Abstract
Human cytomegalovirus (HCMV) infection results in either productive or latent infection, the latter enabling life-long viral persistence. Monocytes support latent infection but become permissive to productive infection upon differentiation into macrophages. These differentiation-driven differences have been largely attributed to chromatin-mediated repression of the viral genome. Using metabolic labeling of newly synthesized RNA, we observe markedly lower viral transcription at early stages of infection in monocytes compared to macrophages. Unbiased comparison reveals that this difference is partly explained by inefficient viral entry in monocytes: fewer viruses enter, and correspondingly, fewer genomes reach the nucleus. Indeed, ectopic expression of known HCMV entry receptors in monocytes enhances viral entry and enables productive infection, demonstrating that these cells can support full lytic replication if entry is efficient. We further identify integrin β3 as a differentiation-induced surface protein playing an important role in HCMV entry into macrophages, partially accounting for the observed differences in entry efficiency. Finally, we show that cells receiving fewer viral genomes are the ones that establish latent infection and have the capacity to reactivate. Overall, our findings reveal that entry is a previously unrecognized factor contributing to latent infection in monocytes, adding a critical layer to the paradigm of HCMV latency.