A specific orientation of RNA secondary structures is required for initiation of reverse transcription

Aiyar, Ashok; Ge, Zheng; Leis, Jonathan

doi:10.1128/jvi.68.2.611-618.1994

Cited by 74 publications

(49 citation statements)

References 21 publications

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“…Reconstitution of reverse transcription in vitro. In vitro reverse transcription reactions were performed as described previously (2). Briefly, RNA transcripts (270 nucleotides) bearing either wild-type or mutant sequences (as indicated) were incubated with a 28-nucleotide RNA primer representing the 3Ј end of tRNA Trp and with AMV RT in a 30-l reaction volume containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl 2 , 3 mM dithioerythreitol, 1 mM (each) dATP, dGTP, and dTTP, and [␣-32 P]dCTP (0.1 mM; 6.7 Ci/mmol).…”

Section: Methodsmentioning

confidence: 99%

Multiple biological roles associated with the Rous sarcoma virus 5' untranslated RNA U5-IR stem and loop

Miller

Morris

et al. 1997

J Virol

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The 5 untranslated region of Rous sarcoma virus (RSV) RNA is a highly ordered structure involved in multiple processes in the viral replication cycle. One of these structures, referred to as the U5-IR stem, is located immediately upstream of the 5 end of the primer binding site. Disruption of its base pairing results in a decrease in initiation of reverse transcription (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991). In the present study, the length of the U5-IR stem structure has been extended by insertions of different sequences which decrease the efficiency of reverse transcription, in vivo and in vitro. Reverse transcription is rescued partially by placing single-stranded bulges into the middle of the extended duplexes. Nucleotide substitutions or insertions into the loop region of the U5-IR stem also decrease the efficiency of reverse transcription, suggesting that these sequences may specifically interact with reverse transcriptase. Surprisingly, all of the extended stem mutations cause significant RNA packaging defects. In contrast, nucleotide insertions or base substitutions in the U5-IR loop do not affect RNA packaging. These data indicate that the reverse transcription initiation complex and RNA packaging apparatus are influenced by the same region of RSV RNA and that each process is differentially sensitive to changes in sequence and/or secondary structure.

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Section: Methodsmentioning

confidence: 99%

Multiple biological roles associated with the Rous sarcoma virus 5' untranslated RNA U5-IR stem and loop

Miller

Morris

et al. 1997

J Virol

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“…Initiation of reverse transcription is primed by a tRNA whose 3' end is complementary to the so called 'primer binding site' (PBS) (for a review see Marquet et al, 1995). In addition to this 'general' PBS-tRNA interaction, evidence has recently accumulated for virus-specific interactions between the primer tRNA and the genomic RNA of avian retroviruses (Aiyar et al, 1992(Aiyar et al, , 1994, human immunodeficiency virus type 1 (HIV-1) (Isel et al, 1993 and the yeast retrotransposon Tyl (Wilhelm et al, 1994;Friant et al, 1996). These virus-specific interactions are required for efficient replication (Aiyar et al, 1992;Wilhelm et al, 1994;Wakefield et al, 1996), suggesting that they are directly involved in the initiation of reverse transcription of retroviruses and retrotransposons.…”

Section: Introductionmentioning

confidence: 99%

Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

et al. 1996

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We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV‐1) RNA and HIV‐1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV‐1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV‐1 RT are not very different for the two complexes, RT dissociates approximately 200‐fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre‐formed primer‐template‐RT complexes is reduced by a approximately 50‐fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV‐1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post‐transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

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“…For example, it has been proposed that during the initiation of reverse transcription, a specific interaction occurs between an A-rich loop located upstream of the PBS in the HIV-1 genome and the anticodon of tRNA 3 Lys and that dethiolation of mcm 5 S 2 U at position 34 in the anticodon of tRNA 3 Lys destabilizes this interaction (12). An interaction between regions upstream of the PBS and the T⌿C loop has also been proposed to exist in non-HIV retrovirus (1,2). Furthermore, in vitro studies using platinum cross-linking (5,6) and nuclease digestion studies (21,26) have also indicated that HIV-1 RT interacts with the D, anticodon, and T⌿C loops of tRNA 3 Lys .…”

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confidence: 98%

Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription

Huang

Shalom

et al. 1996

J Virol

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tRNA 3Lys is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA 3 Lys has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA 3 Lys gene was changed from UUU to CUA (tRNA 3 Lys Su ؉ ) or, in addition, G-73 was altered to A (tRNA 3 Lys Su ؉ G73A). COS-7 cells were transfected with either wild-type or mutant tRNA 3 Lys genes, and both the wild-type and mutant tRNA 3 Lys produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA 3 Lys was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA 3 Lys . Short RT extensions of wild-type and mutant tRNA 3 Lys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA 3 Lys Su ؉ has the same ability as heat-annealed wild-type tRNA 3 Lys to prime RT and competes equally well with wild-type tRNA 3 Lys for priming RT. tRNA 3 LysSu ؉ G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA 3 Lys for priming RT. However, the priming abilities of wild-type and mutant tRNA 3 Lys are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA 3 Lys Su ؉ contains both endogenous, cellular wild-type tRNA 3 Lys and mutant tRNA 3 Lys . When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA 3 Lys is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA 3 Lys act as primers for RT. These results indicate that the tRNA 3 Lys Su ؉ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.

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A specific orientation of RNA secondary structures is required for initiation of reverse transcription

Cited by 74 publications

References 21 publications

Multiple biological roles associated with the Rous sarcoma virus 5' untranslated RNA U5-IR stem and loop

Multiple biological roles associated with the Rous sarcoma virus 5' untranslated RNA U5-IR stem and loop

Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription

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