Onconase, a cytotoxic and antitumor RNase inhibits viral replication in chronically HIV-1-infected human cells under sub lethal concentrations. Cellular tRNA has been implicated as the target for onconase action. We have recently shown that onconase cleaves selectively at GG residues in the UGG context in the variable loop and D-arm of the tRNA substrates. We therefore examined onconase cleavage specificity in in vitro transcribed tRNA(Lys3) which is the primer for HIV-1 reverse transcription but does not have UGG anywhere in its sequence. Onconase was found to cleave tRNALys3 predominantly at the GG residues in the GGG triplet present in the variable loop. Mutations at this site did not effect onconase cleavages. Interestingly thus, onconase seems to cleave predominantly in the variable loop of tRNA (Lys3) regardless of the sequence context implying possible contribution of even structural determinants for its selective cleavages. (C) 2007 Elsevier Inc. All rights reserved.