Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34 + hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of resistant and permissive cell types, and the cellular features characterizing the latter, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ∼7000 individual cells at day 1 post-infection using the 10× genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit – Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells permissive to CMV replication, and provide a possible rationale for their preferential infection.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

13 14 Short title: single-cell analysis of CMV-infected myeloid cells 2 15 ABSTRACT 16 Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We 17 previously showed that only a small subset of myeloid cells differentiated from CD34 + hematopoietic 18 stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these 19 populations. The exact identity of susceptible and resistant cell types, and the cellular features 20 characterizing permissive cells, however, could not be dissected using averaging transcriptional 21 analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes 22 of ~ 7000 individual cells at day one post-infection using the 10X genomics platform. We show that 23 viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be 24 the main target of cellular restriction mechanisms. We further show that viral replication occurs in a 25 small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of 26 cells expressing markers of Colony Forming Unit -Granulocyte, Erythrocyte, Monocyte, 27 Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, 28 CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell 29 proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-30 related genes. While expression levels of the former are maintained in infected cells, the latter are 31 strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be 32 due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts 33 from viral effectors, rather than to the absence of specific restriction factors. Together, these findings 34 identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a 35 possible rationale for their preferential infection. 3 AUTHOR SUMMARY37 Myeloid cells such as monocytes and dendritic cells are critical targets of CMV infection. To identify 38 the cellular factors that confer susceptibility or resistance to infection, we profiled the transcriptomes of 39~ 7,000 single cells from a population of semi-permissive myeloid cells infected with CMV. We found 40 that viral RNAs are detectable in the majority of the cells, but that marked expression of CMV lytic 41 genes occurs in only a small subset of cells transcriptionally related to a cluster of CFU-GEMM 42 progenitors that express similar amounts of transcripts encoding interferon-related anti-viral factors as 43 the rest of the population but higher levels of transcripts encoding proteins required for energy, RNA, 44 and protein production. We thus conclude that the preferential infection of CFU-GEMM cells might be 45 due to the pre-existing presence of an intracellular environment conducive to infection onset, rather ...