DNA M ETHYLATION

One very important finding was the absence of DNA methylation from the entire viral genome at day 5 post de novo infection, when the cells had already adopted a latent expression profile. These results demonstrate for the first time that DNA methylation is not a principal requirement for the establishment of latency at least in infected endothelial cell lines in vitro. However, these findings stand in stark contrast to the widely accepted traditional

model that DNA methylation within the promoter region of the obligate lytic transactivator Rta (ORF50) to maintenance of the latent state (Chen et al., 2001). Therefore, the differences between the Chen Study and our study need to be discussed in further detail.

Chen and colleagues analyzed DNA methylation at the Rta promoter region in PEL cells (BCBL1) and not in endothelial cells which may be different. However, when examining this promoter region at late stages of latency in the cell lines that had adopted a common methylation profile, this region was discovered to be free of DNA methylation in SLKP as well as PEL derived BCBL1 and AP3 cells. A possible explanation for the different results obtained for the Rta promoter methylation in BCBL1 cells may be that Chen and colleagues established a different sub-clone of BCBL1. Interestingly, the DNA methylation pattern at the ORF50 promoter detected in HBL6 cells was similar to that described by Chen and colleagues, thus indicating that this particular promoter methylation profile can at least principally evolve and furthermore may represent an important repressive mark under specific circumstances in vivo. The possibility remains that presence or absence of DNA methylation at the ORF50 promoter, though not an absolute requirement, nevertheless may have a more subtle impact on the stability of latency in PEL cells. Although we have not observed any evidence for such a model, future studies should therefore investigate, whether cells harboring Rta promoter methylation (e.g. HBL6) are perhaps less susceptible to lytic reactivation than PEL cell lines without this particular methylation profile (e.g. BCBL1). In addition to the finding that DNA methylation is not a pre-requisite for latency establishment during de novo infection of endothelial cells, it could be demonstrated that methylation of the Rta promoter is also not a general requirement for maintenance of latency during any stage of latent infection.

We could additionally show by our analysis of histone modifications that the processes leading to the establishment of latency are regulated by histone modifications rather than DNA methylation (see section 5.2).

At this point it might be concluded that DNA methylation is generally not relevant for maintenance and stability of KSHV latency. However, this conclusion cannot be drawn for a number of reasons: The development and existence of distinct methylation patterns which were highly similar between the analyzed cells of different origin (endothelial SLKP and

different PEL derived B-cell lines) strongly indicates that these represent general hallmarks of latent KSHV genomes at late time points of infection in vivo as well as in cell culture.

However, we could demonstrate that DNA methylation evolves over time at regions, which are devoid of activating histone modifications and therefore, the observed DNA methylation patterns very likely are a simple consequence of the histone modification profile. Regardless of them being a cause or consequence, such patterns might still reinforce the latency program.

For example, when compared to SLKp cells which harbor methylated episomes, de novo infected SLK-5dpi cells were indeed found to display elevated levels of lytic gene expression and a higher number of spontaneously reactivated cells (Figs. 4-11 and 4-16). This could be explained indeed by missing DNA methylation in early latency. However, the generally low transcript levels together with the very low numbers of lytic cells even in SLK-5dpi cultures impede any absolute conclusion. A comparative study regarding the capability of lytic reactivation in SLKP and in SLK-5dpi cells may lead to a better insight into the importance of DNA methylation in early latency. However, one complicating factor to this approach is that SLK cells do not respond to chemical reagents that induce the lytic cycle in PEL cells. It has been shown that overexpression of Rta leads to lytic reactivation as well in SLK cells (Bechtel et al., 2003) and thus may represent an alternative approach. However, this experimental setting is also compromised since ectopic Rta expression circumvent activation of the endogenous Rta which may represent a very critical step in lytic induction.

