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UC San Francisco Electronic Theses and Dissertations UCSF UC San Francisco Electronic Theses and Dissertations Title High-throughput generation of transmissible antivirals Permalink https://escholarship.org/uc/item/6176r6zv Author Notton, Timothy Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California High-throughput generation of transmissible antivirals by Timothy Norton DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY k Bioengineering in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO ii Copyright 2017 by Timothy Notton iii Abstract High-throughput generation of transmissible antivirals by Timothy Notton Doctor of Philosophy in Bioengineering University of California, San Francisco Leor Weinberger, Chair Effective population-level control of viral infections faces significant challenges including: how to therapeutically target the highest-risk populations, circumvent behavioral barriers, and overcome pathogen persistence and resistance mechanisms. A potential solution to overcome these barriers is the use of transmissible antivirals such as defective interfering particles (DIPs) or recently-proposed therapeutic interfering particles (TIPs). These trans- missible antivirals are molecular parasites and transmit by `piggybacking' on wildtype viral replication. By competing effectively for pools of common goods produced by the wildtype virus, DIPs/TIPs can interfere with wildtype virus replication and reduce viral loads in patients. As obligate parasites, TIPs would transmit via the same risk factors and transmis- sion routes as wildtype viruses, automatically reaching high-risk populations, and thereby substantially limiting viral transmission even in resource-poor settings. At present, methods to generate such transmissible antivirals are ad hoc and rely on either expert knowledge to rationally design transmissible antivirals or a laborious and often lengthy process of prospecting for rare, spontaneously-occurring subgenomic mutants. Thus, while deletion mutants of human viruses are desired for use as viral vectors, live-attenuated vaccines, and transmissible antivirals, modern technologies to generate them at scale are not available. iv We introduce a new tool to overcome this barrier: a high-throughput method to gener- ate diverse libraries of barcoded viral deletion mutants (> 105 unique mutants) at modest expense in a period of fewer than 5 days. The method is scalable and cyclical: viral strains with multiple deletions can be obtained by iterating the process. As proof of concept, we demonstrate the construction and screening of libraries of > 23; 000 barcoded deletion mu- tants of HIV-1 and > 90; 000 barcoded deletion mutants of Zika virus (ZIKV). Through repeated in vitro passage and deep sequencing of the pooled viral mutants, we are able to comprehensively map the cis-acting elements of HIV-1 and ZIKV at single base resolution. Moreover, we are able to track the prevalence of each barcoded deletion mutant through in vitro passage, and identify a subset of deletion mutants that are efficiently mobilized and amplified by the wildtype virus. For HIV-1, our results recapitulate empirical reports of cis-acting elements in the litera- ture. We identify four cis-acting regions in the HIV-1 genome which could not be comple- mented in trans: (1) 50 LTR through the matrix domain of Gag, (2) cPPT/CTS, (3) RRE through SA7, (4) PPT through 30 LTR. Thus the minimal proviral size of an HIV-1 vector with two intact LTRs is approximately 2.6 kbp. For ZIKV, we identify two cis-acting regions: (1) 50 UTR though C, (2) NS2A through the 30 UTR. We show that deletions which induce frameshift of the common open reading frame do not persist, in agreement with a basic mechanism of flaviviral replication. These results suggest a model where Pr, M, E, and NS1 can be provided in trans, but not C, NS2A/B, NS3, NS4A/B, and NS5. Finally, we use the information garnered in the HIV-1 screen to construct a library of transmissible antivirals. Use a modular cloning strategy, we assemble a combinatorial library of multiply-deleted mutants that are composed of a subset of adaptive HIV deletion mutants. We find that mutants with deletions of the accessory region vif {vpu, when combined with v gag/pol deletions, interfere with wildtype virus replication and transmit efficiently in single round studies. These results suggest a model of interference where transcriptional asymmetry allows this subset of deletion mutants to compete effectively for a common pool of capsid proteins provided by the wildtype virus. Taken in whole, we show that we have developed a framework for generating transmissible antivirals from first principles. The method is of general use in virology, where the technology can be used to generate live-attenuated vaccines, viral vectors, and replicons, as well as to understand fundamental principles of viral replication and genetics in diverse viral systems. It is of particular interest in emerging viral infections, where therapies must be quickly generated and deployed. vi To Mom & Dad Who gave me my first two microscopes. vii Acknowledgments I'm indebted to many. Foremost, to Ashley, for her love and gracious sacrifices to accommodate me. For the rest of the SC family who insisted on their names in print, here you are: K.I., S.P., S., F.B., V., L.H.