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Role of hepatitis C virus (HCV) transactivating proteins in the pathogenesis of HCV-induced liver lesions and hepatocellular carcinoma (HCC)

Final Activity Report Summary - TRANS-HCV (Role of hepatitis C virus (HCV) transactivating proteins in the pathogenesis of HCV-induced liver lesions and hepatocellular carcinoma (HCC))

Chronic HCV infection is responsible for chronic hepatitis, cirrhosis and end-stage liver disease, and hepatocellular carcinoma (HCC, primary liver cancer). Although cirrhosis is an important factor in the development of HCC in HCV-infected patients, it is likely that viral proteins play a role in carcinogenetic mechanisms, although this role is still unclear. In addition, HCV is a genetically variable virus: this diversity arises from an error prone RNA-dependant RNA polymerase lacking a correcting mechanism. This particular property gives rise to the distribution HCV in so-called quasispecies. Viral quasipecies are composed of a complex, unstable mixture, of genetically distinct but closely related variants, arising from the same inoculum. The random production in any given hepatocyte of novel HCV proteins carrying new or enhanced functional properties may trigger cellular transformation which could subsequently lead to the development of HCC. The core and NS5A proteins of HCV have been demonstrated to encode transactivation properties, and are currently strong candidates for playing a crucial role in such mechanisms.

We therefore hypothesise that: (i) HCV core and NS5A quasispecies variants, naturally produced during the course of chronic infection, possess different levels of transcriptional activities; and that (ii) proteins with the highest intrinsic transactivation properties, even if they belong to minor quasispecies populations, could interact with apoptotic and cell cycle mechanisms and have transforming effects on hepatocytes, leading to HCC development.

In this context, the objectives of the research were:
-To characterise the natural genetic variability of core and NS5A variants and the putative functional impact of such variability on their in vitro transcriptional activities.
-To study the consequences of expression of core and NS5A proteins with different levels of intrinsic transactivation properties, both ex vivo and in vivo.
-To understand the role of transactivation properties carried by HCV proteins in the modification of apoptosis processes and carcinogenesis.

The results we gathered suggested the existence of a selective environment in HCC tissue resulting in HCV NS5A variant compartmentalization between tumoural and non-tumoural adjacent tissues. For the first time, we demonstrate the existence of a genetic compartmentalization within the same infected organ at a single timepoint during infection. From these original results arise two hypotheses. On the one hand, HCV variants detected within tumours could have been selected and/or evolved after cellular transformation to adapt to the particular tumoural environment (i.e. fast replicating hepatocytes). On the other hand, a particular HCV variant may induce cellular transformation and then remain in the growing tumour. In order to address the different hypotheses we have cloned the different HCV variants isolated from the normal and tumoural compartments. Using these constructs, we tested the NS5A transactivation properties (using a yeast model) and the HCV replication capacity of the corresponding NS5A variants (using a sub-genomic replicon system).

We were able to demonstrate the existence of a functional compartmentalization of HCV variants in HCC tissues. Two hypotheses may be drawn from these observations: 1) HCV variants carrying high transactivation levels were responsible for the development of HCC by transactivating genes involved in carcinogenesis, and had since replicated in the tumour; 2) HCV variants with high transactivation properties were better fit to the tumoural environment (e.g. highly replicative hepatocytes) and were selected against other variants. We investigated the latter hypothesis by testing the effect of NS5A transactivation properties on HCV replication using the sub-genomic replication system. These set of experiments will answer the broader question: are the NS5A transactivation properties important for the HCV life cycle? Our results demonstrated that the transactivation level of a given NS5A sequence is correlated to the HCV replication capacity and that the NS5A NLS is partly required for the modulation of HCV replication in vitro, thus validating the hypothesis. We are currently investigating the other hypothesis using pan-genomic approaches.

The technologies used in this project, including truly novel technical approaches, will allow us to understand the potential role HCV itself may play in the occurrence of liver cancer.