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. 2018 Dec 7;430(24):5137-5150.
doi: 10.1016/j.jmb.2018.08.029. Epub 2018 Sep 7.

Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex

Aaron T Coey  1 , Kevin P Larsen  1 , Junhong Choi  2 , Daniel J Barrero  3 , Joseph D Puglisi  3 , Elisabetta Viani Puglisi  4
Affiliations

Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex

Aaron T Coey et al. J Mol Biol. .

Abstract

The initiation of reverse transcription in human immunodeficiency virus-1 is a key early step in the virus replication cycle. During this process, the viral enzyme reverse transcriptase (RT) copies the single-stranded viral RNA (vRNA) genome into double-stranded DNA using human tRNALys3 as a primer for initiation. The tRNA primer and vRNA genome contain several complementary sequences that are important for regulating reverse transcription initiation kinetics. Using single-molecule Förster resonance energy transfer spectroscopy, we demonstrate that the vRNA-tRNA initiation complex is conformationally heterogeneous and dynamic in the absence of RT. As shown previously, nucleic acid-RT interaction is characterized by rapid dissociation constants. We show that extension of the vRNA-tRNA primer binding site helix from 18 base pairs to 22 base pairs stabilizes RT binding to the complex and that the tRNA 5' end has a role in modulating RT binding. RT occupancy on the complex stabilizes helix 1 formation and reduces global structural heterogeneity. The stabilization of helix 1 upon RT binding may serve to destabilize helix 2, the first pause site for RT during initiation, during later steps of reverse transcription initiation.

Keywords: HIV-1; RNA dynamics; protein and RNA interactions; reverse transcriptase; single-molecule FRET.

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Figures

Figure 1.
Figure 1.
Overview of reverse transcription initiation in the 5’-UTR. (a) Reverse transcription initiates from the 5’-UTR of the vRNA genome. (b) The vRNA PAS and anti-PAS sequences flank the stem loop containing the PBS where reverse transcription initiates. (c) Sequence and predicted secondary structure for the vRNA used in single-molecule experiments; numbering is for the NL4.3 isolate. (d) Secondary structure of the tRNALys3 primer.
Figure 2.
Figure 2.
Global architecture of the +1 HIV-1 reverse transcriptase initiation complex. (a) The vRNA and tRNA form an extended PBS helix which occupies the RT binding cleft. H1 and the elongated tRNA protrude from the polymerization active site and RNase H domain respectively. H1 rests on top of the occupied RT binding cleft. (b) Secondary structure of the vRNA and tRNA as fit to cryo-EM electron density maps. (c) Schematic of constructs used for surface immobilization for smFRET experiments.
Figure 3.
Figure 3.
Single-molecule experimental design for measuring RT binding to the vRNA-tRNA complex. (a) Two different RNA oligonucleotides are annealed to the vRNA to promote formation of either an 18 bp PBS helix or 22 bp PBS helix which extends into the RNase H domain of RT. (b) Design of mutant vRNAs with variable PBS helix lengths in complex with full-length tRNA.
Figure 4.
Figure 4.
Representative traces from single-molecule experiments using the 18 nucleotide (a), 22 nucleotide (b), and full-length tRNA constructs (c). As the size of the tRNA construct is increased, RT binding lifetimes also increase. (d) Zoom of long-lived RT binding events shows similar high/low FRET exchange as previously reported.
Figure 5.
Figure 5.
Single-molecule experimental design for measuring the stability of H1 in the presence and absence of RT (1 μM) using the three different tRNA constructs. (a) Schematic of dyelabeling scheme for measuring H1 stability. (b) Representative trace from these experiments which shows two FRET states corresponding to H1 formation (high FRET) and H1 melting (low FRET). (c) Histograms of population densities for H1 stability experiments using all three tRNA constructs in the presence and absence of RT (1 μM).
Figure 6.
Figure 6.
Single-molecule experimental design for monitoring the global fold of the initiation complex in the presence and absence of RT (1 μM). (a) Schematics of dye-labeling schemes used to monitor global RNA conformation. (b) Histograms of population densities for both FRET labeling schemes in the presence and absence of RT (1 μM).
Figure 7.
Figure 7.
Model of the early steps of reverse transcription initiation. (a) In the absence of RT, the vRNA-tRNA is structurally dynamic as indicated by multiheaded arrows. Dynamics between formed and melted H1 and the tRNA 5’ end are observed by smFRET. The formation of an extended PBS helix destabilizes H1, and dynamics are still observed between the vRNA 5’ and 3’ ends and the tRNA 5’ end. (b) The binding of reverse transcriptase is stabilized by the extended PBS helix which can make contacts within the RNase H domain, and the dynamic properties of the RNA/RNA complex decrease. Reverse transcriptase binding facilitates refolding of the tRNA into an elongated state and stabilizes H1. (c) The stabilization of H1 may serve to induce strain on H2 which is the RT pause site during the process of initiation.
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