Human cytomegalovirus (HCMV) infects over 70 percent of the population worldwide and like other herpesviruses, it establishes a lifelong latent infection that upon reactivation can have devastating consequences in the very young, very old, or immunosuppressed. Recent research has demonstrated possible causal roles for HCMV in atherosclerosis, autoimmunity and tumorigenesis. In vitro studies have shown that certain HCMV proteins are mutagenic and that specific breaks in chromosome 1 are induced when fibroblasts are infected during S-phase. S-phase infected fibroblasts undergo mitosis and divide, and therefore it has been hypothesized that viral episome segregation during mitosis could result in one of the daughter cells lacking the HCMV-induced block to cell cycle progression and permitting the genetic damage to propagate. Further, in contrast to its interaction with fibroblasts, HCMV replicates in some endothelial cells without blocking cell cycle progression, providing another opportunity for propagation of virus-induced damage to the cellular genome. The overall objective of this application is to test the hypothesis that HCMV-induced damage to the genome of a host cell can be propagated to progeny cells, potentially dysregulating cell growth.
Aim 1 : To characterize specific HCMV-induced genetic damage in human cell types in which cell cycle progression is not inhibited by HCMV, specifically, aortic endothelial cells.
Aim 2 : To investigate and document the propagation of HCMV-induced genetic damage by (1) developing a """"""""tagging"""""""" method to identify cells infected with HCMV or daughter cells thereof, irrespective of viral genome or gene product maintenance or concentration and (2) isolating cells identified to have lost viral genomes for further analysis of replication ability, growth kinetics and genetic damage. Cells will be cloned to encode a mCherry sequence that is preceded by a loxP-flanked STOP sequence such that infection with cre-encoded HCMV will induce cleavage of the STOP sequence and mCherry will be expressed.
This application seeks to document HCMV's ability to induce and propagate genetic damage in human cells, which may lead to cancer development and other diseases. ? ? ?
