Nucleocapsid Protein Locally Melts The Iib Region Of Hiv-1 Rre Rna, But Rev Protein Does Not: A Single-molecule Spectroscopic Study

Abstract

The focus of this work is to develop a single molecular-level understanding of how the binding of two important viral proteins of human immunodeficiency virus type-1 (HIV-1), Rev and nucleocapsid (NC) proteins, locally change the secondary structures of the IIB stem-loop region of the Rev-Responsive Element (RRE) of the HIV-1 viral genomic RNA. Rev and NC represent two types of nucleic acid-binding proteins in HIV with distinct structures, behaviors and functionalities. Rev is a sequence-specific RNA-binding protein that binds to stem IIB and other regions of the RRE, facilitating nuclear export of unspliced HIV RNAs. In contrast to Rev, NC is a multifunctional protein that plays a role in almost every step of the retroviral life-cycle. NC can bind both DNA and RNA hairpin structures using its CCHC-type zinc fingers and basic domains. Here we systematically investigate how the binding of Rev and NC on the 42-nt IIB RNA hairpin locally changes the RNA secondary structures using a single-molecule fluorescence resonance energy transfer (SM-FRET) approach. A series of RNA and DNA oligonucleotides containing appropriate dyes have been designed to probe the local melting of the IIB RNA hairpin using a single-molecule oligonucleotide annealing assay. Our study shows that NC locally melts the IIB RNA hairpin but Rev does not, illustrating how two proteins that use dramatically different nucleic acid recognition motifs can give rise to very different secondary structural changes upon RNA binding. This SM-FRET-based approach provides a unique way to gain insight into the secondary structural change of HIV viral RNAs induced by protein binding with minimal interference from protein aggregation.