Observing Changes to HIV-1 Rev Oligomerization Using Fluorescence Resonance Energy Transfer

Neville
1Department

1 Bethel ,

Bhargavi

2 Jayaraman ,

Alan

2 Frankel

of Biomedical Engineering and Physics, Stony Brook University, Stony Brook, NY 2Department of Biophysics and Biochemistry, University of California San Francisco, CA

Goal
Although Rev is known to bind to the RRE, it is not known whether or not RRE remodels Rev oligomerization interactions. We are investigating whether or not this remodeling occurs. Fluorescence Resonance Energy Transfer (FRET)
90000 Fluorescence Intensity (RFU) 70000 60000 50000 40000 30000 20000 10000 0
y = 2665.5x R = 0.9999

Rev plays an essential role in the HIV-1 life cycle:

Preliminary Measurements
Linear Range of Alexa Fluor Fluorescence
23500 Fluorescence Intensity (RFU) 23000 22500 22000 21500 21000 20500 20000 19500 19000
Alexa-Dabcyl Alexa-Unlabled Rev Alexa-Buffer

Time for Equilibration of Rev Dimers

80000

0
en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer

10 20 30 Alexa Fluor Concentration (M)

40

2 Time (Hours)

Regular Fluorescence
FRET is distance dependent. Denoted by equation below where E=1 means total quenching of fluorescence. Can be used to see if Rev molecules shift upon binding to RRE. If they move closer, we should see a decrease in fluorescence.

FRET

Fernandes et al. 2012

HIV-1 Protein Rev Dimer

Rev Response Element

Lakowicz et al. 2006

The lowest concentration of Alexa Fluor labeled Rev that could be measured was found to be 10 nm. This was the point where the top left graph became nonlinear. The top right figure indicates that the time needed to equilibrate is three hours. According to the data shown in bottom right figure we found the KD for this Rev dimer to be 680 nM.
Valeur et al. 2001

Dissociation Constant for Rev Dimers

0.14

Percent Quenching

0.12 0.1 0.08 0.06 0.04 0.02 0 -0.02 Alexa-Dabcyl Rev Alexa-Unlabeled Rev

Log(Concentration(nM))

Preparing Rev
Fluorescence Intensity (RFU)

The Effect of RNA Titrations on Dimer Fluorescence

2850 2800 2750 2700 2650 2600 2550 2500 2450 2400 2350

Mutate
Daugherty et al. 2010 Daugherty et al. 2010

Label

Rev Dimer

Rev N30C

Alexa FluorDabcyl Labeled Rev N30C

Control

3'U 0.25X

3'U 0.5X

3'U 1X

IIB 0.5X

IIB 1X

IIB 2X

The Jellyfish Model

Rev Response Element (RRE) Fragment

Labeling Rev
Needed to optimize percent labeled Rev and final Rev concentration. Several conditions were tested during multiple labeling reactions: Concentration of reducing agent (0mM - 5 mM) Molar excess of dye (5X - 10X) Incubation time (2hr - 12hrs) Incubation temperature (4C - 25C) pH of reaction buffer (7.0 or 7.5) Optimized Labeling Conditions

Sorting Through the Chaos

Rev dimers were titrated with three type of RRE fragments of varying concentrations. RRE fragment 3U is the larger fragment. It has two sites for Rev dimer attachment; one for each molecule. At the concentration of Rev molecules, there would be an equal number of sites to Rev molecules. Stem IIB are stems that have one site for attachment. At equal concentration to Rev molecules these fragments would have the equal number of binding sites to Rev molecules. Meaning all molecules will be bound to RRE fragments.

Discussion and Conclusions

PD-A Percent of Alexa labeled Rev molecules that are in dimers with Dabcyl labeled Rev. Ii Fluorescence intensity of Alexa labeled Rev in absence of Dabcyl labeled Rev If Predicted fluorescence intensity after mixing The decrease in fluorescence upon binding to 3U at 1X and 2X concentration indicate that the FRET pair moved 3.7 This indicates that the Rev dimer does remodel upon binding to the RRE To further confirm Rev dimer remodeling, more experiments should be done where controls such as titrating with other RNA strands should be done.

Three Rev Dimers

Multiple Rev molecules serve as a bridge that connects the unspliced RNAs to nuclear export proteins.

Daugherty et al. 2010

Two Crm-1 Molecules (Nuclear export proteins)

pH 7.5 2mM reducing agent Incubate for three hours at room temperature. 5X molar excess of dye

References
Fernandes J, Jayaraman B, Frankel A. (2012) The HIV-1 rev response element: An RNA scaffold that directs the cooperative assembly of a homo-oligomeric ribonucleoprotein complex. RNA Biol. 2012 Jan 1;9(1) Daugherty MD, DOrso I, Frankel AD. A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA. Mol Cell 2008; 31:824-34 Valeur, Bernard. 2001. Molecular Fluorescence: Principles and Applications Wiley-VCH, p.48 Lakowicz, Joseph R. Principles of Fluorescence Spectroscopy. Vol. 1. Boston (MA): Springer, 2006. PDF

Acknowledgements
Many thanks to Beth Adams, Julia Clark, Peter Taylor and Flora Rutaganira for their guidance and support during this research project.