RESUMO
HCMV establishes latency in myeloid cells. Using the Kasumi-3 latency model, we previously showed that lytic gene expression is activated prior to establishment of latency in these cells. The early events in infection may have a critical role in shaping establishment of latency. Here, we have used an integrative multi-omics approach to investigate dynamic changes in host and HCMV gene expression and epigenomes at early times post infection. Our results show dynamic changes in viral gene expression and viral chromatin. Analyses of Pol II, H3K27Ac and H3K27me3 occupancy of the viral genome showed that 1) Pol II occupancy was highest at the MIEP at 4 hours post infection. However, it was observed throughout the genome; 2) At 24 hours, H3K27Ac was localized to the major immediate early promoter/enhancer and to a possible second enhancer in the origin of replication OriLyt; 3) viral chromatin was broadly accessible at 24 hpi. In addition, although HCMV infection activated expression of some host genes, we observed an overall loss of de novo transcription. This was associated with loss of promoter-proximal Pol II and H3K27Ac, but not with changes in chromatin accessibility or a switch in modification of H3K27.Importance.HCMV is an important human pathogen in immunocompromised hosts and developing fetuses. Current anti-viral therapies are limited by toxicity and emergence of resistant strains. Our studies highlight emerging concepts that challenge current paradigms of regulation of HCMV gene expression in myeloid cells. In addition, our studies show that HCMV has a profound effect on de novo transcription and the cellular epigenome. These results may have implications for mechanisms of viral pathogenesis.
RESUMO
We used the Kasumi-3 model to study human cytomegalovirus (HCMV) latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP, flow sorted for green fluorescent protein-positive (GFP+) cells, and cultured for various times to monitor establishment of latency, as judged by repression of viral gene expression (RNA/DNA ratio) and loss of virus production. We found that, in the vast majority of cells, latency was established posttranscriptionally in the GFP+ infected cells: transcription was initially turned on and then turned off. We also found that some of the GFP- cells were infected, suggesting that latency might be established in these cells at the outset of infection. We were not able to test this hypothesis because some GFP- cells expressed lytic genes and thus it was not possible to separate them from GFP- quiescent cells. In addition, we found that the pattern of expression of lytic genes that have been associated with latency, including UL138, US28, and RNA2.7, was the same as that of other lytic genes, indicating that there was no preferential expression of these genes once latency was established. We confirmed previous studies showing that tumor necrosis factor alpha (TNF-α) induced reactivation of infectious virus, and by analyzing expression of the progenitor cell marker CD34 as well as myeloid cell differentiation markers in IE+ cells after treatment with TNF-α, we showed that TNF-α induced transcriptional reactivation of IE gene expression independently of differentiation. TNF-α-mediated reactivation in Kasumi-3 cells was correlated with activation of NF-κB, KAP-1, and ATM.IMPORTANCE HCMV is an important human pathogen that establishes lifelong latent infection in myeloid progenitor cells and reactivates frequently to cause significant disease in immunocompromised people. Our observation that viral gene expression is first turned on and then turned off to establish latency suggests that there is a host defense, which may be myeloid cell specific, responsible for transcriptional silencing of viral gene expression. Our observation that TNF-α induces reactivation independently of differentiation provides insight into molecular mechanisms that control reactivation.
