Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication.
The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.
Arts
E J
EJ
Center for AIDS Research and Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Ghosh
M
M
Jacques
P S
PS
Ehresmann
B
B
Le Grice
S F
SF
D001483:Base Sequence; D018076:DNA, Complementary / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis*; D054303:HIV Reverse Transcriptase; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D004279:DNA, Viral; D012357:RNA, Transfer, Lys; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H