RALPH AND SOPHIE HEINTZ LABORATORY RESEARCH

Herpesvirus Research

Work in the herpesvirus research laboratory, under the direction of Dr. Todd P. Margolis, centers around herpes simplex virus (HSV) infections of the eye. The primary focus of the laboratory is research on the cellular and molecular mechanisms that regulate the establishment and maintenance of latent neuronal infection with HSV. Ongoing research is aimed at documenting the role of neuronal gene expression in the establishment and maintenance of HSV latency, as well as the role of the HSV latency associated transcript (LAT). The ultimate goal of this work is to gain enough of an understanding about the regulation of HSV latent infection that therapeutic interventions can be devised to eliminate latent infection or prevent viral reactivation.

Establishment of Latent Infection with Herpes Simplex Virus.
T.P. Margolis, Y. Imai, K. and K. Apakupakul

The primary focus of this laboratory is to carry out research on the viral and cellular mechanisms that regulate the establishment and maintenance of latent neuronal infection with herpes simplex virus (HSV-1). Previous work carried out in this laboratory and elsewhere suggests that establishment and maintenance of HSV latent infection is heavily dependent on the specific genetic expression of the host neuron. For this reason ongoing research is aimed at further documenting the role of neuronal gene expression in the establishment and maintenance of HSV latency as well as the role of viral LAT in this process. This is being accomplished through a number of different basic strategies. The first strategy has been to use cDNA probes generated from enriched populations of A5+ and IB4+ neurons to probe murine cDNA expression arrays in order to identify host genes that are differentially expressed in these two populations. Differentially expressed neuronal genes are then assayed for their ability to activate or inhibit key regulatory viral genes, and thus influence the outcome of HSV infection (lytic vs latent). A second strategy has been to utilize knock-out mice deleted for specific elements of the immune system to determine the role that GM-CSF EGR-1, and the interferons play in the establishment of latent HSV-1 infection and its preference for A5 neurons. A third strategy, carried out in collaboration with Dr. Philip Krause at the FDA, has been to examine why HSV-1 and HSV-2 preferentially establish latent infection in different subsets of ganglionic neurons (HSV-2 is more commonly seen in KH10 neurons). Through the use of HSV-1/HSV-2 intertypic recombinants we have found that a 600 bp fragment of the viral LAT in both HSV-1 and HSV-2 clearly dictates specificity for the preferential establishment of HSV-1 latency in A5 neurons and HSV-2 latency in KH10 neurons. Additional studies have demonstrated that neither preferential access, nor differential spread of the two viruses is responsible for the differential establishment of latency by HSV-1 and HSV-2. Fourth, in collaboration with Dr. Larry Feldman at UCLA, we are designing and testing a doxycycline regulated system that makes use of constitutively expressed and inducible genes in transgenic mice for inducing expression of specific genes during viral latency in order to determine which viral proteins are required for viral reactivation from latency. The ultimate goal of our work on HSV is to gain enough of an understanding about the regulation of latent infection that therapeutic interventions can be devised to eliminate latent infection or prevent viral reactivation. Finally, we continue to pursue our studies of spontaneous reactivation of HSV-1 in the mouse trigeminal ganglion.

Confocal image of a spontaneously reactivating neuron in a latently infected mouse trigeminal ganglion. Nuclear staining (blue), neuron specific staining (red), HSV-1 specific staining (green).

The AIRE Gene and Inflammatory Eye Disease.
T.P. Margolis, M. Anderson, E. Strauss and N. McNamara

Recent studies suggest that mutations in the AIRE gene may play a role in susceptibility to systemic autoimmune disease. Furthermore, mice that have had the AIRE gene “knocked out” develop autoimmune retinitis, keratitis and dacryoadenitis. We are currently carrying out studies to determine whether mutations in the AIRE gene are risk factors for developing ocular disease in patients with Sjögren’s syndrome and HLA-B27 associated iritis. To date we have enrolled thirty study patients with HLA-B27 disease and found no mutations in the AIRE gene of these individuals. Work on a similar cohort of Sjögren’s patients is underway.

Molecular Diagnostics
T.P. Margolis, K. Apakupakul, A.V. Cevallos

In the past we developed molecular assays to aid in the diagnosis of HSV, VZV, CMV, and toxoplasmosis eye diseases and have moved these assays into the clinical laboratory at the Proctor Foundation. We are currently working on developing and testing similar assays for HHV6 and HHV7. This work is being carried out in collaboration with Dr. Jeff Cohen and the NIAID in Bethesda.