Using simian virus 40 (SV40) as a model for infections by DNA viruses, the proposed research will contribute to our understanding of the relationship between chromatin remodeling through histone modification and the activation of an incoming viral genome for early transcription.
The specific aims of the research are: 1. To determine the genomic organization of nucleosomes containing modified histones in SV40 chromosomes early in infection; 2. To determine if SV40 chromosomes containing modified histones serve as substrates for regulatory factors; and 3. To determine whether individual regulatory sequences or combinations of regulatory sequences can label chromatin with a specific """"""""histone code."""""""" SV40 chromatin from cells infected with wild-type strain 776 virus or viral constructs containing various combinations of potential regulatory sequences introduced into a chromatin reporter virus which we have developed will be isolated at 30 minutes or 3 hrs post-infection. The SV40 chromatin will be analyzed either as intact chromosomes or following sonication or micrococcal nuclease digestion to generate suitably sized fragments of chromatin by a combination of chromatin immunoprecipitation (ChIP) assays and competitive, multiplex, or real-time PCR. By analyzing mononucleosomes generated by micrococcal nuclease digestion, the specific location of chromatin containing modified histones on the SV40 genome will be determined. Antibodies will be used which recognize hyperacetylated histones, methylated histones, and viral structural proteins, all of which we have shown to be associated with SV40 chromosomes during this time period in preliminary studies, and antibodies to other forms of histone modifications and transcription factors known to be associated with early transcription. Many DMA viruses, although not SV40, are significant human pathogens. Understanding the events occurring during the first hours of infection by DNA viruses may ultimately lead to therapeutic interventions designed to limit the horizontal spread of these viral infections. ? ? ?
