The contribution of the primer activation signal to differences between Gag- and NCp7-facilitated tRNA(Lys3) annealing in HIV-1.
During tRNA(Lys3) annealing in HIV-1, tRNA(Lys3) binds to both the primer binding site (PBS) and to an 8 nucleotide base-paired sequence upstream of the PBS known as the primer activation signal (PAS). In protease-negative (Pr(-)) HIV-1, the amount of tRNA(Lys3) annealed by Gag is 35% less than that annealed by mature nucleocapsid (NCp7) in protease-positive (Pr(+)) virions. Gag-annealed tRNA(Lys3) also has a reduced ability to initiate reverse transcription, and binds less tightly to viral RNA than NCp7-annealed tRNA(Lys3). Pr(-) virions containing a constitutively single-stranded PAS (2R mutant), show a significant increase in the ability to initiate reverse transcription with little change in the amount of tRNA(Lys3) annealed. However, the 2R mutant does not achieve levels of RT initiation achieved in Pr(+) virions, and tRNA(Lys3) binding to viral RNA remains weak. Wild type levels of initiation and tRNA(Lys3) binding to viral RNA can only be recovered by transient exposure of Pr(-) or Pr(-)2R viral RNA to NCp7. This suggests that in addition to facilitating annealing of tRNA(Lys3) to the PBS and possible denaturation of the PAS, other functions of NCp7 involved in annealing are required. The effect of an inactive protease and/or the 2R mutation upon tRNA(Lys3) annealing and initiation are also observed when the tRNA(Lys3) is annealed in vitro to wild type or mutant viral RNA using either NCp7 or GagDeltap6, indicating a direct effect of the 2R mutation upon tRNA(Lys3) annealing.
Saadatmand
Jenan
J
Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2.
Niu
Meijuan
M
Kleiman
Lawrence
L
Guo
Fei
F
D020029:Base Pairing; D001665:Binding Sites; D015497:HIV-1 / Q000502:physiology*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000502:physiology*
C071884:NCP7 protein, Human immunodeficiency virus 1; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus