Constraints on protein structure in HIV-1 Rev and Rev-RNA supramolecular assemblies from two-dimensional solid state nuclear magnetic resonance.
The HIV-1 Rev protein is required for export of partially spliced and unspliced viral mRNA from nuclei of infected cells, and ultimately for viral replication. Rev is highly prone to aggregation, both in the absence and in the presence of the Rev responsive element (RRE) RNA to which it binds. As a result, the full molecular structures of Rev and Rev-RRE complexes are not known. We describe the results of transmission electron microscopy, atomic force microscopy, and solid state nuclear magnetic resonance (NMR) experiments on pure Rev filaments and coassemblies of Rev with a 45-base RNA sequence representing the high-affinity stem-loop IIB segment of the RRE. The morphologies of Rev filaments and Rev-RNA coassemblies are qualitatively different. Nonetheless, two-dimensional (2D) solid state 13C-13C NMR spectra of Rev filament and Rev-RNA coassembly samples, in which all Ile, Val, and Ala residues are uniformly labeled with 13C, are nearly indistinguishable, indicating that the protein conformation is essentially the same in the two types of supramolecular assemblies. Analysis of cross-peak patterns in the 2D spectra supports a previously developed helix-loop-helix structural model for the N-terminal half of Rev and shows that this model applies to both Rev filaments and Rev-RNA coassemblies. In addition, the 2D spectra suggest the presence of additional helix content at Ile and Val sites in the C-terminal half of Rev.
Havlin
Robert H
RH
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Blanco
Francisco J
FJ
Tycko
Robert
R
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015694:Gene Products, rev / Q000737:chemistry*; D015752:Genes, env / Q000235:genetics*; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry*; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1