Murine gammaherpesvirus 68 (MHV-68),a member of the gammaherpesvirus family,replicates robustly in permissive cell lines and is able to infect laboratory mice.MHV-68 has emerged as a model for studying the basic aspects of viral replication and host-virus interactions of its human counterparts.Herpesvirus genome replication is mediated through a cis-element in the viral genome called the origin of lytic replication (oriLyt).A family of transcription factors,CCAAT/enhancer binding proteins (C/EBPs),assists in oriLyt-mediated DNA replication during gammaherpesvirus reactivation.In this study,we examined the role of C/EBPs in gammaherpesvirus DNA replication during de novo infection,using MHV-68 as a model.We found that C/EBP α and β bind to the CCAAT boxes in the MHV-68 oriLyt core region both in vitro and in vivo,as demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay.A dominant negative form of C/EBPs significantly impaired the lytic replication efficiency of MHV-68 on both the plasmid and genome levels in a replication assay,indicating that functional C/EBPs are required for maximal MHV-68 genome DNA replication.Collectively,our data demonstrate that C/EBPs interact with the oriLyt core region and play an important role in MHV-68 lytic DNA replication during de novo infection.