Human cytomegalovirus (HCMV) enters primary CD34+ hematopoietic progenitor cells by macropinocytosis where it establishes latency in part because its tegument transactivating protein, pp71, remains associated with endosomes and is therefore unable to initiate productive, lytic replication. Here we show that multiple HCMV strains also enter cell line models used to study latency by macropinocytosis and endocytosis. In all latency models tested, tegument-delivered pp71 was found co-localized with endosomal markers and not associated with the seven other cytoplasmic localization markers tested. Like the capsid-associated pp150 tegument protein, we detected capsid proteins initially associated with endosomes but later in the nucleus. Inhibitors of macropinocytosis and endocytosis reduced latent viral gene expression and precluded reactivation. Importantly, we utilized electron microscopy to observe entry by macropinocytosis and endocytosis, providing additional visual corroboration to our functional studies. Our demonstration that HCMV enters cell line models for latency in a manner indistinguishable from its entry into primary cells illustrates the utility of these cell lines for probing the mechanisms, host genetics, and small molecule-mediated inhibition of HCMV entry into the cell types where it establishes latency.
Primary cells cultured in vitro currently provide the highest available relevance for examining molecular and genetic requirements for the establishment, maintenance, and reactivation of HCMV latency. However, their expense, heterogeneity, and intransigence to both long-term culture and molecular or genetic modification create rigor and reproducibility challenges for HCMV latency studies. There are several cell line models for latency not obstructed by deficiencies inherent in primary cells. However, many researchers view cell line studies of latency as physiologically irrelevant because of the perception that these models display numerous and significant differences from primary cells. Here we show that the very first step in a latent HCMV infection, entry of the virus into cells, occurs in cell line models indistinguishably from how it occurs in primary CD34+ hematopoietic progenitor cells. Our data argue that experimental HCMV latency in cell lines and primary cells is much more similar than it is different.