Abstract
Hepatitis C virus encodes an autoprotease, NS2-3, which is required for processing of the viral polyprotein between the non-structural NS2 and NS3 proteins. This protease activity is vital for the replication and assembly of the virus and therefore represents a target for the development of anti-viral drugs. The mechanism of this auto-processing reaction is not yet clear but the protease activity has been shown to map to the C-terminal region of NS2 and the N-terminal serine protease region of NS3. The NS2-3 precursor can be expressed in Escherichia coli as inclusion bodies, purified as denatured protein and refolded, in the presence of detergents and the divalent metal ion zinc, into an active form capable of auto-cleavage. Here, intrinsic tryptophan fluorescence has been used to assess refolding in the wild-type protein and specific active site mutants. We also investigate the effects on protein folding of alterations to the reaction conditions that have been shown to prevent auto-cleavage. Our data demonstrate that these active site mutations do not solely affect the cleavage activity of the HCV NS2-3 protease but significantly affect the integrity of the global protein fold.
Highlights
Hepatitis C virus (HCV) currently infects an estimated 170 million individuals worldwide; in approximately 80% of these cases, the virus establishes a chronic infection that leads to liver cirrhosis and hepatocellular carcinoma [1]
One complication of the analysis of the activity and folding of J4 NS2-3 is the presence of a 30-kDa C-terminal truncation product of the fulllength protease (Fig. 2A) [13], which is detectable with NS3 antisera but not detectable with the FLAG antisera, as the truncation lacks the FLAG-tag located at the C-terminus of the full-length protein
We have demonstrated through comparative analyses of the purified recombinant NS2-3 autoproteases from J4 and JFH-1 isolates that the proteins exhibit similar characteristics in terms of auto-cleavage requirements and efficiency
Summary
Hepatitis C virus (HCV) currently infects an estimated 170 million individuals worldwide; in approximately 80% of these cases, the virus establishes a chronic infection that leads to liver cirrhosis and hepatocellular carcinoma [1]. HCV infection is a growing health problem with an estimated three to four million individuals becoming newly infected each year. HCV infection is the leading indicator for liver transplantation in the developed world research into identifying targets for therapeutic intervention is vital and has been stimulated by the limited ineffective therapy options and the lack of vaccines. HCV is an enveloped virus with a positive sense RNA genome of 9.6 kb consisting of a 5′-untranslated region (UTR) which includes an internal ribosome entry site (IRES), a single open reading frame (ORF) that encodes the structural and non-structural viral proteins and a 3′UTR [2,3,4,5]. Co-translational and post-translational processing of the polyprotein by host signal peptidases and two viral proteases at the endoplasmic reticulum (ER) results in 10 mature virus proteins; these cleavage
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