INTRODUCTION
Human gammaherpesviruses, Kaposi’s sarcoma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV) are closely linked to malignant tumors (
Kieff and Rickinson, 2001;
Moore and Chang, 2001). However, studies of KSHV and EBV are relatively limited due to their restricted host ranges and lack of permissive cell lines that can efficiently support their proliferation. Murine gammaherpesvirus 68 (MHV68) also belongs to the gamma herpesvirus subfamily and shares many similarities with KSHV and EBV, both genetically and biologically (
Efstathiou et al., 1990;
Virgin et al., 1997). MHV-68 replicates robustly in permissive cell lines and produces progeny viruses with a high titer. MHV-68 infection of mice also provides a tractable small animal model for further investigation of the mechanisms of viral infection and the pathogenesis of gammaherpesviruses (
Simas and Efstathiou, 1998).
Like all other herpesviruses, gammaherpesviruses have two life cycles: latency and lytic replication (
Roizman and Pellett, 2001b). During latency, the viral genome replicates synchronously with the host genome and is maintained in host cells in an extrachromosomal manner (
Collins et al., 2002;
Hu and Renne, 2005). During the lytic cycle, the virally encoded DNA replication proteins gather at the origin of lytic replication (
oriLyt) to initiate the replication process, and the newly synthesized viral DNAs are processed at terminal repeats and packaged into progeny virions (
Kieff and Rickinson, 2001;
Mocarski and Courcelle, 2001;
Roizman and Knipe, 2001a).
Previous studies on gammaherpesviruses reveal that both cellular and viral proteins are involved in lytic viral DNA replication. A family of transcription factors, CCAAT/enhancer binding proteins (C/EBPs), assists in the lytic genome replication of EBV and KSHV during viral reactivation (
Wu et al., 2003;
Wang et al., 2004;
Huang et al., 2006). The C/EBP family is composed of six members (C/EBPα–C/EBPζ), which all contain conserved basic-leucine zipper (bZIP) domains at their C-terminus and an activation domain at their N-terminus. Via their C-terminal DNA binding domain, they interact with DNA sequence especially CCAAT motifs in promoter regions to modulate gene expression (
Landschulz et al., 1988;
Ramji and Foka, 2002). Research on EBV and KSHV has shown that C/EBPs play a role in viral DNA replication during reactivation through interaction with the core region of the EBV and KSHV
oriLyts (
Wu et al., 2003;
Wang et al., 2004;
Huang et al., 2006).
Our group has identified two
oriLyts in the MHV-68 genome, which are located toward the right (nt. 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. 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.
RESULTS AND DISCUSSION
C/EBPs bind to MHV-68 oriLyts in vitro
We first used electrophoretic mobility shift assays (EMSAs) to test whether C/EBPs bind to the core region of MHV-68
oriLyts. Oligonucleotides were synthesized for EMSA as follows: L1 + 2 covered CCAAT boxes 1 and 2 present in the MHV-68 left
oriLyt; L3 + 4 covered CCAAT boxes 3 and 4; R1 + 2 and R3 + 4 were designed in the same manner based on the right
oriLyt (Fig. 1A). As controls, Ori-L1 and Ori-L3 were synthesized according to a previous study showing that C/EBPs bind to fragments containing paired CCAAT boxes in the KSHV
oriLyt (
Wu et al., 2003). We transfected 293T cells with expression plasmids for either FLAG-tagged C/EBPα (pHC125B) or C/EBPβ (pHC108C), and prepared nuclear extracts for EMSAs. Our result demonstrated that C/EBPα and β bind to Ori-L1 and Ori-L3, as well as the C/EBP consensus sequence (arrowhead, Fig. 1B, lanes 1, 10 and 13; Fig. 1C, lanes 1, 10 and 13). A complex with similar mobility was observed when oligonucleotides L1 + 2 and L3 + 4 were used (arrowhead, Fig. 1B, lanes 4 and 7; Fig. 1C, lanes 4 and 7), indicating that C/EBPs are able to bind to the core region of the MHV-68 left
oriLyt. To confirm the specificity of C/EBP binding, we conducted supershift and competition experiments. Addition of an antibody against the FLAG epitope successfully supershifted the complex (arrow, Fig. 1B, lanes 2, 5, 8, 11 and 14; Fig. 1C, lanes 2, 5, 8, 11 and 14), suggesting that FLAG-tagged C/EBPs are indeed part of the supershifted complex. Moreover, the intensity of the shifted bands decreased in the presence of unlabeled C/EBP consensus oligos (Fig. 1B, lanes 3, 6, 9, 12 and 15; Fig. 1C lanes 3, 6, 9, 12 and 15), further proving that the interaction between C/EBPs and L1 + 2 or L3 + 4 are specific. Likewise, C/EBPα and β also bound to the core region of the MHV-68 right
oriLyt, though with lower efficiency (Fig. 2C, lanes 3, 4, 9 and 10; and Fig. 2D, lanes 3, 4, 9 and 10). It should be noted that in addition to this complex, other bands were also detected with probe L1 + 2 or L3 + 4. This was presumably caused by non-specific binding of the probe with the nuclear extract because these bands were neither supershifted nor inhibited by competitors.
