Several lines of evidence indicate that rearranged EBV DNA sequences in the HR-1 strain are responsible for the disruption of latency. These regions are designated """"""""het"""""""" (for heterogeneous) DNA. A 36 Kbp stretch of het DNA has been cloned as two EcoRI fragments of 20 and 16 Kbp. The 20 Kbp piece has an arrangement similar to standard EBV DNA except that sequences homologous to BamHI M and BamHI B' which are not normally contiguous are found linked at the left end of the fragment. The 16 Kbp piece shows duplicated EBV DNA in a palindromic structure. Within this palindrome are several rearrangements of EBV DNA, including sequences of BamHI S linked to sequences of BamHI c', and sequences of BamHI W linked to sequences of BamHI d. By expression of the two EcoRI het DNA fragments and subfragments in COS-1 cells at least 5 antigens are identified. At least three antigens are found on the palindromic EcoRI het 16, two of which are represented by 35 KD and 45 KD polypeptides detected by Western immunoblots. This proposal addresses the following specific problems. First, the genome containing het DNA will be mapped by restriction mapping and homology to standard EBV DNA by Southern blotting. The extent of duplication and the exactness of duplication will be determined. This information will yield a physical map of the het DNA genome. Second, the manner in which these genes are maintained, either on a separate molecule of DNA or covalently linked to the standard EBV episome, will be studied by density gradient centrifugation and electron microscopy. If het genes are maintained on a separate molecule of DNA then determination of whether this DNA is packaged separately from standard EBV DNA will be made. Using recombinant DNA techniques and gene transfer the het genes will be expressed in a variety of eukaryotic cells. By Western immunoblotting the protein products encoded by these genes will be determined. The activity of these genes when transferred into lymphocytes will pinpoint their biologic role in the disruption of latency. The het DNA genes provide a unique opportunity to study the genetic and molecular cascade of events responsible for the disruption of latency. These studies will provide a basis of understanding the biochemical events which mediate transition from latency to active viral replication.
