Analyses of herpes simplex virus latency and reactivation at the single cell level.

Herpes simplex virus (HSV) can exist as two types, HSV-1, which is associated with recurrent cold sores, and HSV-2 that causes genital disease. In addition these viruses can sometimes cause severe and often life threatening infections particularly in neonates and the immunosuppressed. Once an individual is infected with these viruses a dormant or latent infection is established in neurons of the nervous system, which cannot be treated using available antiviral drugs. The mechanism by which HSV establishes, maintains and reactivates from this latent state is poorly understood and is the focus of this research. Using methods that allow the identification and purification of latently infected cells from the nervous system of experimentally infected mice, we aim to characterize the nature of latency at the single cell level. Our current research, and work from other research groups, has established that distinct forms of latency exist in the nervous system. In particular our prior research has shown that different levels of virus gene expression may occur prior to latency establishment. Our research aims to determine whether prior virus gene expression is associated with an elevation of latent virus DNA copy number in latently infected neurones and whether such cells correspond to the small number of neurones that are predisposed to reactivation. Using a transgenic mouse system that allows the identification and purification of fluorescent neurons that have experienced different levels of virus promoter activation compatible with latency, we are now in a position to address these questions. Should prior virus gene expression be responsible for the generation of a latent virus state that is predisposed to reactivation, our research has the potential to reveal novel virus targets whose inhibition may prevent or restrict reactivation and recurrent disease. We also wish to utilize this transgenic model system to explore the function of virus encoded latency transcripts. Our preliminary data have shown that these transcripts have an important role in maintaining latency in the nervous system. An important focus of our research will now be to explore how these transcripts function to maintain latency and specifically whether they are involved in suppressing virus gene expression and/or promote survival of latently infected neurones.

The major aims of the proposed research are to define the reactivation propensity and molecular characteristics of neurones latently infected with herpes simplex virus type 1 and to explore the mechanisms by which the HSV latency associated transcripts function to maintain latency. This will be achieved at single cell resolution using Ai6 ZsGreen transgenic reporter mice to facilitate the fluorescent detection, purification and subsequent characterization of latently infected neurones.

The specific objectives are;

1. To determine the latent virus DNA copy number in individual latently infected neurones that have experienced distinct patterns of lytic cycle promoter activity during latency establishment.

2. To investigate the reactivation potential of isolated populations of latently infected neurones in real time to correlate reactivation propensity to evidence of prior lytic cycle promoter activity and latent DNA loads.

3.To investigate the role of the HSV encoded latency associated transcript (LATs) in latency maintenance.

4. To define the role of latency associated miRNA expression and/or LAT encoded anti apoptotic functions in latency maintenance.

5. To develop a system to restore WT virulence and latency characteristics to an HSV genome cloned as a bacterial artificial chromosome to facilitate in vivo studies.

Impact summary;

This research project aims to utilize a novel in vivo strategy to facilitate the identification and isolation of single cells latently infected with herpes simplex virus for biochemical and biological analyses. The primary beneficiaries of this work will be virologists investigating mechanisms of latency establishment, maintenance and reactivation. However, this research will also lay the foundations for impact in the following areas;

Academia; Outside the immediate field of herpesvirology our research has revealed the utility of transgenic reporter mouse models in studies of virus latency/persistence. It should therefore be possible to adopt the system we have developed to studies of other persistent virus pathogens that infect mice or indeed other transgenic reporter animals. The ability to use fluorescent reporter animals also permits the isolation of single cells for subsequent molecular analyses. This may be of particular relevance when considering the known transcriptional heterogeneity that exists even amongst pooled cells of the same type. Thus at present the impact of latent viruses on the host transcriptome has to-date been performed either on bulk cultures of latently infected cells propagated in vitro or on latently infected tissue biopsies that comprise heterogeneous cell types. The ability to isolate single latently infected cells from a complex in vivo setting therefore offers considerable opportunities to investigate both viral and cellular transcriptomes simultaneously allowing direct correlations to be made between the two. A combination of next generation sequencing and recently developed mRNA-seq technologies makes the realization of this goal possible. In this regard we are now in a position to establish collaborations with leading groups based in the UK to apply state-of-the art sequencing and amplification technologies to investigate the latent virus and host transcriptome at the single cell level.

Commercial Sector; One of the aims of the research is to understand the non-uniformity of herpes simplex virus latency and the biological properties of distinct latent states. If limited virus gene expression is compatible with latency then it is important to determine whether such latent cell reservoirs are predisposed to reactivation. Should this be the case then it will be important to determine the nature, extent and function of virus gene products expressed during latent infection. These could represent novel targets for the development of antiviral drugs and novel immunotherapies to combat virus reactivation. Similarly our preliminary data showing that herpes simplex virus mutants deficient for expression of HSV encoded latency transcripts are unable to maintain a stable latent reservoir in vivo indicates that products of these RNAs may also represent valid antiviral targets.

Society; Herpesviruses are highly successful pathogens affecting the vast majority of the worlds population. The ability of these viruses to establish life-long latency and reactivate from immune individuals to disseminate infection accounts for this success. To-date the development of strategies by which to eliminate the latent reservoir from infected hosts or block the earliest stages of reactivation have proven largely intractable. Further basic research to define the molecular basis of virus latency and reactivation will facilitate the development of novel therapeutic approaches in this area. This is the long-term goal and motivation behind the proposed research.

Professional Development; The postdoctoral research associate and technician named on this application will gain expertise in molecular virology and the utilization of animal models in the study of virus infection. These skills would be highly applicable to many areas of biomedical research including the UK biotech industry.