NS5B polymerase is one of the most attractive targets for developing new drugs to block Hepatitis C virus (HCV) infection.
NS5B, the putative HCV RNA dependent RNA polymerase (RdRP), functions in association with viral non-structural proteins and cellular factors to synthesize a negative strand HCV antigenome in vivo. The viral antigenome in turn serves as a template for generation of additional positive strand genomes during replication. NS5B is known to have a highly hydrophobic 21 amino acid carboxylterminal domain that functions as a transmembrane insertion sequence. This NS5B C-terminal domain is essential in vivo for viral replication. Initial studies sought to identify the cellular localization of NS5B, and immunofluorescence studies of mammalian cells (COS7 cells) expressing NS5B from a transfected plasmid, revealed that NS5B is present within fine speckles in the cytoplasm that accumulate in the perinuclear regions. Subsequently, NS5B was found to associate with both membrane and cytosolic fractions, as suggested by the immunoflorescence localization.
The function of NS5B in HCV
The activity of the HCV polymerase, NS5B has been implicated in the persistence of viral infection, and the polymerase itself has been considered to be an immune evasion molecule. One of the hypotheses as to why HCV is successful as a persistent viral infection is that HCV establishes a 'pool' of genetically divergent RNA genomes in an infected individual over time. This viral population is referred to as a 'quasispecies swarm,' which is defined as a closely related self-replicating population that evolves as a single unit, when placed under environmental pressure. Studies indicate that the genetic variation between each distinct HCV genome ranges from about 1-5% in quasispecies. This strategy is hypothesized to benefit the virus by generating a large pool of genetic variants that can quickly adapt to host immunosurveillance, and confer resistance to anti-virals such as interferon-α. This hypothesis was generated to explain in part the observations that in chronically infected chimpanzees and humans, where CD8(+) cytotoxic T lymphocytes fail to clear the HCV infection, there is a higher degree of mutation in viral RNA sequences coding for proteins that contain T cell epitopes. NS5B mediates viral survival by generating mutations enriched in the Hyper Variable Regions (HVR) of E2, the envelope glycoprotein, and NS5A. The residues encoded by the HVR of E2 are very permissive to mutation; thus the HVR sequence may vary by as much as 50% or more in strains isolated from different patients. There is evidence suggesting that mutagenesis of the E2 HVR's mediates escape from the immune system, given that B-cell derived antibodies do not recognize these new epitopes.
Generation of quasispecies is solely the result of NS5B infidelity during the genome replication cycle. Since the mechanism of action of NS5B in HCV's replication is not fully understood, the contribution of other activities to mutagenesis or quasispecies formation (such as the proposed template switching activity of NS5B as described below) have not been uncovered. Thus, my effort has been to understand the biochemical behavior of NS5B. Data indicates that the quasispecies generated by NS5B could mediate mutational escape (from CD8(+) T-cells) by generating viable HCV genomes whose encoded proteins are impaired in binding to MHC (major histocompatability complex) molecules, or that are refractory to proteosomal destruction.
Cilia, S. M. (2009). In vitro biochemical studies of NS5B, the HCV RNA dependent RNA polymerase. University of California, Berkeley.