&J., G., G.J., F., C.B., S.V., B.B.(y), C.(y). To the Notton family|Mom, Dad, Michael, Suzie, Ryan, Kaitlyn, Grant, Kari, and James|for their support and endurance. To Victoria Saykally (Chapters 2, 4, 5, 6) and Cassandra Thompson (Chapter 6), for excellent technical assistance. To all people who have worked on TIPs in the Weinberger Lab (in rough chronological order): Brandon Razooky, Vincent Metzger, Kate Franz, Igor Rouzine, Lisa Bishop, Josep Sardany´es,Colleen Scheitrum, Jac Luna, Grayson Kochi, Renee Ram, Moses Xiu, Anand Pai, Jonathan Klein, Luke Rast, Seung-Yong Jung, Elizabeth Tanner, Anjali Gopal, Victoria Saykally, Cassandra Thompson, Justin Jenkins, Matthew Dalton, and Joshua Glazier. To current members of the Weinberger Lab not previously mentioned: Cynthia Bolovan- Fritts, Hye-in Son, Noam Vardi, Zo¨eSteier, Maike Hansen, Ravi Desai, Sonali Chaturvedi. To Freeman Lan, who shared his work with transposons at the Bioengineering retreat in 2013, and sparked the idea for this work. To Marielle Cavrois and Gilad Doitsh, who generously shared their knowledge of working with HIV in the BSL-3. To JJ Miranda, for several helpful conversations. To three core facilities for their support: Gladstone BSL-3 Core, Gladstone Flow Cytom- etry Core, UCSF Center for Advanced Technology. To the funders of and generous donors to the NIH AIDS Reagent Program. To Kim Osborn, for administrative assistance. viii To Stephen Beverley, Kai Zhang, and Lon-Fye (George) Lye at WUSTL, for providing a strong foundation in science. To Nick Abello and Nick Vasquez for pouring over 1000 plates and their assistance in the BSL-3. To UCSF librarians, for assistance with research and absolution of late fees. To Lisa DiGiorgio-Haag, for providing outstanding care throughout my time at UCSF. To Winnie Wen and Maike Hansen for sharing desk and bench space, often by comman- deering. To Dan Gibson for many instructional papers. To Michael McManus and John Dueber, for sound advice and critique. To Adam Abate and Shawn Douglas, for advice and for serving on my qualifying exam. To Leor, for his constant support and from whom I have learned much, not least of all to seek out and champion good ideas. To all those left unmentioned here, but who nonetheless made this work possible. ix Contents 1 Introduction1 1.1 The need for transmissible and evolvable therapies............... 1 1.2 Omnis viri e cellula ................................ 6 1.3 Viral markets and the emergence of cheaters.................. 7 1.4 Defective Interfering Particles (DIPs)...................... 10 1.5 Design principles of effective TIPs........................ 12 2 High-throughput generation of deletion libraries 15 2.1 Introduction.................................... 15 2.2 A method to produce tagged random deletion libraries ............ 18 2.3 Materials and Methods.............................. 18 2.4 Construction of an HIV-1 random deletion library............... 43 2.5 Construction of ZIKV random deletion libraries................ 57 2.6 Discussion..................................... 64 3 Random deletion library screening theory 69 3.1 Introduction.................................... 69 3.2 Definitions..................................... 69 3.3 The universe of possible deletion libraries.................... 71 x 3.4 Determining the length of barcode cassettes .................. 74 3.5 Frequency of background mutations....................... 77 3.6 Identification of cis-acting elements....................... 78 3.7 Sequencing analysis of deletion libraries..................... 82 4 Screen for HIV-1 therapeutic interfering particles 86 4.1 Introduction.................................... 86 4.2 Materials and Methods.............................. 93 4.3 Results....................................... 99 4.4 Discussion..................................... 119 5 Screen for ZIKV therapeutic interfering particles 125 5.1 Introduction.................................... 125 5.2 Materials and Methods.............................. 128 5.3 Results....................................... 134 5.4 Discussion..................................... 145 6 Construction of
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