Contacts between Reverse Transcriptase and the Primer Strand Govern the Transition from Initiation to Elongation of HIV-1 Reverse Transcription

Abstract

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA 3 Lys , and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primertemplate complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2-hydroxyl group at the 3 end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA 3Lys . The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA 3 Lys . The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix ␣H in the p66 thumb subdomain and the RNase H domain of RT, respectively.Reverse transcription is the central event of the retrovirus life cycle (1-3). Reverse transcriptase (RT) 1 , the viral enzyme that converts the homodimer of single-stranded genomic RNA into double-stranded DNA, is a multifunctional enzyme that possesses RNA-and DNA-dependent DNA polymerase activities, as well as an RNase H function (reviewed in Ref. 3). In addition, complete DNA synthesis requires two strand transfers, which are also mediated by RT (4 -6).Like most replicative DNA polymerases, RT uses RNA primers to initiate DNA synthesis. RT initiates synthesis of the (Ϫ) strand DNA from the 3Ј end of a specific cellular tRNA species that is complementary to the primer binding site (PBS) of the genomic RNA (reviewed in Refs. 7-9). The primer for (ϩ) strand DNA synthesis is a polypurine tract that is resistant to the RNase H activity of RT (10). Evidence for specific initiation of (Ϫ) strand DNA synthesis in retroviruses and retrotransposons has recently accumulated. Complex species-specific interactions have been identified in vitro between the primer tRNA and genomic RNA of avian retroviruses (11, 12), human immunodeficiency virus type 1 (HIV-1) (13,14), and the yeast retrotransposon Ty1 (15). In HIV-1, in addition to the interaction of the 18 3Ј-terminal nucleotides of the replication primer tRNA 3 Lys with the PBS, the anticodon loop, the 3Ј strand of the anticodon stem, and part of the variable loop of the tRNA interact with the genomic RNA (13, 14, 16). The interaction between the tRNA anticodon loop and HIV-1 RNA is required for optimal replication and reverse transcription in infected cells (17)(18)(19)(20). Post-transcriptional modifications of tRNA 3 Lys are required for stabilizing the primer-template interactions (13,14) and for eff...

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

“…More recently, an additional U5-T⌿C interaction has been described (42). A novel interaction between the 5Ј terminus of tRNA 3 Lys and the feline immunodeficiency virus U5-IR loop has also been reported (43). In the case of HIV-1, chemical and enzymatic probing revealed complex interactions between U5 sequences upstream of the PBS and the anticodon loop, the 3Ј portion of the anticodon stem, and part of the variable loop of tRNA 3 Lys (9 -11, 44 -46).…”

“…Human immunodeficiency virus type 1 (HIV-1) 1 DNA synthesis is initiated by annealing of the 3Ј 18 nucleotides (nt) of a cellular tRNA primer, tRNA 3 Lys , to the complementary 18-nt primer binding site (PBS) near the 5Ј end of the viral RNA template. Minus-strand DNA synthesis proceeds until the 5Ј end of the template is copied, generating the first product of reverse transcription, a 200-nt DNA termed (Ϫ) strong-stop DNA ((Ϫ) SSDNA).…”

“…Studies on HIV-1 initiation in vitro indicate that two modes of synthesis are involved in this process: (i) initiation, characterized by a distributive, slow extension of the primer and a high dissociation rate of reverse transcriptase (RT) from the primer-template complex; and (ii) elongation, which follows a transition between incorporation of the sixth and seventh nt and results in a dramatic increase in the processivity and rate of DNA synthesis (1)(2)(3)(4)(5). The inability of HIV-1 RT to initiate (Ϫ) SSDNA synthesis efficiently in vitro may be in part due to the nature of the three-dimensional structure of the initiation complex, which involves extensive intermolecular interactions between the tRNA 3 Lys primer and the RNA template (see below) (6 -11).…”

“…The inability of HIV-1 RT to initiate (Ϫ) SSDNA synthesis efficiently in vitro may be in part due to the nature of the three-dimensional structure of the initiation complex, which involves extensive intermolecular interactions between the tRNA 3 Lys primer and the RNA template (see below) (6 -11). In addition, the efficiency of (Ϫ) SSDNA synthesis appears to be sensitive to the helical conformation of the nucleic acid duplexes that are accommodated by RT; for example, when an 18-nt DNA primer complementary to the PBS (D18) is used, (Ϫ) SSDNA synthesis begins immediately in the elongation mode (1)(2)(3)5).…”

“…The bend occurs where ␣-helix H in the thumb subdomain of RT contacts the bound nucleic acid (15)(16)(17) and is typically associated with a transition from A-to B-form geometry (17). It has been suggested that this structural transition is correlated with the transition from the initiation to elongation mode (3,11).…”