UCSF

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 transmissible 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 transmission 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.

We introduce a new tool to overcome this barrier: a high-throughput method to generate diverse libraries of barcoded viral deletion mutants (> 1E5 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 mutants 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 literature. We identify four cis-acting regions in the HIV-1 genome which could not be complemented in trans: (1) 5' LTR through the matrix domain of Gag, (2) cPPT/CTS, (3) RRE

through SA7, (4) PPT through 3' 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) 5' UTR though C, (2) NS2A through the 3' 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 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.