HIV and HBV infection as models of viral DNA integration and mechanisms of viral-associated carcinogenesis

My PhD was focused on two viruses, both responsible of persistent infection: HIV and HBV. The two viruses show high worldwide prevalence and both are associated with high morbidity and mortality. HIV establishes a persistent infection through different mechanisms, first of all with the integration of its DNA into the host genome, thus forming the provirus. However, a consistent part of the viral DNA within the infected cells is unintegrated (circular or not). The project following presented aims to analyse the kinetic of viral replication (measured as p24 production) and HIV DNA integration in primary lymphocytes (CD4+ ad PBMC) and Monocyte-derived Macrophages (MdM) in presence and absence of Integrase inhibitors (INIs). HIV DNA quantification (as proviral, unintegrated and 2-LTR) was assessed at different time points. The presented data reconfirmed, as previously reported in literature, the different kinetic of HIV replication in lymphocytes (CD4+ T cells and PBMCs, much faster) compared to macrophages (more slow), and suggested the presence of a different kinetic of HIV DNA integration between the two cellular systems, too. Both INIs efficiently inhibit the viral replication and the integration of HIV DNA. However, a little but consistent amount of HIV DNA was observed at 30 days post treatment in macrophages, which are the main reservoirs of the infection. By analysing the composition of this little HIV DNA amount, we observed a very little difference between the two drug which could be probably explained by the well-described different kinetic of dissociation of the DTG-enzyme complex. In conclusion these data could give new information about the different kinetic of HIV replication between lymphocytes and macrophages, suggesting also the implication of a different kinetic of HIV DNA integration. Both the integrase inhibitors efficiently inhibit HIV replication and integration. Nonetheless, in presence of INI, although an almost total inhibition of HIV replication was observed, a little but consistent amount of viral DNA was maintained in macrophages. This evidence could have important implication in the study of HIV persistence and viral rebound after treatment interruption. The data above mentioned were presented at the 12th edition of the European congress on HIV e hepatitis, in Barcelona in 2014 (26-28 March), and a manuscript is in preparation. HBV is another important virus able to establish a persistent infection that usually causes cirrhosis and hepatocarcinoma (HCC). HBV could indirectly (through compensative cellular replication) or directly (through event of random integration or due to the viral proteins) promote carcinogenesis. Among the viral proteins the trans-activating HBV protein (HBx) covers a key role in this sense. The project here presented aims to highlight specific mutations in HBX gene associated with HCC in a group of chronically-HBV infected patients. By analysing the sequences of 75 HBV chronically infected patients, we observed the mutation F30V prevalently in the HCC group. This is located within the N-terminal region of HBx which seems to be involved in the negative regulation of the HBx trans-activation function. By analysing the results obtained in vitro, we observed that the mutation would reduce the viral replication, probably due to its localization in a domain within the N-terminal region known to be involve in the dimerization of the protein. HBx protein, both wt and mutated, did not alter the cycle progression of exposed cells. On the contrary, the mutation could to be associated with less cellular susceptibility to apoptotic death related to what observed in presence of the HBx wt. This observed cell survival could probably promote the maintenance of aberrant cellular clones thus favouring the appearance of tumour. Of consequence, considering the obtained results, it is possible to propose that F30V mutation could interfere with HBV replication and can have a role in HBV-driven carcinogenesis by reducing the rate of apoptosis. However, further studies are required in order to understand the potential role of F30V as HCC prognostic factor in HBV chronically infected patients.