Another interesting observation was that sub-clones within the SLK_P bulk population were revealed to carry slightly different patterns of DNA methylation within ORF21 by bisulfite sequencing and the COBRA assay. Thus it can be assumed that subtle differences exist among individual episomes and that, together, these mixed profiles contribute to the global pattern obtained by MeDIP on microarray. This finding should be evaluated by bisulfite sequencing of more sub-clones at different loci. However, it allows the interesting hypothesis that the exact route leading to the late latency specific signature of DNA methylation is not predefined, but that undefined pressures result in the establishment of a minimal CpG dinucleotide methylation pattern resulting in the common profile. In this scenario the question would then be how much (random) methylation at a specific region would be sufficient to alter and/or repress transcriptional activity.

The DNA methylation of KSHV genomes in cell culture experiments represented a very slow process, but this may be fundamentally different in vivo. It may be possible that in general primary B-cells have a higher tendency to methylate KSHV DNA than SLK cells but this has to be experimentally evaluated. Future studies have to address then, how the DNA

methylation profile and especially the differences between the analyzed profiles influence lytic reactivation in vivo.

Since latency is a hallmark of herpesviruses, it may be of interest to investigate, whether other herpesviruses use DNA methylation to regulate the latency program. Interestingly, in contrast to KSHV, DNA of latent herpes simplex virus 1 (HSV1) has been described not to be extensively methylated in vivo after infection of the spinal ganglia (Dressler et al., 1987), thus bringing forward the argument that DNA methylation is not generally a requirement of latent herpesvirus infection in vivo. If this would hold true, the question would remain why HSV1 is not subject to DNA methylation. A possible explanation may be that the HSV1 host cells of the dorsal root or trigeminal ganglia are non-dividing and fully differentiated. Hence, they would be presumed to have a stable DNA methylation profile and therefore may have low levels of methyltransferases. However, this hypothesis is speculative and needs to be experimentally proven before drawing any conclusion. Furthermore, it has to be considered that the latent state of HSV1 differs fundamentally from KSHV latency: There is no need for HSV1 to express any viral protein at this stage, e.g. to ensure episome persistence and only latency-associated transcripts (LATs), which lack protein coding information, are present during HSV1 latency (Kent et al., 2003) until the lytic cycle is induced. In contrast, KSHV needs expression of viral factors to ensure the propagation of the replicated episomes during mitosis. Thus it would be interesting to investigate the evolution of DNA methylation on viral episomes in primary cells infected with KSHV in vivo to explore the influence of an intact immune system therein. However, to date no procedure exists to isolate and enrich the rare fraction of KSHV infected cells from healthy donors.

The possibility of a general role for CpG methylation during late stages of latent KSHV infections may be substantiated by investigating further herpesviruses of the same sub-family.

Indeed, EBV and MHV68 have also been reported by different studies to be subject of this epigenetic modification (Gray et al., 2010; Kalla et al., 2010; Yang et al., 2009). In contrast to KSHV, methylation of EBV genomes has been described to emerge within 4 weeks post de novo infection in cell culture (Kalla et al., 2010). However, at day 5 post infection with EBV,

when cells have already adopted a latent infection state, there was also no DNA methylation detectable, thus indicating that like in the case of KSHV the primary gene silencing step is due to another mechanism. Investigations of emerging histone modifications in early EBV infection may shed light on this process. A biological meaning of the observed DNA methylation in EBV has been described regarding completion of the viral life cycle by demonstrating that methylation of target promoters of the transactivation protein BZLF1 is

necessary to emerge prior to efficient lytic replication, since the protein has higher affinity to methylated binding sites than to their unmethylated counterparts (Bergbauer et al., 2010).

