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The impact of HCV diversity on replication fitness and protease inhibitor sensitivity in HIV infected patients with chronic HCV

Final Report Summary - HCV/HIV-PI (The impact of HCV diversity on replication fitness and protease inhibitor sensitivity in HIV infected patients with chronic HCV)

The HCV protease inhibitors inhibit HCV replication by blocking the action of the HCV serine protease. This serine protease is formed by the union of the amino-terminal domain of the HCV NS3 protein and the HCV NS4a. The genetic variability characteristic of chronic HCV infection makes drug resistance a concern in the clinical development of HCV inhibitors.

The main objectives of this grant proposal were:
1. To sequence HCV NS3 from multiple NS3 isolates of both HCV mono-infected and HCV/HIV co-infected patients. To detect the genetic variation specific to the isolates of individual patients and to analyse the effect of HIV infection on HCV diversity.
2. To clone the HCV NS3 from the patient isolates into a subgenomic HCV replicon.
3. To develop a transient cell-based assay in order to evaluate NS3 from the clinical isolates.
4. To assess the replication fitness of the NS3-cloned replicons with that of the parental laboratory-optimised replicons.
5. To assess the efficacy of protease inhibitors targeting the NS3 serine protease in repressing the different NS3-cloned replicons and the effect of individual mutations in conferring resistance to anti-HCV drugs.

Since writing the project in 2007 several advances in this line of investigations have been achieved. A HCV clone (Jc1) capable of replication and infection in vitro and in vivo is now widely available; the clone is derived from a Japanese fulminant hepatitis C patient, infected with HCV of genotype 2a. We are now capable of studying the complete replication cycle of HCV within cell culture and without adaptive mutations.

Deliveries:
1. NS3 protease region was sequenced from various patients' samples to compare the HCV NS3 protease variation in the presence or absence of HIV coinfection.
2. Cloning of the NS3/4a region from genotype 2a, and 1b into the Jc1/GNN HCV infectious clone was achieved to produce various chimeras of the NS3 protease region and NS4a domains.
3. In vitro transcription, infection assays, RNA quantification, titration assays and measurement of protein production were used to compare replication fitness of chimeras with the wildtype HCV virus, all these assays were learnt at the outgoing host and then set up at the returning host.
4. Infected/transfected cells were passaged over 40 days comparing wildtype with chimeric constructs for replication, infectivity and protein production, sequences were analysed for adaptive mutations.

Results:
1. Sequence analyses of the NS3 protease region, comparing samples with or without HIV coinfection showed no statistical difference and no mutations were found that caused resistance to the NS3/4a protease inhibitors.
2. Nine chimeras from two independent genotypes were successfully cloned into the Jc1 plasmid using various domains of the NS3 protease region and the NS4a, these were fully sequenced and then the transcribed RNA electroporated into hepatoma cells and tested for replication.
3. All chimeras using the J6 (genotype 2a) domains gave raise to replicating and infectious virus.
4. Of the Con1 (genotype 1b) chimeras only the chimera with the NS3 protease region, and NS4a cofactor and acidic domain gave rise to a replicating virus. In the four day kinetic assay where only replication was analysed, this chimera resulted in a lower replication yield compared to the Jc1 wildtype and did not give rise to infectious virus. Only after passaging the chimera virus-containing cells did the virus increase in viral load and prove to be infectious to naive cells.
5. The chimera virus was sequenced at day 0, day 3, day 14, day 30 and day 40 post electroporations, only one mutation was found at position P654S of the NS3 helicase domain positioned between the two cloned domains. This mutation was found at day 3 and did not revert over the 37 days in culture suggesting adaptation properties.
6. Construction of the genotype 1a chimera is still ongoing but hampered by technical problems.

Discussion:
Although no significant differences were found between the sequences of NS3 protease of HIV coinfected and HCV chronic monoinfected patients, a robust replicating/infectious HCV virus that expresses genotype 1 NS3 protease will be an enormous contribution to discovering new protease inhibitors and confirm resistance from genotype 1 patients. In this current project a fully replicating and infectious virus was achieved using the NS3 protease region and two domains of the NS4a (NS4a cofactor and acidic domain) from the genotype 1b prototype virus (Con 1). Moreover only one adaptive mutation was discovered to enhance the replication to similar levels of the wildtype Jc1 virus (genotype 2a).

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