PDTC prevents reactivation of latent cytomegalovirus both in vivo and in vitro

Abstract

Reactivation of cytomegalovirus (CMV) from latency results in significant morbidity and mortality in immunocompromised patients. Drug-related toxicity and viral gene mutations leading to resistant virus highlight the need for alternative therapies ideally aimed at the initial steps of the reactivation process. Immediate Early (IE) gene expression is an important first step in CMV reactivation. Our laboratory has focused on the molecular mechanisms of the initial events that trigger reactivation from latency. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant that has been reported to inhibit NFκB in several in vitro systems. We have previously shown using a transgenic in vivo system that PDTC inhibits TNF-induced expression of Human CMV Immediate Early (IE) promoter transgene expression. Our aim was to fully characterize the kinetics of gene expression of an in vitro model of Murine CMV reactivation that would lend itself to experimental manipulation, and to determine if PDTC could inhibit reactivation in such a model. Methods: Spleen explants from latent Balb/c mice were cultured, and harvested at various time points. For PDTC studies, spleens were divided into 2 halves [1/2 with no drug (control), and the other 12 with PDTC]. 3 groups were studied with concentrations 20 μM, 50 μM, and 100 μM. Spleens were harvested at day 16. RT-PCR and Southern Blot for expression of IE-1, IE-3, E-1 (Early), gB (Late) and β-actin gene expression were performed. Infectious virus was assessed by plaque assays. Results: IE-1 and IE-3 gene expression was detected between days 8 and 10. E-1 was detected at days 12 to 14, and gB at day 16. Infectious virus was recovered at day 17. PDTC inhibited reactivation, and this inhibition was concentration dependent, occurring in the presence of 100 μM PDTC, and not at lower concentrations. IE, E and Late gene expression was absent and no infectious virus was recovered. Conclusions: PDTC prevents reactivation of MCMV in an in vitro model of latency. These data are consistent with our previous data utilizing transgenic mice and suggest that TNF induced NFκB activation may play an essential role in CMV reactivation from latency. Finally, these data support the use of this in vitro model as a relevant tool to study the molecular mechanisms of viral reactivation.