Virus Entry is a Major Determinant of HCMV Latency in Monocytes
Human Cytomegalovirus (HCMV) infection can result in either productive or latent infection, the latter being the basis for the virus life-long persistence. Intriguingly, monocytes, which support latent infection, become permissive to productive infection upon differentiation to macrophages. However, the molecular factors explaining these differentiation-driven differences are not fully understood and have been so far attributed to chromatin-mediated repression of the viral genome. Here, by using metabolic labeling of newly synthesized RNA early in monocyte and macrophage infection, we discover a major early block in viral gene expression, and viral transcripts are barely detected in infected monocytes. By unbiasedly analyzing the changes between monocytes and their differentiated counterparts, we reveal that the levels of several cell surface proteins involved in HCMV entry are upregulated upon monocyte to macrophage differentiation, and correspondingly we uncover HCMV entry into monocytes compared to macrophages is extremely inefficient. Remarkably, ectopic expression of a canonical HCMV entry receptor in monocytes facilitates productive infection of these cells, demonstrating that given efficient viral entry, monocytes, like macrophages, have the capacity to support productive infection. Among the cell surface proteins that are upregulated upon monocyte differentiation are several integrins, which we show play an important role in HCMV entry into macrophages, partially explaining the differences in viral entry. Overall, our findings reveal that a previously unrecognized major barrier for productive infection in monocytes is entry, adding a critical layer to the paradigm of HCMV latency. Overall design: Slam-seq: We directly measure early transcription of viral genes using slam-seq on primary and THP1 monocytes and macrophages.  RNA-seq: 1. We performed RNA-seq on primary monocytes, THP1 monocytes and Kasumi-3 myeloid progenitor cells, as well as on macrophages derived from the same cells. 2. We sequence THP1 control and THP1 overexpressing PDGFRa.  CRISPR-screen: THP1-PDGFRa monocytes were transduced with the GeCKO CRISPR/Cas9 genome-wide knockout library (targets 19,050 human genes with three guides per gene and one thousand non-targeting gRNA as controls). We infected these cells with HCMV-GFP and at 2 days post infection sorted them to GFP-bright and GFP-dim populations, reflecting productive and non-productive infection, respectively. gRNAs were sequenced and aligned using MAGeCK.