Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase

The catalytic domain of all retroviral integrases contains an Asp,Asp-35-Glu (D,D-35-E) motif with precisely 35 amino acids between the second aspartate and the glutamate. We have now made several mutations designed to alter the length or flexibility of a mobile loop within this 35-amino-acid spacer region in full-length Rous sarcoma virus integrase. Surprisingly, most of the mutants had enzymatic activity, including ones that shortened or lengthened the loop by up to 6 amino acids. Several size mutants exhibited the two biologically relevant activities of integrase in reactions with Mn²⁺, although they were inactive with Mg²⁺. No viruses containing integrase with an altered length, however, replicated in cell culture, and these viruses were blocked at the integration step. Thus, the conserved 35-amino-acid spacing is not absolutely required for enzymatic activity, but the correlation between infectivity and Mg²⁺-dependent activity supports magnesium as the metal cofactor used by integrase in vivo.