In the HIV viral integration procedure, 3’-processing of the viral DNA by the integrase enzyme is an essential first step which is followed by the integration of viral DNA into the host genome. In 3’-processing, the integrase cleaves the backbone of the DNA substrate on the 3’ end of a conserved CA dinucleotide motif and inserts a helix between the two DNA strands, forcing them apart (Hare, S., 2012). Our study confirms that the presence of a G-amino group is crucial for 3’-processing. Substituting inosine for G in the CA step removes this amino group and results in loss of enzyme activity. Further work showed that the presence of a terminal duplex segment is not required for 3’-processing. Additional substrate modifications are studied in order to evaluate the actual importance of the CA step.