The mechanism of viral gene silencing during HSV latency is not understood. While transcription of the viral IE genes is silenced, an RNA referred to as latency associated transcript (LAT) is transcribed. LAT is considered a stable intron. Viral protein associated with the LAT transcription unit has not been identified. This application describes the use of HSV mutants to investigate HSV gene expression during latency. Mutant viruses with as many as three IE genes have been deleted. These IE genes are ICPO, 4, 22, and 27.
The specific aims of this proposal are as follows: (i) To determine the genetic basis of IE gene promoter silencing and its role in the establishment of latency. (ii) To characterize cis-acting elements associated with the ICPO promoter during latency or reactivation. (iii) To determine the role of the LAT intron in silencing ICPO expression (iv) To characterize cis-acting elements associated with two LAT promoters designated LAP1 and LAP2. (v) To determine the role of chromatin and DNA methylation in promoter silencing during latency. During infection of neurons with IE triple mutants of HSV, the viral ICPO promoter remains active for 2 to 4 weeks in contrast to wild type HSV. Since ICP4 deleted mutants still silence ICPO transcription, the applicant proposes that the ICP27 and ICP22 viral gene products have a role in silencing ICPO promoter activity. To further investigate these observations, promoter switching experiments whereby the ICPO promoter is substituted by either the ICP4, 27, or 47 promoters is proposed. The mutant viruses will be inoculated into the hippocampus of rat brain and the activity of the promoters relative to the number of viral genome equivalents will be determined by RT-PCR. Mutant viruses with the ICPO gene substituted by a reported gene (lacZ) will be analyzed to determine if the ICPO gene product autoregulates its own promoter. The effects of the level of ICP4, 27, and 22 gene expression on ICPO gene expression will be determined by using a regulation system for gene expression consisting of a Gal4 DNA binding domain-VP16-RU486 fusion described by the O'Malley lab that is induced by the hormone progesterone. In the second specific aim, the cis-acting elements in the ICPO promoter that are required for escape from silencing and reactivation from latency will be investigated. Site-directed mutagenesis of potential cis-acting elements and repeat sequences, 5' and 3' truncations and internal deletions will be used to analyze the promoter. The putative cis-acting elements in the ICPO intron will also be analyzed. Latency will be established by the corneal scarification method using mice and the number of latent genomes in the trigeminal ganglia will be determined by quantitative PCR. The applicant proposes that the transcription factor E2F may have a role in ICPO promoter activity. In the third specific aim, the role of the LAT RNA in regulating the level of ICPO RNA will be analyzed. Since the two viral RNAs have complementary sequences, the LAT RNA may target the ICPO RNA for degradation. Mutant viruses that fail to express the 2 kb LAT RNA may fail to downregulate ICPO RNA levels whereas over expression of the 2 kb LAT RNA may efficiently downregulate. To extend this line of research, the applicant proposes to use a yeast system with selectable markers and reporter genes. The yeast system could be used to determine the regions of the LAT RNA required for hybridization to ICPO RNA and, subsequent destruction of ICPO RNA. In the fourth specific aim, the functional elements of the two latency active promoters, LAP1 and LAP2, will be subjected to site-directed mutagenesis. Both common cis-acting sites and brain specific cis-acting sites will be mutagenized. An interesting approach will be to use transcription factor Brn knock-out mice to determine the effects of the brain specific transcription factors and cis-acting elements. In the fifth specific aim, the viral chromatin and the methylation state of the viral DNA will be analyzed with an emphasis on the ICPO and LAP regions. Chromatin arrangement and/or DNA methylation may have a role in silencing IE gene transcription. The differences in the chromatin profile or methylation profiles between wild type virus and IE deletion virus will be determined. Differences in nucleosome arrangement will be analyzed by a nuclease sensitivity assay. Differences in the CpG sequence methylation will be analyzed.
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