Summers, Michael FSingh, Karndeep2025-02-132025-02-132024-01-0112946http://hdl.handle.net/11603/37654The human immunodeficiency virus type-1 (HIV-1) RNA genome’s 5'- untranslated region (5'-Leader, 5'-L) serves as a master regulator for numerous viral replication processes within a cell such as genome dimerization, splicing, packaging, and translation initiation. This 5'-Leader adopts two conformations dependent on the transcriptional start site usage: 5'-capped RNAs beginning with one guanosine (Cap1G) adopt the dimeric conformation whereas 5'-capped RNAs beginning with two or three guanosines (Cap2G and Cap3G, respectively) adopt a monomeric conformation. Published work from our laboratory revealed that Cap1G leader RNAs sequester the 5'-cap through a coaxial stacking of two 5'-hairpins – the TAR and polyA hairpin. This prevents binding to the human eukaryotic translation initiation factor 4E (eIF4E) cap binding protein – the initial recognition step in cap/eIF4E dependent translation of HIV-1 mRNAs. For the monomeric RNA transcripts, the 5'-cap is exposed and accessible for the recruitment and binding of eIF4E. Therefore, the exposure and sequestration of the 5'-cap (a 7- methylguanosine triphosphate) within the HIV-1 5'-Leader dictates the capture of the RNAtranscripts by cap-dependent translation machinery for translation of viral proteins or genome packaging by the Gag polyprotein, respectively. While it is well-established that cap-dependent translation serves as the primary mechanism of HIV-1 genome translation in eukaryotes, the molecular nature of interactions between Cap3G RNAs and eIF4E remain unknown. Electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC) experiments reveal that the human eIF4E binds to the HIV-1MAL Cap3G RNAs at least 2.5-fold tighter than the 5'-cap, suggesting that RNA elements influence binding of the human eIF4E. Using nuclear magnetic resonance (NMR) spectroscopy with a variety of selectively labeled 1H-, 15N-, and 13C-labeling schemes, we worked to characterize the first three-dimensional structure of a structured 5'-capped RNA – a HIV- 1MAL Cap3G RNA oligo (~13 kDa) – bound to the human eIF4E (~22 kDa). The 5'-cap forms a pi-pi stacking interaction with two tryptophan residues (W56 and W102) while residues of the TAR hairpin forming electrostatic interactions with eIF4E’s exposed charged residues to orient the RNA oligo out of the 5'-cap binding pocket. Surprisingly, the unstructured residues of the polyA region at the 3'-end interact with lysine residues of eIF4E. Our findings reveal that HIV-1 5'-Leader’s structural elements influence the recruitment and binding of eIF4E, suggesting an additional quality control mechanism that HIV-1 uses to ensure the translation of its Cap3G mRNA transcripts into viral proteins.application:pdfThis item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu or contact Special Collections at speccoll(at)umbc.edu5'-Capped RNAs5'-CappingeIF4EITCNMRProtein-RNA ComplexText