TRANSCRIPTION FACTORS IN THE REGULATION OF HSV-1 LATENCY

University Of Pennsylvania, Philadelphia PA

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Abstract

Description (provided by applicant) During latency, HSV-1 replication is blocked at the level of viral Immediate Early (IE) and Early (E) gene expression. This restriction of replication may be caused by the differentiated state of neurons, in which cellular factors required for viral gene expression are down-regulated, or transcriptional repressors are present. In addition, chromatin structure is generally repressive to gene activity. There is evidence that, in the latent state, the HSV-1 genome may be generally silenced because of nucleosomal association. Our previous work has shown that reactivation signals induce certain novel cellular genes, which may in turn up-regulate HSV IE and E genes, culminating in reactivation of HSV-4 from latency. The long-term goal of the proposed research is to define the functional role of the identified cellular factors, and their interplay with viral factors and alteration of chromatin structure, in causing reactivation of HSV-1 from latency in neurons. Our general methods will be to evaluate the role of cellular factors in the induction of the HSV-1 gene activity during viral infection, and during establishment of viral latency and reactivation. Our specific methods will be, first, to use quantitative RNA and protein detection analyses, as well as histology studies, to examine the correlation between up- or down-regulation of cellular genes, and reactivation of HSV1 in mouse trigeminal ganglia. Second, we will examine the functional interaction of the candidate factors with the viral genome, to obtain direct evidence that these factors influence HSV-1. Third, we will test the effect of the identified genes in HSV-1 reactivation using two model reactivation systems, murine trigeminal ganglia (TG) and NGF-differentiated PC12/HepG2 cell co-cultivation, to determine whether the candidate genes have a causal role in reactivating the viral genome. We will also determine whether mouse null mutants in these cellular genes alter HSV-1 latency or reactivation. Finally, we will use multiple approaches to study possible chromatin changes that occur during reactivation. We will focus on a transcriptional co-activator, called Bcl-3, that is up-regulated more than 5-fold in neurons during re-activation of HSV-1. Bcl-3 interacts with a DNA binding protein NFKB, and a histone modifying factor Tip60. In addition, there exist putative NFKB binding sites in the IE gene, IPCO, expression from which is required for HSV-1 reactivation from latency. We hypothesize that the protein complex NFKB/Bcl-3/Tip6O binds to the ICPO gene promoter to alter its chromatin structure and to initiate RNA synthesis, and then ICPO protein initiates further HSV-1 gene expression that is critical for reactivation from latency.