Research

Reverse Transcription

Reverse transcription is a central event in the retroviral life cycle, allowing the RNA genome being reverse-transcribed into DNA by the viral reverse transcriptase enzyme. In murine leukemia virus (MLV), we showed that (Miller et al., Nature 2014) the reverse transcriptase enzyme initiates from a highly specific RNA-RNA complex, which is formed between a primer molecule, a specific tRNA packaged into the virion from the host cell during virus assembly, and a region at the 5' end of genomic RNA called the U5 Primer Binding Site (U5-PBS). The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements.

Ribosomal Recoding

It has been well known that viruses employ ribosomal recoding — either by frameshifting or stop codon read-through — to regulate expression levels of various proteins. This not only ensures translation of proteins encoded in different frames but also yields very precise ratios of the proteins. We previously demonstrated (Houck-Loomis et al., Nature 2011) that the frequency of ribosomal readthrough in murine leukemia virus (MLV) is regulated by a dynamic mechanism, in which a recoding signal on the mRNA is maintained in a pre-existing, protonation-dependent equilibrium between two functional conformers: an active conformer that is permissive for recoding, and an alternate, inactive conformer that terminates translation. Importantly, the pKa of the equilibrium is such that at physiological pH the populations of the active and inactive form correlate with the critical levels of recoding and termination, respectively. We are currently working on determining recoding mechanisms in several other viral systems.

Additionally, recent research from other labs has identified many ribosomal recoding candidates in eukaryotic systems. As such, we are currently studying ribosomal readthrough systems in Drosophila and humans.

Transcriptional Regulation

To function properly, RNA polymerase requires the positive transcription elongation factor P-TEFb. When not needed, P-TEFb is sequestered by the non-coding RNA 7SK. This sequestration can only occur in the presence of HEXIM protein and is mediated by the overall structure of the 7SK RNA.

7SK, a 331 nt RNA, is predicted to have 4 stem-loops (SLs). The structure of SL4 has already been solved in the lab and was published in 2010 by Durney & D’Souza. We are currently using NMR to learn more about the full 7SK molecule.

Internal Ribosomal Entry Sites

We recently described (Imai, et al., Nature Structural & Molecular Biology 2016) a mechanism that explains how viral Internal Ribosome Entry Sites (IRESs) are structurally designed to exploit host translation factors. This study provided the first structure of an IRES domain that interacts with initiation factors to assemble ribosomes. Our study showed how a conserved stretch of adenines in the J-K region of Encephalomyocarditis virus folds into a pentaloop and acts as a dual-sided docking station for basepair receptors present in J and K stems. This “adjuster module” thus precisely defines the relative orientation of the subdomains, and allows the HEAT-1 domain of eukaryotic initiation factor 4G (eIF4G) to dock onto the pre-organized motifs, with very little reorganization of the RNA upon binding. Mutations to the adjuster module, which does not make any protein contacts, to subtly change the orientation of the domains, leads to a complete loss of protein binding. This study thus illustrated how a virus adapts its RNA structure to acquire an affinity for a target protein of the host cell.

In addition to viral IRESs, we are also interested in IRESs in eukaryotic systems.