Host factors impact in early phases of HIV-1 infection

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DESCRIPTION (provided by applicant): The obligated insertion of the HIV-1 genome into the host chromosomal DNA is one of the features that differentiates retroviruses from other viruses. In the nucleus, viral and cellular factors cooperate in order to ensure the correct integration of viral DNA in the host chromatin. Moreover repair of the insertion site is fundamental in order to achieve a productive infection. Among the several DNA repair pathways the cell is equipped with, some have been tested both in vitro as well as in vivo for their ability to promote integration gap repair. We have demonstrated the interaction of integrase with two different cellular processes, a proteasome degradation pathway (N-end rule) and a DNA repair pathway (post-replication repair/translesion). These two pathways share the activity of the key molecule, Rad6. We have shown by mutagenesis of integrase, that elements that determine its N-end rule dependent stability, are significant in HIV-1 infection. The aim of this proposal is to investigate to what extent the two pathways influence the early steps of HIV-1infection with special emphasis dedicated to integration. Initially we will determine which phase within the early steps of viral life cycle is inhibited by mutations in the first position of integrase, since this residue is key to its degradation. Furthermore we will analyze the contribution of Rad6 to both integrase stability as well as to the phenotype observed in the context of the viral mutants. DNA repair is essential for HIV-1infection. To date the post-replication DNA repair pathway has not yet been implicated in retroviral integration other than by our observation that there is a direct interaction between one of its principal components, hRadl 8, and integrase, hRadl 8 also binds hRad6. The role hRadl 8 in HIV-1 replication will be studied by infection of cells overexpressing this protein or molecules that inhibit its endogenous expression. The steps mostly affected by these treatments will be identified by real-time PCR, and the effect on the actual DNA gap repair at the insertion junctions will be assessed as well. The results of the experiments described in this proposal are expected to provide us with a set of information about the influence of these two intercrossing cellular pathways on HIV-1 replication. Moreover we will learn whether interfering with the Nend rule and post-replication DNA repair/translation can eventually render the host cell resistant to infection, and thus qualify the two pathways as potential targets for anti-retroviral therapy.