Recurrent herpes, due to reactivation of herpes simplex virus type 1 (HSV-1) from latency in the trigeminal ganglia (TG) can cause herpes simplex encephalitis (HSE) and herpes stromal keratitis (HSK). HSE is the most common cause of sporadic lethal encephalitis in immune competent individuals. Similarly, HSK is the most common cause of corneal blindness due to an infectious agent. The HSV-1 latency associated transcript (LAT), the only viral gene abundantly expressed during latency, is involved in both the virus'neurovirulence and its reactivation phenotype, since various mutants within this genomic locus affect one or the other or both. Our long term goal is to understand the molecular mechanisms behind HSV-1 neurovirulence, latency, and reactivation, which should lead to improved clinical therapies directed against herpes encephalitis and corneal blindness. Recently, 8 "LAT" microRNAs (miRNAs) were reported. Most overlap important viral genes known to affect neurovirulence and/or reactivation. In addition, some of the miRNAs affect expression of these genes. We hypothesize that LAT miRNAs influence neurovirulence and/or the reactivation phenotype. Testing this hypothesis requires construction of LAT miRNA KO mutants, which is complicated by the fact that the miRNA sequences overlap critical HSV-1 genes. Simple deletion of the miRNA sequences would disrupt these viral genes and significantly impact viral functions unrelated to the miRNAs. By making use of codon redundancy to not alter the amino acid sequence of an overlapping gene, we changed 21 of the 75 nts in one of the precursor miRNAs (the H2 miRNA). This was done on both a wt (LAT(+)) and a LAT(-) genomic background. The H2 KO mutants expressed no H2 miRNA. In preliminary studies both H2 KO mutants had increased neurovirulence in mice, and the LAT(-) H2 KO mutant also had increased reactivation. Reactivation results for the wt-H2 KO are not yet available. These preliminary studies strongly suggest that the H2 miRNA is normally involved in decreasing HSV-1 neurovirulence and reactivation. We also found that the LAT miRNAs do not require the LAT promoter for their expression, suggesting that one or more LAT miRNAs may account for LAT promoter deletion mutants'ability to still support some reactivation. If, as expected, we find that one or more of the LAT miRNAs significantly affects neurovirulence (or the spontaneous reactivation phenotype), manipulation of those miRNAs would be a useful target for future clinical interventions.
Our Specific Aims i nclude: 1) Test the hypothesis that LA miRNAs influence HSV-1 neurovirulence by constructing LAT miRNA KO mutants and analyzing them in vivo. 2) Test the hypothesis that the LAT miRNAs influence the HSV-1 reactivation phenotype. 3) Test the hypothesis that the mechanism(s) by which LAT miRNAs influence neurovirulence and/or reactivation is to alter expression of viral genes involved in neurovirulence and reactivation.

Public Health Relevance

Recurrent herpes simplex virus type 1 (HSV-1) can cause herpes simplex encephalitis (HSE), the most common cause of sporadic lethal encephalitis in immune competent individuals, and herpes stromal keratitis (HSK), the most common cause of corneal blindness due to an infectious agent in developed countries. We have strong preliminary results showing that at least one of the newly discovered HSV-1 LAT miRNAs acts to increase HSV-1 neurovirulence and also alters virus reactivation. This proposal is directed at confirming this finding and determining if other HSV-1 LAT miRNAs also alter HSV-1 neurovirulence and/or spontaneous reactivation, and if so investigating the mechanism(s). The knowledge gained here will be critical for the longer term goal of developing efficacious therapeutic clinical interventins to prevent or decrease herpes simplex encephalitis and herpes stromal keratitis. page 1of 1

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56AI098985-01A1
Application #
8730998
Study Section
Virology - B Study Section (VIRB)
Program Officer
Challberg, Mark D
Project Start
2013-09-13
Project End
2014-08-31
Budget Start
2013-09-13
Budget End
2014-08-31
Support Year
1
Fiscal Year
2013
Total Cost
$420,596
Indirect Cost
$147,629
Name
University of California Irvine
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697