Study of HIV-2 primer-template initiation complex using antisense oligonucleotides

TY - JOUR

T1 - Study of HIV-2 primer-template initiation complex using antisense oligonucleotides

AU - Boulmé, F

AU - Freund, F

AU - Gryaznov, S

AU - Nielsen, P E

AU - Tarrago-Litvak, L

AU - Litvak, S

PY - 2000/5

Y1 - 2000/5

N2 - HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1 RNA, no direct evidence of interactions with the U-rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV-1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre-primer binding site (the upstream region contiguous to the HIV-2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A-rich loop of viral RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A-rich region in HIV-2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV-2 reverse transcription.

AB - HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1 RNA, no direct evidence of interactions with the U-rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV-1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre-primer binding site (the upstream region contiguous to the HIV-2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A-rich loop of viral RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A-rich region in HIV-2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV-2 reverse transcription.

KW - Base Sequence

KW - HIV-1

KW - HIV-2

KW - Molecular Sequence Data

KW - Nucleic Acid Conformation

KW - Oligonucleotides, Antisense

KW - RNA, Viral

KW - Templates, Genetic

M3 - Journal article

C2 - 10785403

SN - 0014-2956

VL - 267

SP - 2803

EP - 2811

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

IS - 9

ER -