|dc.description.abstract||Hepatitis C virus NS5A is a multi-functional viral protein essential for viral replication and assembly, although the exact role the protein plays in the viral lifecycle remains unclear. A vast array of functions have been attributed to NS5A in recent years, despite the lack of enzymatic activity. NS5A has been found to interact with over 130 host proteins including many which are central to cellular signaling pathways. NS5A is composed of three domains separated by regions of low complexity. All three domains perform important functions in the viral lifecycle. Domains I and II are essential for viral replication whereas domain III is required for viral assembly. However, the role that NS5A and its individual domains may play in modulating viral translation remains controversial. Previous studies have utilized translation reporter systems that do not accurately reflect the role of the viral 3´-UTR in modulating viral translation. We and others have shown that NS5A binds to the poly-U/UC region of the 3´-UTR. In addition to serving as the initiation site for negative strand synthesis the 3´-UTR functions to significantly enhance viral translation. The mechanism of translation enhancement remains unclear but may involve long range RNA-RNA interaction with the IRES, the binding of cellular proteins which stimulate translation and/or the recycling of ribosomes. Therefore, the protein-RNA interaction between NS5A and the poly-U/UC region has the potential to modulate viral translation. Therefore we set out to determine the role of NS5A and its individual domains in modulating viral translation and the role of the NS5A-poly-U/UC region interaction in this modulation.
Utilizing monocistronic RNA reporters which contain the viral 5´- and 3´-UTRs and an internal Renilla luciferase reporter gene, we determined that NS5A specifically down-regulates viral translation in a dose-dependent manner through a mechanism dependent upon the presence of the poly-U/UC region in the viral 3´-UTR. Furthermore, we have re-tested the effect using full-length HCV genomic RNA reporters. These results suggest that NS5A is able to interfere with the stimulation of viral translation exerted by the 3´-UTR. This down-regulatory function of NS5A may function in mediating a switch from translation to replication, a step required in the lifecycle of a positive sensed RNA virus. Having established a role for NS5A in modulating viral translation, we then aimed to determine which region of NS5A was responsible for this effect. We found that each of NS5A domains was capable of this modulatory effect on viral translation independently. Although surprising, this finding is not entirely unexpected as each domain has been shown to retain the ability to bind to the poly-U/UC region.
Within NS5A domain I we identified a 61 aa. region sufficient for translation down-regulation. Furthermore, we have identified a number of positively charged residues within this region involved in the modulation of viral translation, in particular arginine 112 (R112). This residue has previously been found to be at the domain I dimer contact interface and to form an intermolecular hydrogen bond with glutamic acid 148 (E148). We found that mutations R112A and E148A individually negate the ability of domain I to modulate viral translation and these mutations impede the formation of domain I dimers. Additionally, the R112A mutation appears to affect the ability of domain I to interact with the poly-U/UC region of the viral 3´-UTR alluding to the possible mechanism of translation modulation. Finally this mutation was lethal in an HCV subgenomic replication, confirming the link between NS5A dimerization, RNA binding and viral replication. These results collectively point to a crucial role for the NS5A arginine 112 residue in the modulation of HCV lifecycle by NS5A.
Within NS5A domain II, we identified a 47 aa. region sufficient for translation modulation. Through the mutation of positively charged amino acids within this region, we found that lysine 312 (K312) was essential for this effect. The ability of this domain to modulate viral translation was completely lost when K312 was mutated within a full domain II protein fragment. The mechanism behind this modulation remains unclear but the 47 aa. region identified has been previously found to contain a region proposed to make contact with poly-U RNA and the K312 residue was suspected to interact directly with such RNA. Furthermore, this region interacts with the host protein cyclophilin A, an interaction that enhances the RNA binding ability of domain II. These findings strongly suggest that domain II modulates viral translation by binding within the poly-U/UC region.
While investigating the modulation of viral translation by NS5A domain III we determined that the C-terminal 31 aa. are sufficient for the effect of this domain on viral translation. Through alanine scanning mutagenesis we identified glutamic acid 446 (E446) as playing a key role in the modulatory function of this region. Within a domain III protein fragment mutation of this E446 residue abolishes the modulatory function of this domain towards HCV translation. The mechanism behind this modulation and the role of E446 in this effect remains to be determined.
These findings suggest that in addition to being essential for viral replication and assembly, NS5A has an important role in modulating viral translation through a mechanism requiring the poly-U/UC region of the viral 3´-UTR. Furthermore, each domain of NS5A appears to contribute to this effect. These results support the description of NS5A as a multi-functional protein and the further characterization of its functions may aid in the development of novel antivirals targeting the numerous functions of this complex, and at times puzzling, viral protein.||en_US