Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase.
Packaging of the genomic RNA dimer and replication primer tRNA(Lys,3) into HIV virions are required for the production of infectious virus. The initiation of reverse transcription necessitates the annealing of tRNA(Lys,3) to the primer binding site (PBS) of HIV RNA by nucleocapsid (NC) protein. In this report the interactions of replication primer tRNA(Lys,3) with various forms of reverse transcriptase (RT) and nucleocapsid protein have been analyzed by ultraviolet light (UV) cross-linking and gel retardation assays. We show that of the three forms of RT studied, p66/p51, p66 and p51, only the heterodimer p66/p51 can tightly and stably interact with tRNA(Lys,3). Tight interactions between tRNA(Lys,3) and nucleocapsid protein, either NCp15 or NCp7, were found to take place within the anticodon domain. Interestingly enough, primer tRNA(Lys,3) can interact with RTp66/p51 and NCp15 to form a high molecular weight complex in which RTp66/p51 appears to enhance the binding of NCp15 to tRNA(Lys,3). These findings favor the notion that the RT enzyme and NC protein co-operate to select and package primer tRNA.
Barat
C
C
LaboRetro INSERM, Ecole Normale Supérieure, Lyon, France.
Schatz
O
O
Le Grice
S
S
Darlix
J L
JL
D000163:Acquired Immunodeficiency Syndrome / Q000378:metabolism*; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000378:metabolism; D014466:Ultraviolet Rays; D014758:Viral Core Proteins / Q000378:metabolism*; D014764:Viral Proteins; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C060295:p15 gag protein, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase