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Short Communication |
1 Transplant Division, Department of Surgery, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA
2 Department of Microbiology and Immunology, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA
3 Department of Surgery, University of Michigan Medical School, Ann Arbor, MI, USA
Correspondence
Michael Abecassis
mabecass{at}nmh.org
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ABSTRACT |
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; Kurz et al., 1999; Reeves et al., 2005). ie gene expression is controlled by the major immediate-early promoter/enhancer (MIEP/E) (Dorsch-Hasler et al., 1985). Activation of transcription factors which bind to the MIEP/E (Mocarski & Courcell, 2001) is likely to play an important role in reactivation of ie gene expression. In addition, recent studies indicate that epigenetic factors which control access of the chromatin to transcription factors are also important in controlling CMV latency and reactivation (Meier, 2001; Reeves et al., 2005). We have used a spleen explant model for reactivation of virus (Jordan & Mar, 1982) to further investigate the regulation of murine CMV (MCMV) latency and reactivation.
The CMV ie genes are the first set of genes expressed during productive infection and encode transcriptional transactivator proteins whose expression is required for progression to later stages of virus replication (Mocarski & Courcell, 2001). In spleens, of which approximately 90 % were negative for ie gene expression prior to explant, induction of ie gene expression generally did not occur until 9–10 days in culture (Fig. 1). Release of infectious virus in the medium was rarely detectable until approximately 12 days after explant (Fig. 2). CMV is a slowly replicating virus and plaques are detectable 3–5 days after infection of permissive fibroblasts in vitro. These studies suggest that induction of ie gene expression is the rate-determining step in reactivation and that, once induction of ie gene expression has occurred, reactivation recapitulates productive infection in vitro.
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). To determine whether TNF is required for reactivation ex vivo, we infected mice deficient in both TNF receptors (TNFR) (B6;129S-Tnfrsf1atm1ImxTnfrsf1btm1Imx mice, The Jackson Laboratory) with MCMV and tested the ability of the virus to reactivate from latency in explants. Because the TNFR-deficient mice were generated on a B6;129S background and B6 mice are resistant to MCMV infection due to NK cell recognition of the m157 protein, we infected TNFR-deficient mice with the 
m157 strain of MCMV (Bubic et al., 2004). No differences were observed in the ability of this virus to replicate in wild-type (WT) or TNFR-deficient mice during acute infection (data not shown). Spleens from three WT or TNFR-deficient mice latently infected with m157 were explanted and monitored for the production of virus. No difference in the ability of MCMV to reactivate from explants of WT or TNFR-deficient mice was observed (data not shown).
Recent studies with human CMV-infected cells indicate that changes in histone modifications are associated with reactivation from latency (Meier, 2001; Reeves et al., 2005). Activation of genes silenced through epigenetic mechanisms has been achieved by incubation with 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of de novo DNA methylation, and/or with the histone deacetylase inhibitor trichostatin A (TSA) (Chen et al., 2001; El Kharroubi et al., 2001; Grassi et al., 2003; Magdinier & Wolffe, 2001; Masucci et al., 1989). We therefore investigated the effect of adding TSA, 5-aza-dC or both to cultures of spleen explants from latently infected mice (Fig. 2a
, lanes E). In this and additional experiments, induction of ie gene expression was observed in 1 of 21 explants cultured for 3 days without addition of drug and 1 of 6 explants cultured with TSA alone (
2 analysis, not significant). Incubation for 3 days in the presence of 5-aza-dC alone or the combination of 5-aza-dC and TSA induced ie gene expression in 2 of 6 and 12 of 15 of the cultures, respectively. The effect of 5-aza-dC alone is statistically significant (2 analysis, P<0.05), and the effect of TSA and 5-aza-dC together is greater than that of 5-aza-dC alone (2 analysis, P<0.05). These studies demonstrate that 5-aza-dC can induce ie gene expression and that 5-aza-dC and TSA act synergistically to enhance transcriptional reactivation of ie gene expression.
The kinetics of virus production in medium from explants of nine mice treated with or without 5-aza-dC and TSA was analysed at 7, 10 and 12 days post-explant (Fig. 2c). Consistent with the observation that ie gene expression is not detectable until 9–10 days post-explant (Fig. 1), virus was rarely detectable prior to 10 days in untreated cultures, but increased exponentially starting at 12 days post-explant. Treatment of explants with 5-aza-dC and TSA caused a statistically significant increase in virus production over untreated controls at 7 and 10 days post-explant (t-test, P<0.05). Thus, treatment of explants with 5-aza-dC and TSA results in more rapid reactivation of both ie gene expression and virus production.
