Abstract
The packaging signal of HIV-1 RNA contains a stem-loop structure, SL1, which serves as the dimerization initiation site for two identical copies of the genome and is important for packaging of the RNA genome into the budding virion and for overall infectivity. SL1 spontaneously dimerizes via a palindromic hexanucleotide sequence in its apical loop, forming a metastable kissing dimer form. Incubation with nucleocapsid protein causes this form to refold to a thermodynamically stable mature linear dimer. Here, we present an NMR structure of the latter form of the full-length SL1 sequence of the Lai HIV-1 isolate. The structure was refined using nuclear Overhauser effect and residual dipolar coupling data. The structure presents a symmetric homodimer of two RNA strands of 35 nucleotides each; it includes five stems separated by four internal loops. The central palindromic stem is surrounded by two symmetric adenine-rich 1-2 internal loops, A-bulges. All three adenines in each A-bulge are stacked inside the helix, consistent with the solution structures of shorter SL1 constructs determined previously. The outer 4-base pair stems and, proximal to them, purine-rich 1-3 internal loops, or G-bulges, are the least stable parts of the molecule. The G-bulges display high conformational variability in the refined ensemble of structures, despite the availability of many structural restraints for this region. Nevertheless, most conformations share a similar structural motif: a guanine and an adenine from opposite strands form a GA mismatch stacked on the top of the neighboring stem. The two remaining guanines are exposed, one in the minor groove and another in the major groove side of the helix, consistent with secondary structure probing data for SL1. These guanines may be recognized by the nucleocapsid protein, which binds tightly to the G-bulge in vitro.
Highlights
Two identical copies of genomic RNA are packaged in the viral particles of HIV-1.2 The non-covalent dimer formed by these two RNA strands is involved in several stages of the viral life cycle, such as reverse transcription, recombination, RNA packaging; its integrity is important for optimal viral infectivity
A similar effect is achieved by incubation of kissing dimers at 37 °C together with NC protein, in a process possibly reflecting maturation of genomic RNA in vivo
Delineating structure of SL1 is important for understanding dimerization and packaging of HIV-1 RNA, and it is potentially valuable for structure-based development of therapeutic drugs interfering with these processes
Summary
Sample Preparation—35-nt RNA (SL1) was synthesized by in vitro transcription with T7 RNA polymerase (14, 15), purified by PAGE and electroelution, and dialyzed. RDC constants were measured by comparing separations between the bottom-right and bottom-left components in the non-decoupled constant time 1H-13C TROSY-HSQC spectra (23) acquired in isotropic and liquid crystalline solutions To minimize overlap, these experiments were set up such that only one multiplet component appeared in each spectrum; the two spectra containing the bottom-right and bottom-left components were collected in an interleaved fashion. The miniCarlo program is specialized software for modeling, energy minimization, and Metropolis Monte Carlo simulation of nucleic acids It uses generalized helical parameters of nucleic bases as internal coordinates. To pass the DYANA structures to miniCarlo, helical parameters were calculated from the atomic coordinates as described (30). Restraints used in miniCarlo calculations included distance restraints as described above, sugar pucker restraints (these were used only during high-temperature Monte Carlo simulations), and RDC restraints. The refined structures, chemical shifts, and NMR restraints will be deposited to PDB (code 2GM0) and BMRB
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