Molecular imaging of human gammaherpesvirus lytic gene expression in vivo

Molecular imaging is a useful tool for monitoring gene expression in vivo. The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with a number of malignancies. Developing techniques for detecting and quantifying human gammaherpesvirus lytic infection in vivo would help us to evaluate tumor location and tumor cell response to therapeutic agents better. I first demonstrate that the EBV thymidine kinase (TK) can be used as a reporter gene for in vivo functional imaging with the [125I]-FIAU reporter probe. In vitro cell uptake and biodistribution studies show that uptake and retention of FIAU is highly specific for cells that express the EBV TK. Planar gamma imaging of tumor-bearing SCID mice shows high levels of [125I]-FIAU-derived radioactivity localized in EBV TK positive tumors but not in tissues that do not express the EBV TK. Some bladder, stomach and thyroid accumulation were also seen due to the normal metabolism of [ 125I]-FIAU. Since the EBV TK is not normally expressed in tumor cells, pharmacologic agents are needed to induce EBV lytic gene expression. I show in a series of western blots, real time reverse transcription PCR, and methylation specific PCR analyses that inducing EBV lytic infection with histone deacetylase inhibitors in certain cell lines requires pre-treatment with a DNA methyltransferase inhibitor. Reversal of the dense CpG methylation of promoter regions such as the Zta promoter in these cell lines leads to a dose- and time-dependent increase in lytic antigen expression. I also developed a high performance liquid chromatography-based assay utilizing the activity of the EBV TK to quantify lytic induction. Finally, I show that imaging techniques can be applied to detecting lytic gene induction in vivo. Lytic infection in EBV- and KSHV-positive cells, induced in vitro with azadC and butyrate, respectively, show higher uptake of [14C]-FIAU than in uninduced cells or cells that do not harbor the viruses. Planar gamma imaging with [125I]-FIAU was able to detect EBV and KSHV kinase activity in tumor-bearing SCID mice. These results indicate that molecular imaging can be a highly useful tool in monitoring human gammaherpesvirus lytic infection noninvasively and repeatedly in vivo.