FRET-based

detection and
quantification
of HIV-1 Virion
Maturation

Sarca Anamaria Daniela

March 1st 2021
 • HIV-1 brief introduction
Outline • Virion core morphology
Background • Objective

• HIV-1 Gag-iFRET construct

Materials & • Production conditions
Methods

• Single virion images

• Quantification of mature and immature virions
Results • Testing of a protease inhibitor

• Achievements
• Potential applications
Discussion • Conclusion
 • HIV-1 brief introduction
Outline • Virion core morphology
Background • Objective

• HIV-1 Gag-iFRET construct

Materials & • Production conditions
Methods

• Single virion images

• Quantification of mature and immature virions
Results • Testing of a protease inhibitor

• Achievements
• Potential applications
Discussion • Conclusion
 HIV-1 epidemic
36.2 million
38.0 million
1980s – today People Living with HIV
(PLWH)
Total infections:
[1.7 million in 2019] 1.8 million
~75.7 million (0–14 years)
[55.9 million
–100 million]
32.7 million

Global HIV & AIDS statistics — 2020 fact sheet

Background Materials & Methods Results Discussion
 Human
Immunodeficiency
Virus (HIV)
Disease: Acquired Immune
Deficiency Syndrome (AIDS)
Thomas
Family: Retroviridae Splettstoesser

Species: HIV-1; HIV-2

Background Materials & Methods Results Discussion

HIV-1
Replication
Cycle

Engelman A, Cherepanov P. Nat Rev Microbiol. 2012 Mar 16; 10(4): 279–290
Background Materials & Methods Results Discussion
HIV-1 Maturation

Protease
Inhibitor

HIV-1
Protease

Sundquist WI, Krausslich HG. Cold Spring Harb Perspect Med. 2012 Jul; 2(7): a006924
Background Materials & Methods Results Discussion
HIV-1 Core – capsid (CA)

240 CA + 12 CA
hexamers pentamers

~ 1500 CA
molecules/core

• Fullerene cone shape

• Sealed
Engelman A, Cherepanov P. 2012 Mar 16; 10(4): 279–290
Background Materials & Methods Results Discussion
Study Objective Immature

Morphology Electron Microscopy

determination ✓ High-resolution Mature

❖ Few particles; user dependent

❖ Static

OBJECTIVE Fluorescence microscopy

o Analyse immature and mature morphologies
o Faster, capable of semi-automated large scale quantification
o Capable of live imaging
Background Materials & Methods Results Discussion
 • HIV-1 brief introduction
Outline • Virion core morphology
Background • Objective

• HIV-1 Gag-iFRET construct

Materials & • Production conditions
Methods

• Single virion images

• Quantification of mature and immature virions
Results • Testing of a protease inhibitor

• Achievements
• Potential applications
Discussion • Conclusion
HIV-1 Gag-iFRET construction
HIV-1 genome
(NL4-3)

HIV-1 Gag- Fluorescence

Resonance
interdomain Energy
FRET Transfer
HIV-1 Gag-iFRET

Maturation
(Gag cleavage
by HIV protease)

Background Materials & Methods Results Discussion

HIV-1 Gag-iFRET∆Pro – protease
deficient control

HIV-1 Gag-iFRET∆Pro

Our positive FRET signal control:

▪ Same construct but with dysfunctional protease → iFRET∆Pro
▪ Should result in an immature population of virions

Background Materials & Methods Results Discussion

 Virion production FRET imaging
0:1
1:0
iFRET
iFRET∆Pro
:
:
NL4-3
NL4-3∆Pro
Infectivity Assays Dilute 800x
1:1
1:10 NL4-3 + control
1:20 iFRET labeled
iFRET∆Pro labeled 8well
HEK293T chamber slide
± Darunavir
ON 4C
24h p24 adjusted
Collect supernatant
Nikon
TZM-bl A1R MP+
reporter cells - Argon Laser
2h
Ultracentrifuge - PFS
25,000rpm
48h - 60x oil

Viral pellet
Viral Luciferase Assay
Transmission Concentrate Image Analysis
Electron Microscopy MATLAB R2016b
Background Materials & Methods Results Discussion
 • HIV-1 brief introduction
Outline • Virion core morphology
Background • Objective

• HIV-1 Gag-iFRET construct

Materials & • Production conditions
Methods

• Single virion images

• Quantification of mature and immature virions
Results • Testing of a protease inhibitor

• Achievements
• Potential applications
Discussion • Conclusion
293T cells produce labeled viral proteins

Background Materials & Methods Results Discussion

Virions incorporated the iFRET construct

HIV-1 Gag-iFRET
labeled virions are
processed similarly
to parental NL4-3
CFP
YFP

