Identification and functional characterization of the left origin of lytic replication of murine gammaherpesvirus 68.
Murine gammaherpesvirus 68 (MHV-68) replicates robustly in cell culture, providing a model for studying viral genome replication during de novo infection of tumor-associated herpesviruses. We have previously identified a 1.25-kb origin of lytic replication (oriLyt) for MHV-68. To further investigate the molecular mechanism of viral genome replication, we first fine-mapped essential cis-elements from this oriLyt fragment using a transposon-mediated high-density mutagenesis method. The result provided information for us to identify a second oriLyt located towards the left end of MHV-68 genome using a de novo infection-replication assay. We further characterized this left oriLyt by scanning deletion analysis and site-directed mutations, and showed that several CCAAT motifs are essential for oriLyt function, whereas an AT-rich region enhances replication. However, GC-rich repeats are not important cis-element. Moreover, we identified a cellular transcription factor, NF-Y, which binds to CCAAT boxes in EMSA and associates with oriLyt in ChIP assay. Using a dominant negative expression plasmid, we demonstrated that NF-Y plays an important role in mediating MHV-68 genome replication during de novo infection.
Gong
Danyang
D
Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, P.R. China.
Qi
Jing
J
Arumugaswami
Vaithilingaraja
V
Sun
Ren
R
Deng
Hongyu
H
D000818:Animals; D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D016679:Genome, Viral; D006566:Herpesviridae Infections / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D050436:Regulatory Elements, Transcriptional; D018741:Replication Origin; D018144:Rhadinovirus / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D014412:Tumor Virus Infections / Q000821:virology; D014779:Virus Replication
D023081:CCAAT-Binding Factor; D004279:DNA, Viral