Folding and Structural Ensemble of the Nascent HIV-1 RRE RNA

The human immunodeficiency virus (HIV) precipitates the onset of acquired immunodeficiency syndrome (AIDS), which is responsible for over 600.000 deaths annually. Many aspects of the HIV life cycle are governed by ribonucleic acids (RNAs). The so-called Rev response element (RRE) is one such regulatory RNA found in HIV. Because the genome of the virus is so small (just 9000 bases) it is required to be processed differently than the genome of the host cell after it is transcribed into RNA. During transcription, the genomic code is copied from the deoxynucleic acid (DNA) genome, which is the long- term information storage in the cell, into RNA which is then processed and used as a blueprint for the assembly of new viral particles. Thus, the HIV virus cannot take over the host cells machinery for this process. On the contrary, it needs to avoid the processing, called splicing, by the host cell in its entirety. The RRE RNA helps the virus achieve this goal by assembling a protective protein shell around the HIV RNA that protects it from the enzymes that need to be avoided. Thus, the RRE is crucial for the replication of HIV. To this day, many aspects of the RRE RNA remain elusive: it has been shown to be highly dynamic, i.e. it is present in different secondary structures (i.e. some RRE molecules have different base pairing patterns than others). So far, little is known about the purpose of this flexibility. Furthermore, all RNAs are transcribed sequentially (just like a chain is made from segments) from individual building blocks in the cell. At some point during this process, the RRE forms its intricate structure. However, how this structural folding process works has never been investigated. This research project aims to elucidate how the elongating RRE RNA chain is folded into its functional structure and what the critical steps are that trigger the folding of the RNA. The most important structural features may just be present for a short period of time during this process. This knowledge could potentially be used to develop drugs. Just like one wrong folding move destroys an entire origami sculpture, drugs that sequester these crucial intermediates could result in an RRE that is folded into a non-functional structure that inhibits viral replication. Furthermore, the project aims to investigate elusive RRE structures that are present only in small quantities, so-called excited states. Such excited states have been shown to be functionally important in many other regulatory RNAs. These goals will be addressed using state-of-the-art techniques such as nuclear magnetic resonance (NMR) spectroscopy which, similar to MRI in medicine, utilizes the magnetic properties of the atomic nuclei to investigate molecular dynamics as well as SHAPE-MaP where chemical modification of RNAs is used to investigate their secondary structures.