Background Materials & Methods Results Discussion

HIV-1 Gag-iFRET labeled virus is infectious

HIV-1 Gag-iFRET

requires helper parental

provirus to achieve
infectivity

*p < 0.05
Background Materials & Methods Results Discussion
Electron microscopy confirms labeled
virion morphology Number of
96 93 99 Counted
particles
100%
17.7 18.2
iFRET 80% Mature

60%
Immature
100
iFRET∆Pro 40% 82.3 81.8

20%

NL4-3
0%
iFRET iFRET∆Pro NL4-3

Background Materials & Methods Results Discussion

Single virion imaging of HIV-1 Gag-iFRET
CFP YFP FRET
YFP -FRET Donor- -FRET Acceptor- Low Ratio view High

Background Materials & Methods Results Discussion

Image processing and particle localization
X Y
3.75 185.75
YFP Binary image
MATLAB 10.63157 243.2105
R2016b 14 349

Subtract background Particle 20 168

20.5 101.5
Exclude large particles Location
Identifier 21.25 108.25
22.5 433
25.5 83.5
28 357
… …
1µm

Background Materials & Methods Results Discussion

 Data extraction and FRET calculation
CFP YFP FRET*
intensity intensity Calculated
6608 4464 0.403179

X Y YFP
FRET =
108.25 168 CFP+YFP

CFP emission Particle counts:

(FRET donor)
2000 – 20,000/
Experiment
YFP emission
(FRET acceptor) (21 images)
*Preus S, Wilhelmsson LM, Advances in Quantitative
FRET-Based Methods for Studying Nucleic Acids, ChemBioChem, 2012
Background Materials & Methods Results Discussion
As virions complete maturation, the FRET
signal shifts to the left

Kernel curve

Background Materials & Methods Results Discussion

 Quantification strategy of immature HIV-1
Gag-iFRET labeled virions
Particle density

Background Materials & Methods Results Discussion

iFRET imaging application – protease
inhibitor testing
Protease inhibitor
(Darunavir)

dose-dependent
sensitivity

*Darunavir
IC50 = 1 - 8.5 nM

*De Meyer, et al. (2005) Antimicrob Agents Chemother, 49(6), 2314-2321

Background Materials & Methods Results Discussion
Protease inhibitor: maturation & infectivity

Background Materials & Methods Results Discussion

Protease inhibitor: EC50 and IC50

Darunavir
IC50 = 1 - 8.5 nM

Our study:
EC50 = 7 nM
95% CI (3.9 - 12.1)

IC50 = 2.8 nM
95% CI (0.88 – 8.3)

Background Materials & Methods Results Discussion

 • HIV-1 brief introduction
Outline • Virion core morphology
Background • Objective

• HIV-1 Gag-iFRET construct

Materials & • Production conditions
Methods

• Single virion images

• Quantification of mature and immature virions
Results • Testing of a protease inhibitor

• Achievements
• Potential applications
Discussion • Conclusion
HIV-1 Gag-iFRET labeling achievements

✓ Distinguish between immature and mature particles

✓ Large scale quantification ~10,000particles/experiment

✓ Protease inhibitor – detect dose-dependent changes in immature virions

Background Materials & Methods Results Discussion

HIV-1 Gag-iFRET labeling applications

➢ Determine efficiency of maturation inhibitors (that affect Gag processing)

➢ Determine maturation rates of HIV-1 in various other settings

- mutations in HIV-1 components

- different cell types

➢ Real-time visualisation of the budding and maturation process

Background Materials & Methods Results Discussion

Conclusion

HIV-1 Gag-iFRET is a powerful new addition to the molecular toolkit, a

tool that opens the gates to maturation studies in a fluorescence
microscopy setting and will help enrich our knowledge of this step of
the HIV-1 replication cycle.

Background Materials & Methods Results Discussion

Acknowledgements
Kyoto University Department of Infectious Disease
and Vaccines, MRL, Merck & Co.
Hirofumi Fukuda
Hiroyuki Matsui Luca Sardo
Kotaro Shirakawa
Akifumi Takaori-Kondo The Walter Reed Army Institute of
Research, US Military HIV Research
Tokushima University Program

Kazuki Horikawa Taisuke Izumi

Thank you

Questions
References

(1) Thomas Splettstoesser (www.scistyle.com)

(2) Global HIV & AIDS statistics — 2020 fact sheet
(3) Engelman A, Cherepanov P. Nat Rev Microbiol. 2012 Mar 16; 10(4): 279–290
(4) Sundquist WI, Krausslich HG. Cold Spring Harb Perspect Med. 2012 Jul; 2(7): a006924.
(5) Yu ZH, Dobro MJ, Woodward CL, et al. Jour of Mol Biol. 2013; 425:112-23
(6) Preus S, Wilhelmsson LM, Advances in Quantitative FRET-Based Methods for Studying
Nucleic Acids, ChemBioChem, 2012
(7) De Meyer, et al. (2005) Antimicrob Agents Chemother, 49(6), 2314-2321