Protein-Nucleic Acids Interactions in viral systems
Our laboratory is dedicated to the structural and functional investigation of protein-nucleic acids complexes present in viruses responsible for infectious diseases, using mass spectrometry as the main analytical technique. In mature retroviral virions, genetic information is encoded by two homologous strands of genomic RNA packaged in a dimeric form. During the assembly of human immunodeficiency virus type 1 (HIV-1), the crucial processes of strand recognition, dimerization, and packaging are mediated by the interaction of the nucleocapsid protein p7 (NC) with a highly conserved stretch of genomic RNA located near the 5’-long terminal repeat, referred to as the packaging signal, or Psi-RNA. The structure of the complete RNA-protein complex is exceedingly large to be determined directly by nuclear magnetic resonance (NMR) and appears to be too flexible for X-ray crystallography. For these reasons, we have developed an alternative method based on solvent accessibility probes, chemical crosslinking, and photo-activated crosslinking to obtain information about the molecular contacts between the stable secondary structures formed by the folding of Psi-RNA, or between these structures and NC. Fourier transform mass spectrometry (FTMS) was utilized to reveal the sequence position of the crosslinked bases and amino acid residues, which provide the spatial constrains necessary for the molecular modeling of the complete Psi-site. This method was initially tested with excellent results on the ribosomal frameshifting pseudoknot of the mouse mammary tumor virus (MMTV), which provided a ~3 Å root mean square deviation (RMSD) from the corresponding NMR structure, and on the feline immunodeficiency virus (FIV) pseudoknot, for which there is currently no NMR or X-ray structure.
The structural determinants of binding and the factors affecting the interactions between the nucleocapsid protein and viral RNA can also be studied by taking advantage of the soft nature of electrospray ionization (ESI) mass spectrometry, which preserves in intact form the non-covalent complexes of NC with the different stem-loop hairpins present on Psi-RNA. Using this technique, the binding stoichiometry was directly obtained with no need for the curve fitting procedures necessary for other spectroscopic or calorimetric techniques. Further, first time for protein-RNA systems, the solution-phase dissociation constants were determined for the different complexes directly by ESI-FTMS. Their respective values were found to be in very close agreement with those measured by isothermal calorimetry. A similar approach was recently employed to demonstrate that the addition of aminoglycosidic antibiotics in solution can inhibit the binding of NC to the Psi-RNA stem-loop 4 (SL4) more efficiently than to stem-loop 3 (SL3). In a series of competitive binding experiments, we are now applying ESI-FTMS to identify the best possible inhibitor in vitro among a family of available aminoglycosidic structures. This method promises to allow for very rapid and efficient screening of combinatorial libraries of widely different composition and structures, which could yield new drug candidates to alleviate the increasing problem of drug resistance in AIDS therapy.