The observation that C/EBPα and β bound to the MHV-68 left and right
oriLyt core regions is in agreement with previous studies of KSHV and EBV (
Wu et al., 2003;
Huang et al., 2006). In KSHV, the left and right
oriLyts share an almost identical 1.15-kb sequence organized in opposite directions. A previous study demonstrated that each KSHV
oriLyt harbors three C/EBP binding regions, as detected by probes Ori-L1, Ori-L3, and Ori-V in EMSAs. However, the specific shifted bands representing C/EBP-DNA complexes were somewhat smeared, and non-specific shifts were also detected (
Wu et al., 2003). For comparison, we included Ori-L1 and Ori-L3 in our EMSAs. Our results demonstrated that C/EBPs bound to the CCAAT boxes in the MHV-68 left
oriLyt region (L1 + 2 and L3 + 4) with similar efficiency to KSHV Ori-L3 but with higher efficiency than KSHV Ori-L1, although non-specific bands were also detected (Fig. 1B and 1C, lanes 4, 7, 10 and 13).
C/EBPs bind to oriLyts in the MHV-68 genome during de novo infection
To examine whether C/EBPs bind to the MHV-68 oriLyt core region of the viral genome in vivo, chromatin immunoprecipitation (ChIP) assays were performed. Cells transfected with FLAG-tagged C/EBPα or β were infected with MHV-68, and DNA-protein complexes were cross-linked by formaldehyde and used for ChIP assays. The resulting DNA fragments were amplified with specific primers toward the MHV-68 left or right oriLyt core region. PCR products of the expected length were detected when the anti-FLAG antibody was used for immunoprecipitation (Fig. 3, top 2 panels, lanes 3 and 6) but not when control IgG was used (Fig. 3, top 2 panels, lanes 2 and 5). Moreover, PCR reactions using primers specific for the cellular beta-actin coding sequence or the intron region of the MHV-68 rta gene, which do not contain C/EBP binding sites, yielded no products (Fig. 3, bottom 2 panels, lanes 2, 3, 5 and 6). These results demonstrate that both C/EBPα and β are able to bind to the oriLyt core region in the MHV-68 genome during de novo infection.
Two CCAAT boxes in the MHV-68 left oriLyt are necessary for C/EBP binding
Previous functional studies demonstrate that CCAAT boxes are crucial
cis-elements for
oriLyt-dependent DNA replication during KSHV reactivation (
Wang et al., 2004). 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
cis-elements 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. 1A). The oligonucleotides were biotin-labeled and used for EMSAs as described above. Anti-FLAG antibody was again introduced to confirm the specificity of binding. Our results demonstrate that both C/EBPα and C/EBPβ were able to bind to L1 + m2 and L3 + m4 with similar affinity compared to wild type sequences, whereas the binding affinities to Lm1 + 2 and Lm3 + 4 were much reduced, suggesting that CCAAT box 1 and especially box 3 are important for mediating binding of C/EBPs to the left
oriLyt (Fig. 2A and 2B). Intriguingly, the CCAAT boxes in the right
oriLyt were largely dispensable for C/EBPα or β binding when compared to those from the left
oriLyt (Fig. 2C and 2D). Together, these results suggest that CCAAT boxes 1 and 3 from the left
oriLyt play a pivotal role in the binding of C/EBPs
in vitro.
