Heterologous viral promoters incorporated into the human cytomegalovirus genome are silenced during experimental latency.

Human cytomegalovirus (HCMV) lytic phase gene expression is repressed upon entry into myeloid lineage cells where the virus establishes latency. Lytic infection is not initiated because the tegument-delivered transactivator protein pp71 fails to enter the nucleus and inactivate the Daxx-mediated cellular intrinsic defense that silences the viral genome. When pp71 is expressed de novo in THP-1 monocytes, it localizes to the nucleus, inactivates the Daxx defense, and initiates lytic infection. We speculated that replacing the native viral promoter that drives pp71 expression with one that is highly and constitutively active in myeloid cells would permit pp71 de novo expression upon infection and that this newly expressed pp71 would accumulate in the nucleus, inactivate the intrinsic defense, and initiate the cascade of lytic gene expression. Surprisingly, we found that this promoter was still subject to normal silencing mechanisms in THP-1 monocytes and primary CD34(+) cells, two independent myeloid lineage cells. A second constitutively active heterologous viral promoter located in a different region of the HCMV genome was also silenced in THP-1 and CD34(+) cells. Furthermore, these two independent heterologous viral promoters inserted into three different regions of the HCMV genome in three different viral strains all required prior expression of the viral immediate early proteins for activation in fibroblasts. From this, we conclude that incorporation within the HCMV genome impacts the proclivity of heterologous viral promoters to initiate transcription. These observations have mechanistic implications for the expression of viral genes and transgenes during both lytic infection and latency.