An in vitro system for studying murid herpesvirus-4 latency and reactivation.
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 5' 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.
May
Janet S
JS
Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK.
Bennett
Neil J
NJ
Stevenson
Philip G
PG
D000818:Animals; D002460:Cell Line; D004279:DNA, Viral; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D016366:Open Reading Frames; D018144:Rhadinovirus / Q000235:genetics / Q000502:physiology*; D014775:Virus Activation; D017735:Virus Latency
D004279:DNA, Viral