Identification and functional characterization of the left origin of lytic replication of murine gammaherpesvirus 68

Abstract

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.

CCAAT/enhancer binding proteins play a role in oriLyt-dependent genome replication during MHV-68 de novo infection

“…By EMSAs, the second CCAAT box of Ori-L1 is essential for C/EBP binding, whereas the first CCAAT box is dispensable (Wu et al, 2003). Our group revealed that CCAAT boxes are essential ciselements for the function of the MHV-68 left oriLyt (Gong et al, 2009). To investigate whether the CCAAT boxes in the MHV-68 oriLyt core region are also important for C/EBP binding, we designed eight oligonucleotides based on L1 + 2, L3 + 4, R1 + 2, and R3 + 4; each of them contained mutations in one CCAAT box (Fig.…”

“…We first cloned the CHOP10 sequence into pCMV-HA to generate pCMVHA-hCHOP10 and confirmed the expression of hCHOP10 by western blotting (data not shown). We then co-transfected pMOL (bearing the 1.1-kb MHV-68 left oriLyt) (Gong et al, 2009) and pCMVHA-hCHOP10 (or pCMV-HA as a vector control) into 293T cells. Twenty four hours later, we infected cells with MHV-68 to provide trans-factors required for DNA replication and prepared total cellular DNA for Southern blotting.…”

“…25,695-26,883, amplified by PCR) of MHV-68 DNA into the pGEM-T vector (Promega), as described previously (Gong et al, 2009). To generate plasmid pCMV-hCHOP10, human CHOP10 was amplified by PCR from 293Tcell cDNA and cloned into pCMV-HA (Clontech).…”

“…100,724-101,975) and left (nt. 25,695-26,883) ends of the genome (termed the right and left oriLyt, respectively) (Deng et al, 2004;Gong et al, 2009). The two oriLyts of MHV-68 share several conserved cis-elements, including two pairs of CCAAT boxes, an AT-rich palindrome, and a GC-rich repeat region.…”

“…The two oriLyts of MHV-68 share several conserved cis-elements, including two pairs of CCAAT boxes, an AT-rich palindrome, and a GC-rich repeat region. We further analyzed the left oriLyt in detail through deletion mutation and site-directed mutagenesis and found that the CCAAT boxes are indispensable for the function of the left oriLyt (Deng et al, 2004;Gong et al, 2009). In the current study, we aimed to test whether C/EBPs bind to the core region of MHV-68 oriLyt and, if so, whether such interactions play a functional role in mediating MHV-68 lytic genome replication.…”