ESI-FT-ICR investigation of the non-covalent interactions between the HIV-1 nucleocapsid protein p7 (NC) and the U5: AUG duplex

摘要

The 5'-untranslated region (5'-UTR) of human immunodeficiency virus type 1 (HIV-1) is a non-coding region of genomic RNA, which mediates very critical steps of the virus life cycle, including reverse transcription, splicing, translation, dimerization, and packaging. Its structure is believed to assume two possible conformations in which the various functional domains are alternatively protected or exposed. The Branched Multiple Hairpin (BMH, Fig. 1) conformer contains regulatory motifs that participate in dimerization, recognition, and packaging mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein. The Long Distance Interaction (LDI, Fig. 1) conformer lacks such motifs, but presents instead an intact primer-binding site (PBS) that could serve to initiate reverse transcription. The possibility that 5'-UTR may change conformation in response to specific cellular events has prompted suggestions that it may act as a riboswitch regulating the different activities of the virus lifecycle. We have recently implemented nanoflow electrospray ionization (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to investigate the chaperone activities of NC in the isomerization of the dimerization initiation signal (DIS) of HIV-1. We are now investigating its ability to bind and rearrange selected motifs that define the alternative BMH and LDI conformers. One of the target substrates corresponds to the U5-AUG complex formed exclusively in BMH by the specific annealing of nt 104-114 of the U5 region and nt329-339 of the AUG domain (Fig. 1). This long-range pairing interaction is absent in LDI, therefore any dynamics between the two-riboswitch conformations are expected to involve the formation/elimination of this duplex structure. The other target consists of the extended Psi-domain ((PSI)~(E), Fig. 1) formed exclusively in LDI by the annealing of complementary regions surrounding the SL3 domain of the genome's packaging signal.