NS3 helicase

Maga G, Gemma S, Fattorusso C et al. (2005) Specific targeting of hepatitis virus C NS3 helicase. Biochemistry 44 9637-9644... 

Rationale for the Selection of HCV NS3 Helicase as a Target for Antiviral Drug Development... 

The RNA helicase activity of the full-length NS3/4A enzyme (8) and of the NS3 C-terminal helicase domain has been demonstrated (10,11,17-19) by the unwinding of double-stranded RNA (dsRNA) molecules. The directionality of RNA helicase unwinding is 3 to 5 with respect to the template strand (8,19). Unlike most other helicases, the HCV NS3 helicase is capable of unwinding both RNA and DNA homoduplex and heteroduplex molecules (19). In common with other helicases, NS3 helicase requires divalent cations (Mg2+ or Mn2+) in conjunction with the hydrolysis of nucleoside triphosphates (NTPs) to provide the energy source for unwinding (11,12). 

Preugschat, F., Averett, D. R., Clarke, B. E., and Porter, D. J. T. (1996) A steady-state and pre-steady-state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain. J. Biol. Chem. 271, 24,449-24,457. 

Tai CL, Chi WK, Chen DS, Hwang LH (1996) The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol 70 8477-8484 Tong X, Chase R, Skelton A, Chen T, Wright-Minogue J, Malcolm BA (2006) Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034. Antiviral Res 70 28-38... 

Fig. 3 A ribbon diagram of the HCV NS3/4A protease ICU1 (Yao et al, 1999). The serine protease domain is shown in cyan with the catalytic triad highlighted in yellow, and the helicase domain is...

One exciting approach is the development of short sequences of RNA that bind specifically to HCV helicase and/or the protease activity found in the same hepatitis C virus-encoded non-structural protein, NS3, and inhibit helicase at sub-micromolar concenttations (Umehara et al. 2005). These molecules could provide the basis for developing potent helicase inhibitors with improved pharmacotherapeutic properties. 

Frick DN (2007) The hepatitis C virus NS3 protein a model RNA helicase and potential drug target. Curr Issues Mol Biol 9 1-20... 

Townsend L, Devivar R, Turk S, Nassiri M, Drach J (1995) Design, synthesis, and antiviral activity of certain 2,5,6-trihalo-l-(beta-d-ribofuranosyl)benzimidazoles. J Med Chem 38 4098 105 Turlure F, Devroe E, Silver PA, Engelman A (2004) Human cell proteins and human immunodeficiency virus DNA integration. Front Biosd 9 3187-3208 Umehara T, Fukuda K, Nishikawa F, Kohara M, Hasegawa T, NisUkawa S (2005) Rational design of dual-functional aptamers that inhibit the protease and helicase activities of HCV NS3. J Biochem 137 339-347... 

Fig. 2.2 (A) Structure of full-length NS3 including the N-terminal protease domain (bottom) and C-terminal helicase domain (top). The NS4A peptide (purple) is covalently attached to the N-terminus of NS3 (see text). Within the protease domain the N- and C-terminal -barrels are at the right and left, respectively. The zinc atom is visible at the bottom left. [98]. (B) Surface view of the NS3 protease domain showing compound (1) bound at the relatively shallow active site (See also Fig. 2.6) [42].

NS3 proteinase (helicase) (Love etal, 1996), NS5B lalq (NS3), lc2p,... 

Helicases unwind dsDNA and are classified 5 ->3 or 3 ->-5 according to their ability to unwind DNA adjacent to either a 5 or 3 ssDNA overhang. An assay has been developed to determine whether this preference also indicates unidirectional translocation on ssDNA. Using oligonucleotides which have either a 3 - or 5 -biotin bound to streptavidin, it was found that 5 3 helicases displaced streptavidin from the 3 -end but not from the 5 -end. Similarly, 3 - 5, such as the helicase NS3 from HCV, displaces streptavidin from the 5 -end only. ... 

In this chapter, the development of a 96-well plate increasing signal helicase assay will be described. The authors have used this assay to detect inhibitors of hepatitis C virus (HCV) NS3/4A RNA helicase. 

Fig. 1. Model for HCV RNA replication. The diagram shows the putative formation and function of the HCV replication complex that involves the RNA-dependent RNA polymerase (RdRp) together with the NS3/4A multifunctional enzyme. The RdRp catalyzes the formation of phosphodiester bonds between adjacent nucleotides, and the helicase separates the two RNA strands after synthesis.

The authors expressed and purified a /// -tagged NS3/4A enzyme (26) for use in the helicase assay. Please note that the author s helicase assay is versatile and can be used be optimized for use with other DNA or RNA helicase... 

Fig. 5. The helicase high-throughput assay can detect both DNA and RNA unwinding activity and gives similar results to the traditional gel-based helicase assay. (A-E) Gel-based assay showing unwinding of RNA and DNA heteroduplex and homoduplex substrates by action of 5-120 fmol of HCV NS3/4A enzyme. The positions of the dsNA and ssNA are labeled.

In these studies, a His-tagged form of the HCV NS3/4A helicase was used (26). All enzyme aliquots in enzyme dilution buffer (buffer III) were stored at -80°C. 

Johansson, A., Poliakov, A., Akerblom, E., Wiklund, K., Lindeberg, G., Winiwarter, S., Danielson, U. H., Samuelsson, B., Hallberg, A. Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase/NTPase) a comparative study of different C-terminals. Bioorg. Med. Chem. 2003, 77(12), 2551-2568. 

The N-terminal domain of yeast eIF4A is structurally similar to RecA, two DNA helicases (Per and Rep), and the RNA helicase NS3 from hepatitis C virus. Despite differences in amino acid sequence and substrate specificity, these domains share a common fold involved in ATP hydrolysis (Bird et al., 1998). The domains have a core structure composed of five parallel (3-strands connected by a-helices within an a/p motif. In eIF4A, the helicase core consists of -strands S2, S3, and S5-S7 and a-helices H4, H5, and H7-H9. In most extant helicase structures, polypeptide regions flanking the core have either additional secondary structure elements or entire domains that are specific to the biochemical functions to these related molecules. The core domain establishes a scaffold upon which residues involved in ATP binding and hydrolysis are located (motifs I, la, II, and III). 

Figure 7.4b Target macrocycle 14 (yellow) overlaid with ciluprevir (blue). Green full length NS3/4A purple helicase.

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