Abstract
Hepatitis C virus (HCV) is susceptible to cyclosporine (CsA) and other cyclophilin (CypA) inhibitors, but the genetic basis of susceptibility is controversial. Whether genetic variation in NS5A alters cell culture susceptibility of HCV to CypA inhibition is unclear. We constructed replicons containing NS5A chimeras from genotypes 1a, 2a and 4a to test how variation in carboxy terminal regions of NS5A altered the genotype 1b CsA susceptibility. All chimeric replicons including genotype 1b Con1LN-wt replicon exhibited some cell culture sensitivity to CsA with genotype 4a being most sensitive and 1a the least. The CypA binding pattern of truncated NS5A genotypes correlated with the susceptibility of these replicons to CsA. The Con1LN-wt replicon showed increased susceptibility towards CsA when proline at position 310P was mutated to either threonine or alanine. Furthermore, a 15 amino acid long peptide fused N terminally to GFP coding sequences confirmed involvement of proline at 310 in CypA binding. Our findings are consistent with CypA acting on multiple prolines outside of the previously identified CypA binding sites. These results suggest multiple specific genetic variants between genotype 1a and 1b in the C-terminus of NS5A alter the CsA susceptibility of replicons, and some variants may oppose the effects of others.
Highlights
200 million people are infected with Hepatitis C virus (HCV), and many of these people will develop severe liver disease, with or without hepatocellular carcinoma, because of persistent HCV infection
To avoid the lower replication capacity associated with certain HCV genotypes and isolate the nonstructural protein 5A (NS5A) variation between genotypes, we constructed NS5A chimeric replicons in the backbone of the commonly used genotype 1b Con1 replicon which displays robust replication capacity
The in vitro transcribed RNAs derived from the Con1bLN-wt, and chimeric replicons containing other NS5A genotypic sequences from amino acid 312 to the NS5A-NS5B cleavage site (Con1bLN-5A1a, Con1bLN-5A2a and Con1bLN-5A4a), were electroporated into Huh7.5 cells and luciferase activity was monitored over a period of five days in the presence and absence of CsA
Summary
200 million people are infected with HCV, and many of these people will develop severe liver disease, with or without hepatocellular carcinoma, because of persistent HCV infection. HCV treatments include pegylated interferon α along with ribavirin and possibly a protease inhibitor. This treatment has problems with toxicity, cost, delivery, and efficacy. Whether naturally occurring variation in NS5A alters cyclophilin susceptibility has been disputed [11], but this variation could explain why an antiviral effect of cyclosporine in transplant patients may be detectable only in selected cases, if at all. In this study we observed varying levels of CsA susceptibility for replicons containing the carboxy terminal regions of NS5A variants of similar lengths from different. Our findings are consistent with CypA acting on multiple prolines outside of the previously identified CypA binding site and suggests that conformations of NS5A adopted in viral replication can vary in cyclophilin inhibitor susceptibility
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