Functional Recognition of the Modified Human tRNA super(Lys3) sub(U)UU Anticodon Domain by HIV's Nucleocapsid Protein and a Peptide Mimic

• Published in: Journal of molecular biology Vol. 410; no. 4; pp. 698 - 715
• Main Authors: Graham, William D, Barley-Maloney, Lise, Stark, Caren J, Kaur, Amarpreet, Stolyarchuk, Khrystyna, Sproat, Brian, Leszczynska, Grazyna, Malkiewicz, Andrzej, Safwat, Nedal, Mucha, Piotr, Guenther, Richard, Agris, Paul F
• Format: Journal Article
• Language: English
• Published: 22.07.2011
• Subjects: Human immunodeficiency virus 1
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2011.04.025

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA super(Lys3) sub(U)UU 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 super(Lys3) sub(U)UU, the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL super(Lys3) were studied. NCp7 bound the hASL) super(L)ys3 sub(UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm) super(5)s super(2U) sub(3)4) and 2-methylthio-N super(6-threonylcarbamoyladenosine at position-37 (ms) super(2)t super(6A) sub(3)7) with a considerably higher affinity than the unmodified hASL super(Lys3) sub(U)UU (K sub(d = 0.28 +/- 0.03 and 2.30 +/- 0.62 mu M, respectively). NCp7 denatured the structure of the hASL) super(L)ys3 sub(UUU-mcm) super(5)s super(2U) sub(3)4; ms super(2t[su per]6A) sub(3)7; [Psi] sub(39 more effectively than that of the unmodified hASL) super(L)ys3 sub(UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K) sub(d) = 0.55 +/- 0.10 mu M) and specificity for the ASL super(Lys3) sub(U)UU-mcm super(5s) super(2)U sub(34; ms) super(2)t[sup er]6A sub(37 comparable to that of NCp7. The peptides recognized a t) super(6)A sub(37-modified ASL with an affinity (K) sub(d) = 0.60 +/- 0.09 mu M) comparable to that for hASL super(Lys3) sub(U)UU-mcm super(5s) super(2)U sub(34; ms) super(2)t[su per]6A sub(37, indicating a preference for the t) super(6)A sub(37 modification. Significantly, one of the peptides was capable of relaxing the hASL) super(L)ys3 sub(UUU-mcm) super(5)s super(2U) sub(3)4; ms super(2t[su per]6A) sub(3)7; [Psi] sub(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) super(L)ys3 sub(UUU have been found to be important determinants of NCp7's recognition prior to the tRNA) super(L)ys3 sub(UUU being annealed to the viral genome as the primer of reverse transcription.)