Recurrent HSV-1 infection as a result of viral reactivation is a major cause of viral induced blindness. Our overall goal is elucidation of the underlying molecular mechanisms behind the HSV-1 latency-reactivation cycle, hopefully leading to development of a means for reducing HSV-1 reactivation and hence the incidence of HSV-1 induced corneal blindness. LAT, the only viral gene abundantly transcribed during latency enhances the reactivation phenotype by blocking apoptosis. Since no LAT protein has been reported, LAT is thought to function via a noncoding RNA. In contrast to this notion, we now have strong evidence for an anti-apoptotic LAT protein (L2) that is expressed during latency. We also have evidence for the first small LAT RNA (RNA-1) that also appears to have anti-apoptosis activity. Since LAT's anti-apoptosis activity is its most important latency related function and since L2 and RNA-1 both appear to have anti-apoptosis activity and are encoded by the functional 1st 1.5 Kb of LAT, we hypothesize that both contribute to LAT's ability to enhance the reactivation phenotype.
Our Specific Aims to pursue these novel innovative findings include: 1. Confirm the hypotheses that L2 (encoded by nts 487-669) and spliced-L2 are authentic LAT proteins with anti-apoptosis activity by: a) Humanizing the L2 ORF nt sequence without changing the L2 amino acid sequence in a plasmid expressing the functional 1st 1.5 Kb of LAT and confirming retention of the plasmid's anti-apoptosis activity;b) Expressing the humanized L2 (h-L2) ORF (60 aa) vs. the h-spliced-L2 ORF (118 aa) in plasmids with no LAT flanking sequences and confirming their anti-apoptosis activity;c) Tagging the C- terminus of L2 and spliced-L2 with myc in separate otherwise wt viruses and determining changes in their expression during acute infection vs. latent infection vs. reactivation. 2. Confirm the hypothesis that L2, spliced-L2, and RNA-1 contribute to LAT's ability to support the wt reactivation phenotype and that at least one of them exerts its main influence during reactivation rather than establishment of latency by: a) Constructing knock out mutants and confirming their reduced reactivation phenotype;b) Constructing mutants that express just h-L2, h-spliced-L2, or RNA-1 and confirming that they have an increased reactivation phenotype compared to LAT(-) viruses;c) Blocking expression/function of L2, spliced-L2, or RNA-1 during establishment of latency vs. reactivation, and determining the effect on the reactivation phenotype. 3. Test the hypothesis that L2, spliced-L2, and RNA-1 interfere with apoptosis via different mechanisms by: a) Confirming that they have differing abilities to block apoptosis induced by different agents/methods;b) Confirming that they block different steps in the extrinsic Fas pathway;c) Determining if they bind to different apoptotic factors, suggesting that they block function of the bound apoptotic factor.
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 application is directed at understanding the molecular mechanisms by which the HSV-1 LAT gene enhances the virus'reactivation phenotype. The knowledge gained here will be critical for the longer term goal of developing efficacious clinical interventions that target LAT and thereby decrease the incidence and/or severity of recurrent ocular herpetic disease.
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