biological dither: hiv’s - virology...
TRANSCRIPT
Biological dither: HIV’s Hardwired Latency Circuit
Leor Weinberger, PhDGladstone Center for Cell Circuitry
Depts. of Pharmaceutical Chemistry, Biochemistry and Biophysics
University of California, San Francisco
Outline
1. HIV’s hardwired latency program:
a noise-amplification circuit
2. Post-transcriptional splicing
attenuates noise & stabilizes HIV
3. Harnessing noise to control HIV fate
Disclosures
Leor Weinberger is a co-founder and chair of
the SAB of Autonomous Therapeutics Inc. (ATI)
Data reported here were not obtained through
any commercial funding
The Ones Who Deserve the Credit
Leor Weinberger, PhDGladstone Institutes (Virology/Immunology)
Dept. of Biochemistry and Biophysics
California Inst. for Quantitative Biology (QB3)
University of California, San Francisco
Single
Molecule
RNA
FISH
Stochastic Fluctuations (Noise) in Gene Expression
CV
Diverse Systems Display Stochastic Fate Commitment
Ab Producing B cells
Duffy et al. Science 2012
Metastasis
Gupta et al. Cell 2011
Stem-cell Reprogramming
Chang et al. Nature 2008
HIV Latency
Weinberger et al. Cell 2005
Bacterial DNA uptake
Suel et al. Science 2007
The HIV Fate Decision
Latency driven by active→resting cell transitioning?
Latency in < 72h in vivo (resting memory formation ~2wks)
(Whitney et al. Nature 2014)
Partially penetrant latency reversal upon T-cell activation
(Ho et al. Cell 2013)
Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015)
HIV is Not Silenced in Primary Cells
during Active-to-Resting Transition
HIV is Not Silenced as Primary Cells
Relax from Activated to Resting
Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015)
Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015)
Tat Feedback is Not Silenced
in Resting Primary T cells
HIV’s Noise-Driven Latency Switch
Weinberger et al. Cell (2005) – … – Razooky et al. Cell (2015)
Outline
1. HIV’s hardwired latency program:
a noise-amplification circuit
2. Post-transcriptional splicing
attenuates noise & stabilizes HIV
3. Harnessing noise to control HIV fate
If noise drives fate selection,
how is fate stabilized?
Hansen et al. Cell 2018
Maike Hansen
Hansen et al. Cell 2018
Post-transcriptional splicing
might generate negative feedback Co-transcriptional Splicing
HIV alternative splicing
No Feedback
Hansen et al. Cell 2018
Post-transcriptional splicing
might generate negative feedback Post-transcriptional Splicing
HIV alternative splicing
Auto-depletion
Feedback
Single-molecule RNA FISH Hansen et al. Cell 2018
HIV splicing is
post-transcriptional
SS
mR
NA
#
US
mR
NA
#
MS
mR
NA
#
A negative feedback ‘overshoot’ in HIV
Early
(Nef-GFP)
Late
(mCh-RRE)
Time (h)
< S
ing
le-c
ell In
ten
sit
y >
Hansen et al. Cell 2018
0 18
1
Post-transcriptional splicing
attenuates noise & stabilizes fate
GF
P
Time (h)
Wild-type HIV-1 Splicing Mutant
Hansen et al. Cell 2018GFP
0h
24h
48h
Re
mo
ve
ac
tiva
tio
n
Outline
1. HIV’s hardwired latency program:
a noise-amplification circuit
2. Post-transcriptional splicing
attenuates noise & stabilizes HIV
3. Harnessing noise to control HIV fate
Screening for Noise
Dar et al. Science (2014)
LTR
mCherry
mCherry
Activator
Noise
Enhancer
85 Noise Enhancers Identified (from 1,600 FDA-approved Compounds)
Noise Enhancers Potentiate LRAs(opposite of a stress response)
Dar et al. Science (2014)
Noise Enhancers Potentiate LRAs in Human Primary CD4+ T Cells
Dar et al. Science (2014)
with
Nina Hosmane &
Bob Siliciano
Hansen et al. Cell 2018
High-Noise Mutant
0h
24h
GFP
+ Noise Suppressor
GFP
Noise Suppressors Attenuate Activators & Stabilize Cell State
Summary
1. Stochastic noise drives an HIV latency switch
1. Fluctuations
can be harnessed
AcknowledgementsGladstone/UCSF
Cynthia Bolovan-Fritts, Ph.D.
Anand Pai, Ph.D.
Noam Vardi, Ph.D.
Winnie Wen, Ph.D.
Igor Rouzine, Ph.D.
Elena Ingerman, Ph.D.
Seung-Yong Jung, Ph.D.
Sonali Chatuverdi, Ph.D.
Elizabeth Tanner, Ph.D.
Maike Hansen, Ph.D.
Timothy Notton, Ph.D.
Ravi Desai
Victoria Saykally
Lab Alumni
Roy D. Dar, Ph.D. (Faculty, UIUC)
Abhyudai Singh, Ph.D. (Faculty, U. Del)
Melissa Teng, Ph.D. (A.S.C.)
Kate Franz (Harvard)
Brandon Razooky, Ph.D. (Rice Lab, Rockefeller)
Luke Rast (Harvard)
Jon Klein (Yale)
Collaborators
Ariel Weinberger (Wyss/Harvard)
Tom Shenk (Princeton)
Michael Simpson (Oak Ridge)
Michelle Arkin (UCSF)
Robert Siliciano (HHMI/Johns Hopkins)
Funding
• NIH Director’s Pioneer Award
• NIH Director’s New Innovator Award
• NIH Avant-Garde Award for HIV
• NIH R01-AI109593, AI09611
• NIH K25-GM083395
• NIH P01AI090935, P50GM081879
• Alfred P. Sloan Research Fellowship
• Pew Scholarship in Biomedical Science
• W.M. Keck Research Excellence Award
• Bill & Melinda Gates Foundation
• California HIV/AIDS Research Program
• DARPA D15AP0024