TNF-α induces reactivation of human cytomegalovirus independently of myeloid cell differentiation following post-transcriptional establishment of latency

We used the Kasumi-3 model to study HCMV latency and reactivation in myeloid progenitor cells. Kasumi-3 cells were infected with HCMV strain TB40/Ewt-GFP, flow sorted for 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 latency was established post-transcriptionally: transcription was initially turned on, and then turned off. We did not find evidence to support a latency model in which viral gene expression is repressed at the outset of infection. 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 in latency. 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 reactivation independently of differentiation. In contrast, LPS did not induce reactivation in Kasumi-3 cells, despite activation of ERK and NF-κB, and did not induce transcription of inflammatory cytokines. We speculate that reactivation occurs in two steps: 1. activation of signal-specific transcription factors that bind to the MIEP; 2. reconfiguration of viral chromatin to allow access to the MIEP. We propose that differentiation is required to render cellular chromatin competent to express inflammatory cytokines that drive reactivation in response to LPS, but it is not required for reactivation in response to TNF-α itself.