Functional Recognition of the Modified Human tRNA Lys3UUU Anticodon Domain by HIV's Nucleocapsid Protein and a Peptide Mimic

Abstract

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA Lys3 UUU as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA Lys3 UUU, the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL Lys3) were studied. NCp7 bound the hASL Lys3 UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm 5s 2U 34) and 2-methylthio- N 6 -threonylcarbamoyladenosine at position-37 (ms 2t 6A 37) with a considerably higher affinity than the unmodified hASL Lys3 UUU ( K d = 0.28 ± 0.03 and 2.30 ± 0.62 μM, respectively). NCp7 denatured the structure of the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 more effectively than that of the unmodified hASL Lys3 UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K d = 0.55 ± 0.10 μM) and specificity for the ASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37 comparable to that of NCp7. The peptides recognized a t 6A 37-modified ASL with an affinity ( K d = 0.60 ± 0.09 μM) comparable to that for hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37, indicating a preference for the t 6A 37 modification. Significantly, one of the peptides was capable of relaxing the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA Lys3 UUU have been found to be important determinants of NCp7's recognition prior to the tRNA Lys3 UUU being annealed to the viral genome as the primer of reverse transcription.