DNA requirements for assembly and stability of HIV-1 intasomes

Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.