Oak Ridge) Igor Rouzine, Ph.D

Biological dither: HIV’s Hardwired Latency Circuit

Leor Weinberger, PhD Gladstone 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, PhD Gladstone 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

HIV Latency Weinberger et al. Cell 2005 Bacterial DNA uptake Suel et al. Science 2007 Stem-cell Reprogramming Chang et al. Nature 2008 Metastasis Gupta et al. Cell 2011 Ab Producing B cells Duffy et al. Science 2012 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) HIV is Not Silenced in Primary Cells during Active-to-Resting Transition

Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015) HIV is Not Silenced as Primary Cells Relax from Activated to Resting

Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015) Tat Feedback is Not Silenced in Resting Primary T cells

Razooky & Pai et al. Cell (2015) Rouzine et al. Cell (2015) 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?

Maike Hansen

Hansen et al. Cell 2018 Post-transcriptional splicing mightHIV generate alternative negative splicing feedback

Co-transcriptional Splicing

No Feedback

Hansen et al. Cell 2018 Post-transcriptional splicing mightHIV generate alternative negative splicing feedback

Post-transcriptional Splicing

Auto-depletion Feedback

Hansen et al. Cell 2018 HIV splicing is

post-transcriptional

mRNA # mRNA

MS mRNA # mRNA

Single-molecule RNA FISH Hansen et al. Cell 2018 A negative feedback ‘overshoot’ in HIV 1

Early

(Nef-GFP)

cell Intensity > Intensity cell - Late

(mCh-RRE) < Single <

0 Time (h) 18 Hansen et al. Cell 2018 Post-transcriptional splicing attenuates noise & stabilizes fate

Wild-type HIV-1 Splicing Mutant GFP

Time (h)

24h Remove

activation 48h

GFP Hansen et al. Cell 2018 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 85 NoiseScreening Enhancers for Noise Identified (from 1,600 FDA-approved Compounds)

mCherry LTR

Noise Enhancer

Activator

mCherry

Dar et al. Science (2014) Noise Enhancers Potentiate LRAs (opposite of a stress response)

Dar et al. Science (2014) Noise Enhancers Potentiate LRAs in Human Primary CD4+ T Cells

with Nina Hosmane & Bob Siliciano Dar et al. Science (2014) Noise Suppressors Attenuate Activators & Stabilize Cell State

High-Noise Mutant + Noise Suppressor

24h

GFP

Hansen et al. Cell 2018 Summary

1. Stochastic noise drives an HIV latency switch

1. Fluctuations can be harnessed Acknowledgements Gladstone/UCSF Cynthia Bolovan-Fritts, Ph.D. Collaborators Anand Pai, Ph.D. Ariel Weinberger (Wyss/Harvard) Noam Vardi, Ph.D. Tom Shenk (Princeton) Winnie Wen, Ph.D. Michael Simpson (Oak Ridge) Igor Rouzine, Ph.D. Michelle Arkin (UCSF) Elena Ingerman, Ph.D. Robert Siliciano (HHMI/Johns Hopkins) Seung-Yong Jung, Ph.D. Sonali Chatuverdi, Ph.D. Elizabeth Tanner, Ph.D. Funding Maike Hansen, Ph.D. Timothy Notton, Ph.D. • NIH Director’s Pioneer Award Ravi Desai • NIH Director’s New Innovator Award Victoria Saykally • NIH Avant-Garde Award for HIV • NIH R01-AI109593, AI09611 Lab Alumni • NIH K25-GM083395 Roy D. Dar, Ph.D. (Faculty, UIUC) • NIH P01AI090935, P50GM081879 Abhyudai Singh, Ph.D. (Faculty, U. Del) • Alfred P. Sloan Research Fellowship Melissa Teng, Ph.D. (A.S.C.) • Pew Scholarship in Biomedical Science Kate Franz (Harvard) • W.M. Keck Research Excellence Award Brandon Razooky, Ph.D. (Rice Lab, Rockefeller) • Bill & Melinda Gates Foundation Luke Rast (Harvard) • California HIV/AIDS Research Program Jon Klein (Yale) • DARPA D15AP0024