Establishing a Laboratory Model of Herpes simplex virus (HSV) Latency Using Compartmented Neuron Cultures

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Alpha herpesvirus infections (e.g. herpes simplex virus 1 and 2; HSV-1 and HSV-2) are among the most common virus infections in the world affecting more than 70% of human population. A typical alpha herpesvirus infection results in life-long latency in the peripheral nervous system (PNS) ganglia. Latent viral genomes can reactivate to start a disseminated infection often leading to herpes blisters, genital lesions or keratitis. The primary infection starts with a productive infection in the epithelial cells of mucosal surfaces. After replication, progeny virus particles mainly invade the dorsal root and trigeminal ganglia (DRG and TG), and other autonomic sympathetic ganglia (e.g. SCG) to establish latency. Establishment of latency, reactivation, and subsequent spread of infection is affected by many cell intrinsic, tissue-specific and systemic factors that are challenging to dissect. The available anti-herpetic drugs target viral DNA replication but have no effect on latent or reactivating viral genomes. Consistent and reproducible laboratory models are required to dissect the molecular mechanisms of latency and reactivation to be able to design novel therapies. Currently used neuron culture models of HSV-1 latency employs dissociated rodent primary neurons with the use of acyclovir to inhibit DNA replication and force the infection into latency. This is not ideal, not only because the virions are directly infecting neuronal cell bodies instead of axons, but also because the viral DNA that incorporated the guanine analogue might not be competent for further replication. Recently, we have developed a reproducible and reactivatable in vitro latency system by infecting isolated axons of compartmented neurons with a low concentration of the veterinary pathogen, pseudorabies virus without the use of drugs. Since the outcome of infection is always silenced under these conditions, we studied mechanisms that enable escape from genome silencing. We discovered two distinct pathways that prevented PRV genome silencing. We will develop this system to explore the molecular mechanisms of latency establishment and reactivation of the human pathogen, HSV-1. Identification of neuronal stress pathways enabling escape from silencing will contribute to designing novel therapeutic strategies to control virus reactivation and related pathologies in patients with recurrent herpesvirus reactivation.