Functional C/EBPs are required for maximal oriLyt-mediated lytic DNA replication during MHV-68 de novo infection
Given the fact that C/EBPs bind to CCAAT boxes in the MHV-68
oriLyt, we next conducted a knock-down experiment to evaluate the functional significance of such interactions during
de novo MHV-68 infection. CHOP10 is a C/EBP family member that heterodimerizes with C/EBPα or β and impairs their DNA binding ability, consequently serving as a dominant negative mutant of C/EBPs. 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. The replication efficiency of pMOL was significantly impaired by CHOP10 (Fig. 4A, lanes 2, 4 and 6) compared to controls (Fig. 4A, lanes 1, 3 and 5), suggesting that C/EBPs play important roles in
oriLyt-mediated DNA replication in this plasmid system.
To further explore the contribution of C/EBPs in mediating MHV-68 lytic genome replication during de novo infection, we transfected 293T cells with pCMVHA-hCHOP10 or pCMV-HA and then infected them with MHV-68. Total cellular DNAs were harvested, and the replication of the MHV-68 genome was analyzed. Southern blotting demonstrated that the replication efficiency of the MHV-68 genome decreased by ~50% in the presence of hCHOP10 (Fig. 4B), indicating that a functional C/EBP-DNA complex is required for maximal MHV-68 lytic genome replication. The reason that the decrease of genome replication efficiency was not as large as in the plasmid replication assay may be two-fold. First, pCMV-hCHOP10 may not have been transfected into and expressed in every cell that was later infected with MHV-68. Second, although DpnI digestion can be utilized to eliminate the input plasmid DNA before Southern blotting, as done in the plasmid assay system (Fig. 4A), such manipulation is futile for viral genomes. Hence, the newly synthesized viral genomes and the input parental genomes cannot be discriminated by Southern blotting, masking the dominant negative effect of CHOP10 to certain extent (Fig. 4B).
In the present study, we demonstrated that C/EBPα and β interact with the MHV-68 oriLyt regions, both in vitro and in vivo, via binding to the CCAAT boxes. By introducing mutations into the CCAAT boxes, we found that CCAAT boxes 1 and 3 in the left oriLyt are crucial for C/EBP binding. Moreover, a “dominant negative C/EBP mutation” (i.e. CHOP10 expression) remarkably inhibited the lytic replication of MHV-68, both in a plasmid system and on the genome level, suggesting that functional C/EBPs are important for left oriLyt-mediated DNA replication of MHV-68 during de novo infection. In comparison, although the left and right KSHV oriLyts identified in a plasmid system share an almost identical 1.15-kb region, Xu et al. reported that on the genome level, the left oriLyt is sufficient to replicate the KSHV genome, whereas the right oriLyt alone fails to propagate the viral genome, indicating that functional variation may exist between the left and right oriLyt regions of gammaherpesviruses. Consistent with these results, we found that although both the left and right oriLyt regions of the MHV-68 genome contain CCAAT boxes, only boxes 1 and 3 in the left oriLyt are important for mediating C/EBPs binding.
Most studies of the lytic replication of EBV or KSHV have been performed using latently-infected cell lines due to the lack of a permissive infection system; little is known about the
de novo infection process. Therefore, although C/EBPs are reported to contribute to the lytic replication of KSHV and EBV during reactivation (
Wu et al., 2003;
Wang et al., 2004;
Huang et al., 2006), it remains to be determined whether such a scenario also takes place during
de novo infection. Fortunately, the robust replication of MHV-68 in tissue culture enabled us to evaluate the role of C/EBPs during
de novo infection for the first time and helps in a more comprehensive understanding of gammaherpesvirus lytic replication.
Nevertheless, because the mechanism of viral lytic replication is complicated, multiple trans-factors, both viral and cellular, may be involved. We previously reported that a ubiquitous cellular transcription factor, NF-Y, binds to CCAAT boxes 1, 3, and 4 in the MHV-68 left
oriLyt, both
in vitro and
in vivo, and plays an important role in mediating lytic viral genome replication during
de novo infection (
Gong et al., 2009). Together with our current findings, we hypothesize that MHV-68 may utilize NF-Y to pre-set the architecture of the
oriLyt and then employ C/EBPs to proceed with DNA replication because NF-Y and C/EBP can interact with each other (
Xu et al., 2006). It is also possible that NF-Y and C/EBP bind to the
oriLyt in a simultaneous manner and facilitate efficient lytic DNA replication. To dissect this, future investigations of the detailed mechanisms governing C/EBP’s and NF-Y’s role in MHV-68 lytic genome replication are required.