Whether this is also true for the KSHV homologue K-bZIP or other virally or host encoded transactivation proteins has to be further investigated. This leads to the interesting hypothesis, that DNA methylation of KSHV episomes likewise to EBV may be more important for lytic replication than for maintenance of latency. Supporting this idea Bechtel and colleagues (Bechtel et al., 2003) have observed that after de novo infection SLK cells are not susceptible to lytic reactivation by chemical inducers, only by ectopic expression of Rta (as mentioned above) which, however, does not lead to production of viral progeny. Interestingly, they demonstrated that 293 cells and telomerase-immortalized endothelial (TIME) cells (in contrast to SLK cells) are susceptible to lytic reactivation by chemical inducers after latency establishment following a de novo infection (Bechtel et al., 2003). If viral episomes were DNA methylated in these cells this would be a strong argument that lytic replication may be enhanced by the presence of DNA methylation. However, this hypothesis does not satisfy how SLK_P cells which have DNA methylated episomes are refractory to lytic cycle induction. Taken together, the facts that the ORF50 promoter is not methylated in cells latently infected with KSHV and that Rta expression is sufficient to induce lytic replication, indicate that KSHV and EBV most likely use different mechanisms during lytic reactivation.

In the case of the murine gamma-herpesvirus 68 (MHV68) viral promoters including the Rta promoter become methylated in laboratory mice already a few month after infection with the virus (Yang et al., 2009; Gray et al., 2010). These findings substantiated the theory that DNA methylation of the Rta promoter regulates lytic reactivation in MHV68, but also in KSHV. As discussed above, the latter is being strongly against by the results of our study, and hence the question arises, whether these viruses have adopted different strategies to prevent Rta expression. Interestingly, when comparing the content and frequency of CpG dinucleotides between the KSHV and the MHV68 genomes, in particular within the Rta promoter regions, it appears that MHV68 exhibits a higher suppression of CpG dinucleotides across the entire genome than KSHV. Observed to expected CpG dinucleotide ratios are 0.82 for KSHV and 0.43 for MHV68 (Figure 5-1). Interestingly, this suppression is even higher within the Rta promoter region of MHV68 with CpG sites often being separated by more than one hundred base pairs. Considering the hypothesis that DNA methylation evolutionary leads to lowered CpG frequencies via the mutational process of spontaneous deamination, the low CpG content suggests that the entire MHV68 genome and in particular the Rta promoter are subject to much more profound DNA methylation in their host compared to KSHV. This

furthermore substantiates that the default mechanism to establishment and maintenance latency in MHV68 most likely is DNA methylation of the Rta promoter, whereas KSHV primarily uses deposition of repressive H3K27-me3 for this purpose. This model does not exclude the possibility that the H3K27-me3 histone modification mark is present on the MHV68 genome and may have an additive effect on latency, a scenario that will have to be experimentally tested in the future. However, it is unlikely that Rta expression in MHV68 is poised by bivalent chromatin, since activating marks are mutually exclusive to DNA methylation and thus the strategies to stabilize latency seem to be fundamentally different between these two gamma-herpesviruses.

Figure 5-1: Suppression of CpG frequency in KSHV and MHV68.

CpG dinucleotides across the viral genomes are symbolized by pink bars. Values on the y-axis represent the ratio of observed to expected CpG-dinucleotides (termed CpG suppression) within shifting calculation windows of 200 base pairs. Lower graphs represent close-ups of the respective ORF50 promoter region within both virus genomes. Mean and standard deviation of ratios are indicated by black horizontal lines and gray boxes.

Taken together, our results clearly demonstrate that DNA methylation is not a pre-requisite for latency establishment or maintenance in KSHV infection. Given the findings that the DNA methylation occurs late during latent infection exactly at H3K27-me3 marked regions that are not activation marked, it most likely represents a passively occuring process that may be explained by the DNMT recruitment capability of PRC1 and PRC2 (described in detail in section 5.3). Furthermore, as MHV68 most likely forces Rta repression and latency by DNA methylation, and as EBV uses DNA methylation to ensure efficient lytic replication (although it does not need DNA methylation for establishment of the latent expression profile) it seems that gamma-herpesviruses follow different strategies to establish latency.