Retroviruses integrate a DNA copy of their genome into host DNA as an obligatory step in their replication cycle. Our work focuses on the molecular mechanism of integration, and in particular on the structure and function of HIV integrase and other proteins involved in retroviral DNA integration. The major impediment to further structural, biophysical and further biochemical studies of retroviral DNA integration is that it has not yet been possible to reconstitute a nucleoprotein complex from purified integrase and DNA substrate that exhibits all the properties of complexes isolated from infected cells. In particular, the complexes are not competent for efficient two-ended integration and aggregate under reaction conditions. We have studied conditions for optimizing two-ended integration in order to obtain nucleoprotein complexes with properties that more faithfully reproduce those of complexes isolated from infected cells. Our recent work has focused on the role of cellular proteins in retroviral DNA integration. Elucidation of the role of host factors in retroviral DNA integration is necessary to gain a more complete understanding of the integration process, and to potentially identify targets other than integrase that allow viral replication to be blocked at the step of DNA integration. We have previously identified a cellular protein (BAF) that blocks self-destructive autointegration of retroviral DNA. We have focused on the mechanism by which BAF blocks autointegration and obtained evidence that supports our hypothesis that BAF blocks autointegration by compacting the viral DNA within the preintegration complex. We have also identified a second cellular protein, lamina-associated-protein-2a (LAP2a), as a component of the preintegration complex (PIC). We have determined the functional domains of LAP2_ that confer the stable association of BAF with DNA by generating a series of deletion mutants lacking a part of the N- or C-terminal domain of LAP2_ as fusion proteins with GST and tested their ability to form a salt-resistant DNA complex with BAF and DNA . Both the LAP2 common domain, which contains LEM domain that interacts with BAF, and the LAP2_-specific domain alone failed to stabilize the BAF/DNA complex in the presence of 400 mM KCl. These data demonstrate that the common domain or _-specific domain alone is insufficient to form a stable complex with DNA and BAF. However independent proteins of the LAP2 common domain and LAP2_-specific domain could together reconstitute the function of LAP2_ in stabilization of the BAF/DNA complex. Since the association of BAF with the PIC regulates the preference for intermolecular integration of the viral DNA, depletion of LAP2a would be expected render the intermolecular integration preference more sensitive to salt-challenge. To test this idea, we used the small interference RNA (siRNA) gene silencing technique to make a stable NIH3T3 cell line with substantially reduced levels of LAP2a from which PICs were isolated. Growth of this cell line was indistinguishable from that of NIH3T3, although other cell lines with greater reductions in LAP2a exhibited impaired growth. We initially asked whether LAP2_ was depleted from PICs made in the LAP2a-knockdown cells. Immunoprecipitation with anti-LAP2a rabbit serum demonstrated that recovery of PICs derived from LAP2_-knockdown cells was significantly decreased compared with PICs from NIH3T3 and siRNA control cells. This data demonstrates that reduced levels of LAP2_ are associated with PICs from LAP2_-knockdown cells. As predicted, although initial PICs from LAP2_-knockdown cells were able to carry out intermolecular integration, this activity was mostly abolished after treatment with 400 mM KCl. In contrast, PICs from NIH3T3 and siRNA control cells still retained intermolecular integration activity after treatment with 400 mM KCl. BAF plays the primary role in compacting the viral DNA withiin the preintegration complex, but its association is much more salt-stable in the presence of LAP2_. In order to determine whether this is biologically relevant we assayed replication of MoMLV in LAP2_ knockdown cells. Replication of MoMLV in these cell lines is delayed, demonstrating a functional role for LAP2_.
Showing the most recent 10 out of 15 publications
