Recurrent ocular herpes simplex virus type 1 (HSV-1), due to reactivation of HSV-1 from latency, is a major cause of viral induced corneal blindness. Our overall goal is the elucidation of the underlying molecular mechanisms behind the HSV-1 latency-reactivation cycle. This should lead to development of a means for reducing HSV-1 reactivation and herpetic corneal blindness. The HSV-1 LAT (latency associated transcript) gene is required for wild type (wt) spontaneous reactivation. LAT promoter knock out (KO) mutants reactivate at ~30% the rate of wt virus. Recently, 6 LAT miRNAs were reported. Three overlap the ICP0 gene in an antisense direction. Three overlap or are near the ICP34.5 gene, also in an antisense direction. We surprisingly found that LAT promoter KO mutants express each LAT miRNA at levels similar to wt virus, even though these KO mutants do not express significant amounts of large LAT RNAs. This suggests that the residual 30% of wt reactivation seen with LAT promoter KO mutants may be due to one or more LAT miRNA. Thus, an important GAP in knowledge is Do one or more of these LAT miRNAs significantly contribute to the spontaneous reactivation phenotype? To rigorously answer this question, LAT miRNA mutants must be constructed and analyzed in vivo. This is the overall goal of this proposal.
Our Specific Aims i nclude: 1. Test the hypothesis that the 3 LAT miRNAs that overlap ICP0 (H2, H7, H8) contribute to LAT s ability to support the wt reactivation phenotype. The spontaneous reactivation phenotype of LAT/ICP0 miRNA KO mutants, made using two different innovative approaches that do not alter the amino acid sequence of the important overlapping ICP0 open reading frame (ORF), will be determined in our rabbit ocular model. If, as expected, the spontaneous reactivation phenotype is reduced, it would confirm a role for one or more of these LAT miRNAs in the latency-reactivation cycle. Mutants that KO individual LAT/ICP0 miRNAs would then be constructed and analyzed to determine the contribution of each miRNA. If the LAT miRNA KO mutants have decreased establishment and/or maintenance of latency, it would indicate that these miRNAs normally increase establishment and/or maintenance of latency. This is a commonly proposed mechanism by which LAT could increase spontaneous reactivation. If the LAT miRNA mutants have increased levels of ICP0, ICP34.5, and/or ICP4 viral gene products, it would indicate that these miRNAs normally decrease expression of these viral genes. This is a commonly proposed mechanism by which LAT could increase establishment and/or maintenance of latency, leading to increased spontaneous reactivation. 2. Test the hypothesis that the 3 LAT miRNAs that overlap (or are near) ICP34.5 (H3, H4, H5) contribute to LAT s ability to support the wt spontaneous reactivation phenotype. The approach and analyses are similar to those in aim 1. In addition, a mutant knocked out for all 6 LAT miRNAs (Δ6KO) will also be constructed and analyzed as above.
In the US recurrent ocular herpes simplex virus type 1 (HSV-1) infection is the leading cause of corneal blindness due to an infectious agent, making ocular HSV-1 a clinically important problem. This proposal is directed at determining if the newly discovered HSV-1 LAT miRNAs enhance the virus? reactivation phenotype and if so investigating the mechanism(s). The knowledge gained here will be critical for the longer term goal of developing efficacious therapeutic clinical interventions to decrease HSV-1 spontaneous reactivation thus decreasing the incidence and/or severity of recurrent ocular herpetic disease.
