Substrate sequence selection by retroviral integrase.
Integration of retrovirus DNA is a specific process catalyzed by the integrase protein acting to join the viral substrate DNA (att) sequences of about 10 bases at the ends of the long terminal repeat (LTR) to various sites in the host target cell DNA. Although the interaction is sequence specific, the att sequences of different retroviruses are largely unrelated to one another and usually differ between the two ends of the viral DNA. To define substrate sequence specificity, we designed an "in vitro evolution" scheme to select an optimal substrate sequence by competitive integration in vitro from a large pool of partially randomized substrates. Integrated substrates are enriched by PCR amplification and then regenerated and subjected to subsequent cycles of selection and enrichment. Using this approach, we obtained the optimal substrate sequence of 5'-ACGACAACA-3' for avian sarcoma-leukosis virus (ASLV) and 5'-AACA(A/C)AGCA-3' for human immunodeficiency virus type 1, which differed from those found at both ends of the viral DNA. Clonal analysis of the integration products showed that ASLV integrase can use a wide variety of substrate sequences in vitro, although the consensus sequence was identical to the selected sequence. By a competition assay, the selected nucleotide at position 4 improved the in vitro integration efficiency over that of the wild-type sequence. Viral mutants bearing the optimal sequence replicated at wild-type levels, with the exception of some mutations disrupting the U5 RNA secondary structure important for reverse transcription, which were significantly impaired. Thus, maximizing the efficiency of integration may not be of major importance for efficient retrovirus replication.