide, while others are more geographically confined (see Figure 1). Genotypes 1a, 1b, 2a, 2b, 2c, and 3a account for more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan, Australia, and New Zealand. Genotype 3a is more common among younger populations. Other subtypes of genotype 3 are highly prevalent in Nepal, Bangladesh, India, and Pakistan. Most infections in Egypt are genotype 4a, and this and other subtypes of genotype 4 are
found in Central Africa. Genotype 5a accounts for about 50% of infections in South Africa. Genotypes 4 and 5 are found only sporadically outside Africa. Genotype 6 isolates are primarily found in Southeast
Asia (8). It should be noted that the genotype distribution can vary significantly among different population groups in the same geographical area. Associations between genotype and mode of transmission also exist; genotype 3, for instance, is more prevalent among intravenous drug users (3). 

HCV genotype may be an important factor influencing the severity of liver disease. Infection with genotype 1b has been associated with more advanced liver disease and the development of both liver cirrhosis as well as hepatocellular carcinoma (1). Observations have also shown that patients with genotype 1b typically respond weakly to interferon therapy (3). However, while differences in pathogenicity and responsiveness to antiretroviral therapy have been reported among the genotypes, the biological impact of these differences still remains incompletely defined. Nevertheless, the current limited knowledge base does not undermine the fact that HCV genotyping is an important factor to consider in the management of treatment for HCV-infection. 

Genomic Organization:
HCV is a linear RNA virus, with a positive-sense single stranded genome of approximately 9600 nucleotides. This single, large open reading frame (ORF) encodes a polyprotein of approximately 3000 amino acids. 

The open reading frame is flanked at each terminus by untranslated regions (UTRs), which are highly conserved among the HCV isolates. The 5' UTR is considered important in initiating translation of the viral genome, while conserved elements within the 3' UTR are essential for RNA synthesis and genome packaging (1). The high degree of genetic conservation among the UTR sequences renders them good targets for antiretroviral therapy.

Hypervariable regions of the ORF encode envelope proteins, which vary from isolate to isolate, and may thus allow the virus to evade the host immunologic response directed at specific envelope proteins. The 3' end of the viral genome codes for non-structural (NS) proteins. The RNA-dependent polymerase through which HCV replicates is encoded by the NS5 region (2). (see Figure 2)
 

Figure 2    Organization of the HCV genome and its associated proteins. Structural genes at the 5' end include the nucleocapsid region (C), and the envelope regions (E1 and E2). The 5' UTR and C regions are highly conserved, while the envelope domain E2/NS1 contains the hypervariable region. At the 3' end are five non-structural regions (NS5). Viral proteins included in various immunoassys and in the recombinant immunoblot assay are presented below their corresponding genes (2).

Because HCV does not replicate via a DNA intermediate, it does not integrate into the host genome. The virus tends to circulate in low-titer, which makes visualization of the viral particles quite difficult. Although in vitro HCV replication remains a challenge, the chimpanzee is proving to be an invaluable experimental animal model (2). 

Structural Proteins:
The hepatitis C virus is a small, enveloped virus. The viral nucleocapsid, consisting of core protein and viral genomic RNA, is enveloped by a lipid bilayer containing two glycoproteins (E1 and E2); this constitutes the structure of the infectious virion. Because the HCV genome contains a single open reading frame, all viral proteins are initially contained within one polyprotein precursor. Cellular peptidases in the lumen of the endoplasmic reticulum (ER) cleave the polyprotein precursor to release three structural proteins: two envelope proteins (E1 and E2) and the core protein (1). 

Translocation of HCV core protein into the nucleus of cells has been suggested as one possible mechanism of cell transformation. More significantly, some studies have shown that the core protein can interact with cellular proto-oncogenes, and thus play an important role in the development of hepatocellular carcinoma (4). 

The fate of the envelope proteins present in the lumen of the ER differs from that of the core protein. A specific sequence of amino acids within the carboxy terminal of the E2 protein has been identified as a signal sequence for the retention of E2 in the endoplasmic reticulum. In the absence of this amino acid sequence, E2 is directed to the cell surface (5). Whether or not this same signal also suffices for the retention of E1 within the lumen of the endoplasmic reticulum has not been conclusively determined as yet. However, retention of the HCV glycoprotein complexes in the ER suggests that HCV budding might occur at this intracellular organelle (5). 

Mechanism of Infection:
The exact mechanism by which HCV enters host cells to initiate infection is not well understood. The lack of a reliable HCV culture system makes it particularly difficult to analyze the different steps in viral infection (1). However, it is known that the E1 and E2 envelope proteins of different HCV isolates exhibit significant genetic heterogeneity. It is possible then, that these proteins play a role in cellular receptor binding, and subsequent fusion of the virus to a host cell. 

A recent study has identified the CD81 cell surface molecule as a potential receptor for HCV (6) (see Glossary for the function of the CD81 molecule). This molecule has four membrane-spanning loops, whose sequences vary amongst species. Between humans and chimpanzees -- the only known species of animals known to support HCV replication -- these protein sequences are conserved. Further support for CD81 as a potential HCV receptor comes from the fact that this molecule is expressed on the membranes of hepatocytes and lymphocytes, both of which are cells that support HCV replication (1, 6). 

Non-Structural Proteins:
The 3' end of the open reading frame encodes seven non-structural (NS) proteins (see Figure 2). These proteins include a protease, a helicase, and an RNA polymerase, enzymes necessary for the replication of viral DNA within the host cell. Particular attention has been focused on the NS5B protein, which is believed to function as the RNA-dependent RNA polymerase. Such enzymes often require a priming mechanism for the initiation of RNA synthesis; the primer being either an exogenous RNA molecule or the 3' end of the RNA folded onto itself. In the case of HCV, it is thought that RNA-dependent RNA synthesis takes place by a copy-back mechanism, in which the 3' nucleotide of the template is used to prime synthesis of the complementary strand (7). The RNA polymerase also has potential as a target for inhibition of viral replication. In the case of the Dengue virus, which also uses an RNA-dependent RNA polymerase, the binding of specific compounds to the polymerase has been shown to inhibit their activity in cell culture, thereby blocking viral replicaton (1). 

References:
1. Forns X & Bukh J. The Molecular Biology of Hepatitis C Virus: Genotypes and Quasispecies. Clinics in Liver Disease  3(4),  1999.

2. Dienstag JL & Isselbacher KJ. Acute Viral Hepatitis: Virology and Etiology:
http://www.harrisonsonline.com/

3. The Canadian Hepatitis Information Network: http://www.hepnet.com

4. Moriya K, Fujie H, et al. The Core Protein of Hepatitis C Virus Induces Hepatocellular Carcinoma in Transgenic Mice. Natural Medicine 4:1065, 1998.

5. Cocquerel L, Meunier J-C, et al. A Retention Signal Necessary and Sufficient for Endoplasmic Reticulum Localization Maps to the Transmembrane Domain of Hepatitis C Virus Glycoprotein E2. Journal of Virology 72:2183, 1998.

6. Pileri P, Uematsu Y, et al. Binding of Hepatitis C Virus to CD81. Science 282:938, 1998.

7. Lohmann V, Korner F, et al. Biochemical properties of Hepatitis C Virus NS5b RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. Journal of Virology 71:8416, 1997.

8. Maertens G, Stuyver L. Genotypes and Genetic Variation of Hepatitis C Virus. The Molecular Medicine of Viral Hepatitis. Chichester, England, John Wiley & Sons, 1997.