Analysis of the sequence of CMV DNAs shows a marked suppression in the frequency of CpG dinucleotides in the ie region, suggesting the presence of DNA methylation and its associated transcriptional silencing (Herman & Baylin, 2003; Honess et al., 1989). In addition, the observation that treatment of explants with 5-aza-dC was sufficient to induce transcriptional reactivation of ie gene expression suggested that latency might be controlled in part through DNA methylation-induced silencing of ie gene expression. We therefore looked directly for evidence of methylation of ie-1 DNA. The greatest deficit in the ratio of observed to expected CpGs in MCMV DNA occurs within exon 4 of the ie-1 gene and in the enhancer region (Fig. 3a, b). Methylation of coding regions does not interfere with transcription (Jones, 1999). We therefore used the bisulfite modification method (Clark et al., 1994) to analyse methylation of CpG dinucleotides in the promoter/enhancer region spanning nt –251 to +42 relative to the ie-1 transcription start site in DNAs from organs of nine latently infected mice (Fig. 3b). Bisulfite treatment converts unmethylated C to U, which are converted to T after PCR amplification. Methylated Cs are protected from conversion. Bisulfite-modified DNAs were amplified by strand-specific nested PCR (Herman et al., 1996), cloned and sequenced. Methylated CpGs remain as CpG, while unmethylated CpGs are converted to TpG. Because incomplete conversion of Cs will result in a false positive, conversion of Cs in non-CpGs is used as an internal control for completeness of the reaction. Only clones in which >95 % of the Cs in non-CpGs are converted were analysed. RNA was isolated from the same organs and analysed for expression of ie transcripts to ensure that the mice were negative for ie gene expression. In many cases, MCMV DNA could not be amplified after bisulfite treatment because the DNA copy number is very low in MCMV-latent mice, and bisulfite treatment results in substantial degradation of the DNA (Raizis et al., 1995). However, we were able to analyse 3–12 clones of 10 independent PCR from DNA isolated from various organs of nine mice. One hundred per cent of the C residues in CpG dinucleotides were converted to T residues in clones in which more than 95 % of the Cs in non-CpGs were converted to T. As positive controls, we analysed four of these DNAs for methylation of the Ant4 promoter, which is highly methylated in adult mouse organs (Rodic et al., 2005). As expected, CpGs in the Ant4 promoter were protected from conversion in bisulfite-modified DNA. These results indicate that CpG dinucleotides in the ie promoter/enhancer region are not methylated in latently infected mice.
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) (Fig. 3c). Since partial digestion of the DNA will result in a false positive, and since the DNA must be amplified by nested PCR in order to detect low-copy-number MCMV DNA in latently infected tissues, it is essential to achieve complete digestion of the DNA. DNAs were therefore digested with a large excess of enzyme, precipitated and redigested prior to amplification. As a positive control to ensure that MCMV DNA could still be amplified after these manipulations, an aliquot of the digested DNA was amplified with primers from regions of MCMV DNA which did not contain these restriction sites (lanes E and I). Undigested DNA was also analysed in parallel as a positive control (lanes P+). In cases where these sites occurred in the Ant4 promoter (HpaII and BstUI), we also amplified DNA from this region as a positive control to demonstrate detection of methylated DNA (lanes A). Although the DNA could be amplified in positive controls, none of the DNAs subjected to digestion with these enzymes could be amplified with promoter-specific primers (lanes P). These data confirm the results of our bisulfite analysis, which demonstrate that MIEP/E DNA is not methylated in latently infected mice.
In addition, we investigated methylation of the intron between exons 1 and 2 and in the region encoding exons 2 and 3 by methylation-sensitive restriction enzyme analysis. No methylation of CpGs in the MluI, SacII or HhaI sites in intron 1 or the BsaAI, HincII or XhoI sites in exons 2 and 3 was observed (Fig. 3d, e; BsaA1 data not shown). Although this method allows analysis of a very limited set of CpG dinucleotides within the gene, our results suggest that methylation of other regions of the ie-1 gene is unlikely.
In this study, we find that transcriptional reactivation of ie gene expression in explants is followed by reactivation of infectious virus with kinetics similar to that observed with infection in vitro. These results suggest that induction of ie gene expression is the rate-determining step in reactivation of the virus. In addition, we find that induction of ie gene expression in explants occurs much more rapidly in the presence of 5-aza-dC and TSA. Although the CpG deficit in the enhancer region and the observation that 5-aza-dC alone can induce ie gene expression would seem to suggest a role for DNA methylation in transcriptional silencing, the promoter/enhancer appears to be unmethylated. The lack of CpGs in the enhancer may be due to a selective pressure in favour of multiple AP-1- and NF-
B-binding sites, which lack this sequence (Fig. 3c). The effect of 5-aza-dC may be due to DNA methylation-independent effects on histone acetylation (Nguyen et al., 2002; Takebayashi et al., 2001). While we cannot exclude the possibility that the effects of 5-aza-dC and TSA in this system are unrelated to epigenetic control of ie gene expression, our studies are consistent with previous studies suggesting that latency is achieved through an active mechanism of transcriptional silencing involving histone modifications (Reeves et al., 2005).
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ACKNOWLEDGEMENTS |
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m157 virus. This work is supported by USPHS grant R01 AI42898 to M. A. |
REFERENCES |
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Received 8 November 2006;
accepted 29 November 2006.
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-actin RNA as previously described (Hummel et al., 2001