MATERIALS AND METHODS
Cell culture and viruses
BHK-21 and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Hyclone) and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin) at 37°C in the presence of 5% CO2. MHV-68 was propagated by infecting BHK-21 cells at a multiplicity of infection (MOI) of 0.05 plaque forming units (PFU)/cell. Viral titers were determined by standard plaque assays.
Plasmids
The pMOL plasmid was constructed by cloning a 1.2-kb fragment (NC_001826, nt. 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). The FLAG-tagged C/EBPα and C/EBPβ expression plasmids, pHC125B and pHC108C, were kindly provided by Dr. S. Diane Hayward (
Huang et al., 2006).
EMSAs
293T cells were transfected with 12 μg pHC125B or pHC108C. Forty-eight hours post-transfection, cells were harvested, and nuclear extracts prepared using a modified method described by Schreiber (
Schreiber et al., 1989). Oligonucleotides were prepared as described in the Biotin 3′ End DNA Labeling Kit (Pierce). DNA-protein binding reactions and EMSAs were performed according to the LightShift Chemiluminescent EMSA Kit (Pierce). For supershift experiments, 1 μL anti-FLAG antibody (Sigma) was added to DNA-protein binding reactions. For competition assays, 100-fold excess unlabeled C/EBP consensus oligonucleotide was added.
Oligonucleotides used in the EMSAs are as follows: C/EBP consensus, 5′-TGCAGATTGCGCAATCTG-3′; Ori-L1, 5′-CGCTGATTGGTTCCCGCTCTGGGCCAATCA GCA-3′; and Ori-L3, 5′-CCGAGATTGGTCGGCCGGATGGGCCAATGGCGA-3′ (
Wu et al., 2003). The sequences of L1 + 2, L3 + 4, Lm1 + 2, L1 + m2, Lm3 + 4, L3 + m4, R1 + 2, R3 + 4, Rm1 + 2, R1 + m2, Rm3 + 4, and R3 + m4 are illustrated in Fig. 1A.
ChIP
Two million 293T cells were transfected with pHC125B or pHC108C, and 24 h later, cells were infected with wild type MHV-68 at a MOI of 5. Twelve hours later, proteins were cross-linked to DNA by adding formaldehyde to a final concentration of 1%, and ChIP assays were performed according to manufacturer’s instructions (Upstate). The primer pairs used are as follows: left oriLyt, 5′-GCTATGTTTGACTTTTCGCTGTTTCG-3′ and 5′-AAGGGGATTTCCAGGTAGAGGGTCTTC-3′; right oriLyt, 5′-AGGGATCCGCCTCCCACCTG-3′ and 5′-CTCTGCCGCATCGCCTCACA-3′; RTA intron, 5′-TTTTCTCAAGGCTTCCTCGTCT-3′ and 5′-GGC ACTGTCAATTTACTGGGCT-3′; and human beta-actin coding region, 5′-GGACTTCGAGCAAGAGATGG-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′.
De novo infection-replication assays and Southern blotting
Plasmids were transfected into 293T cells in 6-well plates with jetPEI (Polyplus). Twenty-four hours post-transfection, cells were infected with wild type MHV-68 at a MOI of 0.1. When > 95% cells showed cytopathic effect (CPE) at 72–96 h post-infection, cells were harvested, and total cellular DNA was extracted. One-twelfth of the DNAs were digested overnight with DpnI and PstI to examine newly replicated plasmid DNA (or PstI alone to examine input DNA) and subjected to Southern blotting. For Southern blotting analysis, digested DNAs were separated on a 0.8% agarose gel in 1×TAE buffer. The gel was treated with 0.25 mol/L HCl, followed by alkaline denaturation and neutralization. DNAs were transferred onto a Hybond-N + membrane (Amersham Pharmacia) via capillary transfer in 10×SSC buffer and immobilized by UV-crosslinking. Southern blotting was performed using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) with a probe against the pGEM-T vector.
Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
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