A chromatin loop controls the expression of anterograde transport genes during HSV-1 latency

Project Summary/Abstract Ocular Herpes Simplex Virus 1 (HSV-1) is a leading cause of blindness in the US and results from repeated reactivation of latent virus from reservoirs established in host sensory neurons. Latent HSV-1 reservoirs are created when virions infect neuronal axons and travel by retrograde transport to the sensory neuron. While the latent stage of HSV-1 is dormant and not associated with disease, latent virus presents a significant clinical challenge since virus in those reservoirs can reactivate, replicate, and transmit virus to new hosts. During a reactivation event, limited numbers of latently infected neurons produce infectious virus that is transported to the cornea by anterograde transport. That virus can then be spread to other hosts. Nonetheless, mechanisms that govern initiation and completion of reactivation events are unknown, impairing the development of novel therapeutics that can deplete reservoirs or prevent reactivation then transmission of virus to new people. In our proposed research we have identified cellular elements and viral elements that are required to start and then complete the process of reactivation from neurons. We have combined a new human neuronal platform with novel technology known as Chromosome Conformation Capture circular-sequencing to identify virally encoded chromatin loops that can further be targeted for disruption to prevent HSV reactivation and transmission. These new methods have allowed us to generate a novel and paradigm shifting hypothesis. We hypothesize that the function of chromatin insulators in the HSV-1 genome silences gene expression to establish latency, while the initiation of reactivation from latency ameliorates the chromatin insulator function to allow gene expression and subsequent reactivation. We have designed three aims that will allow us to 1) fully define the mechanisms for how this happens during latency and in reactivation and 2) to develop novel therapeutics in accordance with the strategic priorities of the NIH HSV strategic plan. Our novel therapeutic arm of this proposal combines mechanistic data with treatment of infections in the eye by depletion of viral proteins using rAAV-delivered siRNAs or editing of viral genomes using rAAV-delivered CRISPRs. Collectively, our proposal will leverage mechanisms involved in reactivation with targeting those mechanisms to therapeutically block reactivation or deplete latent reservoirs.