Does the HIV-1 primer activation signal interact with tRNA3(Lys) during the initiation of reverse transcription?
Reverse transcription of HIV-1 RNA is primed by a tRNA3(Lys) molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3(Lys) and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TPsiC stem of tRNA3(Lys) and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3(Lys) or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3(Lys) or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3(Lys). These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3(Lys) does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs.
Goldschmidt
Valérie
V
UPR 9002 du CNRS, l'Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Ehresmann
Chantal
C
Ehresmann
Bernard
B
Marquet
Roland
R
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis; D015967:Gene Expression Regulation, Viral; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D009843:Oligoribonucleotides; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase