RETROVIRAL INTEGRATION - IN-VITRO HOST SITE SELECTION BY AVIAN INTEGRASE

Viral integrase catalyzes the integration of the linear vir al DNA gen ome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristi c small duplication of host sequences at the site of integration, impl ying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measu re the concerted integration of virus-like plasmid DNA into naked lamb da DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplicat ion, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplicatio n relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustai ned multiple independent integration events producing the same duplica tion size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of GIC and AIT sequence biases was found. Using duplex oligonucleotides as target sub strates, we tested the significance of the 6-bp G/C and A/T pattern fo r site selection. In the context of this assay, which is likely domina ted by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominately into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonuc leotide assays indicated that although host site selection has propert ies in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.