NS5 is a viral protein found in hepatitis C.NS5B has the key function of replicating the HCV’s viral RNA by using the viral positive RNA strand as its template and catalyzes the polymerization of ribonucleoside triphosphates (rNTP) during RNA replication. Several crystal structures of NS5B polymerase in several crystalline forms have been determined based on the same consensus sequence BK (HCV-BK, genotype 1). The structure can be represented by a right hand shape with fingers, palm, and thumb. The encircled active site, unique to NS5B, is contained within the palm structure of the protein. Recent studies on NS5B protein genotype 1b strain J4’s (HC-J4) structure indicate a presence of an active site where possible control of nucleotide binding occurs and initiation of de-novo RNA synthesis. De-novo adds necessary primers for initiation of RNA replication. Current research attempts to bind structures to this active site to alter its functionality in order to prevent further viral RNA replication.
Nonstructural protein 5A (NS5A) is a zinc-binding and proline-rich hydrophilic phosphoprotein that plays a key role in HCV RNA replication. It appears to be a dimeric form without trans-membrane helices. NS5A is derived from a large polyprotein that is translated from the HCV genome, and continues to be processed by nonstructural protein 3 (NS3) viral protease. Despite no inherent enzymatic activity being attributed to NS5A, its function is mediated through interaction with other nonstructural (NS) viral and cellular proteins. NS5A has two phosphorylated forms: p56 and p58, which differ in the electrophoretic mobility. p56 is basally phosphorylated by host cellular protein kinase at the center and near the C terminus, whereas p58 is a form of hyperphorylated NS5A at the center of the serine-rich region. It has been predicted that the N-terminal 30 aa of NS5A form an amphipathic α-helix with a highly preserved feature, which is essential to modulate the association between NS5A and ER membrane. Interestingly, the IFN-sensitivity determining region (ISDR) at the C-terminal of NS5A has been reported to perform strong trans-activating activities, suggesting that NS5A likely functions as a transcriptional activator.
NS5A has three structurally different domains: domain I was demonstrated to be an alternative dimeric structure by crystallography, while domain II and III remained unfolded. Furthermore, the conformational flexibility of NS5A plays an important role in multiple HCV infection stages. It is also possible that NS5A is a critical component during HCV replication and subcellular localization, which may shed light on the poorly understood HCV life cycle. Additionally, NS5A has been shown to modulate the polymerase activity of NS5B, an RNA-dependent RNA polymerase (RdRp). Intriguingly, NS5A may be a RNA binding protein due to the fact that it is able to bind to the 3’UTR of the plus and minus HCV RNA strands. Moreover, NS5A is a key mediator in regulating host cell function and activity upon HCV infection. Therefore, NS5A has been extensively studied in HCV research also due to its capability to regulate the interferon (IFN) response of the host cells. Because NS5A exerts functionally essential effects in regulation of viral replication, assembly and egress, it has been considered a potential drug target for antiviral therapeutic intervention. Indeed, small molecule drugs efficiently targeting NS5A displayed a much higher potency in controlling HCV infection than other drugs. Therefore, NS5A related researches would have important implications in single molecule drug design and pegIFN-free direct-acting antiviral (DAA) combination therapies.
^Rigat, K.; Wang, Y.; Hudyma, T. W.; Ding, M.; Zheng, X.; Gentles, R. G.; Beno, B. R.; Gao, M.; Roberts, S. B. (2010). "Ligand-induced changes in hepatitis C virus NS5B polymerase structure". Antiviral research88 (2): 197–206. doi:10.1016/j.antiviral.2010.08.014. PMID20813137.edit
^Biswal, B. K.; Cherney, M. M.; Wang, M.; Chan, L.; Yannopoulos, C. G.; Bilimoria, D.; Nicolas, O.; Bedard, J.; James, M. N. (2005). "Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors". The Journal of biological chemistry280 (18): 18202–18210. doi:10.1074/jbc.M413410200. PMID15746101.edit
^O'Farrell, D; Trowbridge, R; Rowlands, D; Jäger, J (2003). "Substrate complexes of hepatitis C virus RNA polymerase (HC-J4): Structural evidence for nucleotide import and de-novo initiation". Journal of molecular biology326 (4): 1025–35. PMID12589751.edit
^Biswal, BK; Wang, M; Cherney, MM; Chan, L; Yannopoulos, CG; Bilimoria, D; Bedard, J; James, MN (2006). "Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition". Journal of molecular biology361 (1): 33–45. doi:10.1016/j.jmb.2006.05.074. PMID16828488.edit