an in vitro system for studying murid herpesvirus-4 latency and reactivation一个体外系统为研究鼠科动物herpesvirus-4延迟和复活.pdfVIP

An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation ¤ Janet S. May, Neil J. Bennett , Philip G. Stevenson* Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom Abstract The narrow species tropisms of Epstein-Barr Virus (EBV) and the Kaposi’s Sarcoma -associated Herpesvirus (KSHV) have made Murid Herpesvirus-4 (MuHV-4) an important tool for understanding how gammaherpesviruses colonize their hosts. However, while MuHV-4 pathogenesis studies can assign a quantitative importance to individual genes, the complexity of in vivo infection can make the underlying mechanisms hard to discern. Furthermore, the lack of good in vitro MuHV-4 latency/ reactivation systems with which to dissect mechanisms at the cellular level has made some parallels with EBV and KSHV hard to draw. Here we achieved control of the MuHV-4 lytic/latent switch in vitro by modifying the 59 untranslated region of its major lytic transactivator gene, ORF50. We terminated normal ORF50 transcripts by inserting a polyadenylation signal and transcribed ORF50 instead from a down-stream, doxycycline-inducible promoter. In this way we could establish fibroblast clones that maintained latent MuHV-4 episomes without detectable lytic replication. Productive virus reactivation was then induced with doxycycline. We used this system to show that the MuHV-4 K3 gene plays a significant role in protecting reactivating cells against CD8+ T cell recognition. Citation: May JS, Bennett NJ, Stevenson PG (2010) An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation. PLoS ONE 5(6): e11080. doi:10.1371/journal.pone.0011080 Editor: Maria G. Masucci, Karolinska Institutet, Sweden Received May 4, 2010