The molecular principles governing HCMV infection outcome

Infection with Human cytomegalovirus (HCMV) can result in either productive or non-productive infection, the latter potentially leading to establishment of latency, but the molecular factors that dictate these different infection outcomes are elusive. Macrophages are known targets of HCMV and considered to be permissive for productive infection, while monocytes, their precursors, are thought to support latent infection. Here we reveal that infection of macrophages is more complex than previously appreciated and can result in either productive or non-productive infection. By analyzing the progression of HCMV infection in c and macrophages using single cell transcriptomics, we uncover that the key characteristics that define productive and non-productive cells are the onset of viral gene expression, and activation of Interferon-stimulated genes (ISGs), respectively. We show that the level of viral gene expression, and specifically the expression of the major immediate early factor, IE1, is the principle barrier for establishing productive infection. On the cellular side, induction of ISGs in response to infection surprisingly does not dictate infection outcome, but we find that the cell intrinsic ISG levels is a main determinant of infection outcome. Indeed, intrinsic ISG level is downregulated with monocyte differentiation. We further show that, compared to monocytes, non-productive macrophages maintain slightly higher levels of viral transcripts and are able to reactivate, raising the possibility that they can serve as latency reservoirs in tissues. Moreover, we find that productive infection perturbs macrophage identity and function, likely affecting their immunological role during active infection. Overall, by harnessing the tractable system of monocyte differentiation we decipher the underlying principles that control HCMV infection outcome, and propose macrophages as a new potential HCMV reservoir in tissues. The MARS-seq protocol was used for single cell RNA-seq of primary CD14+ monocytes and PMA-induced monocyte-derived macrophages infected with TB40/E-GFP strain of HCMV at 5, 12 ,24,48, 72 and 144 hours post infection (Experiment 1) and of PMA-induced monocyte-derived macrophages infected with TB40/E-GFP strain of HCMV at 24, 72 and 144 hours post infection, for which at 72 and 144 hours post infection, only GFP-dim cells were sorted for single cell RNA-seq (Experiment 2). 10X Genomics Chromium Single Cell 3? Gene Expression v3.1 kit was used for scRNA-seq of BAL cells, infected with TB40/E-GFP strain of HCMV at 20 and 70 hours post infectionpools (Experiment 3). A modified mcSCRB-seq was used for RNA-seq of BAL macrophages infeced with TB40/E-GFP strain of HCMV at 3 days post infection sorted to GFP-dim and GFP-bright cells (Experiment 4). Bulk RNA-seq libraries were prepared according to Shishkin et al. Nat. Methods 2015, for THP1 cells following CRISPR KO of IRF9 or STAT2 or IGSF8 (Experiment 5).