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