title: Functional recognition of the modified human tRNALys3(UUU) 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(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(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(5)s(2)U(34);ms(2)t(6)A(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(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 μM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(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.
authors.affiliation: Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: William D
authors.initials: WD
authors.lastname: Graham
authors.forename: Lise
authors.initials: L
authors.lastname: Barley-Maloney
authors.forename: Caren J
authors.initials: CJ
authors.lastname: Stark
authors.forename: Amarpreet
authors.initials: A
authors.lastname: Kaur
authors.forename: Christina
authors.initials: C
authors.lastname: Stolarchuk
authors.forename: Khrystyna
authors.initials: K
authors.lastname: Stolyarchuk
authors.forename: Brian
authors.initials: B
authors.lastname: Sproat
authors.forename: Grazyna
authors.initials: G
authors.lastname: Leszczynska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Malkiewicz
authors.forename: Nedal
authors.initials: N
authors.lastname: Safwat
authors.forename: Piotr
authors.initials: P
authors.lastname: Mucha
authors.forename: Richard
authors.initials: R
authors.lastname: Guenther
authors.forename: Paul F
authors.initials: PF
authors.lastname: Agris
mesh_terms: D000595:Amino Acid Sequence; D000926:Anticodon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D006801:Humans; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D013696:Temperature; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D000926:Anticodon; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D019151:Peptide Library; D010455:Peptides; D012357:RNA, Transfer, Lys; D054301:gag Gene Products, Human Immunodeficiency Virus