Rev is a small regulatory protein of HIV-1 that is essential for virus replication.  The biological role of Rev is to control the pattern of viral gene expression by promoting the transition from the early phase of infection, during which small regulatory proteins are expressed, to the late stage, when larger structural proteins are synthesized and assembled into viral particles.  Rev binds to a highly structured RNA region, the Rev response element (RRE), where it forms an oligomeric ribonucleoprotein complex with ~8-10 Rev proteins bound to the RRE.  The most important region of the RRE consists of five stem-loop domains radiating from a central junction, with the core element for Rev binding consisting of a stem loop-structure containing a purine rich bulge region. We use single-molecule fluorescence techniques to visualize the oligomeric assembly of Rev on the RRE (Fig. 1). This approach registers each Rev monomer binding event and provides complete kinetic information on the assembly pathway (Fig. 2).

Figure 1. Rev-RRE complex assembly monitored by TIRF microscopy. Discrete intensity jumps correspond to binding or dissociation of individual Rev monomers labeled with Alexa Fluor-555.

Figure 2. Kinetic description of Rev-RRE complex assemby obtained from dwell time analysis of fluorescence intensity trajectories.

Recent Millar Laboratory Publications

R. L. Lamichhane, J. A., Hammond, R. Pauszek, R. Anderson, E. J. C. van der Schans, E. Pedron, J. R. Williamson and D. P. Millar, "A DEAD-Box Protein Acts Through RNA to Promote HIV-1 Rev-RRE Assembly”, Nucleic Acids Research, 45, 4632-4641 (2017). PMID: 28379444.

J. A., Hammond, R. L. Lamichhane, D. P. Millar and J. R. Williamson, "A DEAD-Box Helicase Mediates an RNA Structural Transition in the HIV-1 Rev Response Element”, J. Mol. Biol. 429, 697-714 (2017). PMID: 28153748.

G. Pljevaljcic, R. M. Robertson-Anderson, E. J. C. Van der Schans and D. P. Millar, "Analysis of RNA Folding and Ribonucleoprotein Assembly by Single-Molecule Fluorescence Spectroscopy", in Spectroscopic Methods of Analysis: Methods and Protocols, Methods in Molecular Biology, Vol. 875, W. M. Bujalowski (Ed.), 271-295 (Springer, 2012).

R. M. Robertson-Anderson, J. Wang, S. P. Edgcomb, A. B. Carmel, J. R. Williamson and D. P. Millar, "Single-Molecule Studies Reveal that DEAD Box Protein DDX1 Promotes Oligomerization of HIV-1 Rev on the Rev Response Element", Jour. Mol. Biol. 410, 959-971 (2011).

S. J. Pond, W. Ridgeway, R. Robertson, J. Wang and D. P. Millar, "HIV-1 Rev Protein Assembles on Viral RNA One Molecule at a Time", PNAS 106, 1404-1408 (2009).