Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes

Miller, Jennifer T.

doi:10.1093/nar/gkh813

Cited by 10 publications

(5 citation statements)

References 55 publications

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“…In Vitro tRNA Transcription tRNA Lys3 was prepared using a DNA template for tRNA transcription, which was PCR amplified using the following oligonucleotides as primers (Miller et al, 2004):…”

Section: Methods Detailsmentioning

confidence: 99%

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation

et al. 2019

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Section: Methods Detailsmentioning

confidence: 99%

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation

et al. 2019

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“…The HIV and MLV U5-PBS RNA samples were also made using methods previously described 21 . tRNA Pro and tRNA Lys,3 DNA templates were constructed through annealing and ligation of oligonucleotides designed to contain a T7 promoter and two 2'-O-methoxy modified nucleotides at the 5' end of the template strand to maintain 3' end homogeneity 31 . For the U5-PBS construct, used for NMR studies a single basepair swap (G96C:C157G) was made to ameliorate the spectral overlap problem that arose due to five consecutive guanosine residues in a row.…”

Section: Sample Preparationmentioning

confidence: 99%

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing

Miller

Yildiz

et al. 2014

Nature

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In order to prime reverse transcription, retroviruses require annealing of a tRNA molecule to the U5-primer binding site (U5-PBS) region of the viral genome 1,2 . The residues essential for primer annealing are initially locked in intramolecular interactions [3][4][5] , and hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements 6 . Here we show that, unlike classical chaperones, the Moloney murine leukemia virus NC uses a unique mechanism for remodeling: it specifically targets multiple structured regions in both the U5-PBS and tRNA Pro primer that otherwise sequester residues necessary for annealing. This high-specificity and highaffinity binding by NC consequently liberates these sequestered residues-which are exactly complementary-for intermolecular interactions. Furthermore, NC utilizes a stepwise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA Pro reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of nontarget RNAs.Retroviruses preferentially employ specific host tRNAs as primers for the first step of reverse Fig. 4a,), with the C 128 base either stacked on A 129 or, in a small population, extruded from the structure ( Fig. 1d and Extended Data Fig. 4c). Interaction with the NC tails alters the equilibria between the two conformations in favor of the minor, destabilized conformations ( Fig. 1d) and hence leads to release of the tenth and eleventh ( +10 G 155 and +11 U 156 ) PBS residues and the C 128 tetraloop residue.The destabilization of the latter is important for cooperative binding of a second NC to the tetraloop (Extended Data Fig. 2a; see Supplementary Info 3). Thus, the positively charged NC tails do not globally destabilize U5-PBS but instead specifically target residues inherently predisposed for destabilization. Furthermore, because PAS residues immediately follow the destabilized internal loop (Fig. 1a), the NC tails also specifically perturb residues +1 G 99 , +2 G 100 , and +3 G 101 (Extended Data Fig. 4k). Interestingly, both NC tails (Ala1-Arg17 and Arg4-Leu56) remain disordered, indicating that destabilization must occur via transient interactions that are nevertheless residue-specific due to the inherent accessibility of the particular RNA residues and the orientation constraints imposed by the zinc finger binding to UCUG 110 (Fig. 1e and see 4 Extended Data Fig. 4l). In live viruses, a G110U mutant designed to liberate the U 145 and +1 U 146 residues and a deletion mutant (DM) designed to sequester them exhibited only 56% and 7%, respectively, of wild-type MLV infectivity (Fig. 1f). While the severe infectivity defect of DM virions confirms the importance of high-affinity NC binding in releasing the 5' end of the PBS, the partial defect observed for G110U virions indicates that tail-...

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“…They favour the formation of the specific initiation complex but do not affect the polymerisation rate of the bound enzyme [61,74]. For example, the thiolation of U34 has been reported to be important for the stability of the initiation complex and for the formation of an efficient initiation complex [45,92].…”

Section: A Post-transcriptional Nucleoside Modifications Of Trna 3 Lmentioning

confidence: 99%

“…The structure of the Lys tRNA 3 / vRNA complex remains controversial probably due to a great variability in the structure of the HIV vRNA depending on the studied isolate [32,74]. In the original model [46,48] Fig.…”

Section: Structure Of the Trna/viral Rna Initiation Complexmentioning

confidence: 99%

Structural Bases of the Annealing of Primer Lys tRNA to the HIV-1 Viral RNA

Tisné

2005

CHR

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To promote the initiation of reverse transcription, the HIV-1 virus uses a host tRNA as a primer, tRNA(3Lys). The annealing of tRNA(3Lys) to the viral RNA requires the breaking of the 3D structure of the tRNA and RNA rearrangements, to form a stable initiation complex recognised by the reverse transcriptase. The annealing is mediated by a viral factor, the nucleocapsid protein. This protein has been studied for a long while to define the role of its different sub-domains and their mode of action. Only recently, a consensus view seems to emerge. The structure of the initiation complex, is still discussed. The goal of this review is to clarify what is known about the formation and the structure of the tRNA/viral RNA complex and the role of the nucleocapsid protein from a structural point of view.

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Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes

Cited by 10 publications

References 55 publications

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation

A structure-based mechanism for tRNA and retroviral RNA remodelling during primer annealing

Structural Bases of the Annealing of Primer Lys tRNA to the HIV-1 Viral RNA

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