Major and minor groove contacts in retroviral integrase-LTR interactions

The 3'-processing activities of HIV-1, HTLV-3, and M-MuLV integrases (INs) with their corresponding U5 end of the viral DNA molecule were examined to define functional group determinants of U5 terminus recognition and catalys is. Nucleotide analogues were incorporated into the U5 terminus to produce conservative modifications in the surface of the major and/or minor grooves to map the hydrogen-bonding contacts required for LTR-IN interaction. Spec ifically, the phylogenetically conserved CA (positions 4 and 3, respectivel y) and the 5'-proximal nucleotide (position 5) were replaced with base anal ogues in plus and/or minus strands. For each integrase, similar major and m inor groove contacts were identified in the guanine and adenine of the cons erved CA/GT. Overall, perturbances in the minor groove resulted in a greate r decrease in 3'-processing activity than the major groove substitutions. A dditionally for HIV-1 and HTLV-2 INs, we observed an increase in the 3'-pro cessing activity with an O-4-MeThy substitution at position 3 of the minus strand. O-4-MeThy may act to destabilize Watson-Crick base pairing and in d oing so provide these INs with a more favorable interaction with the adjace nt scissile bond. At position 5, a substantial divergence among the three I Ns was noted in the functional groups required for 3'-processing activity, thereby supporting the role of this position in providing some level of sub strate specificity.