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Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus, and infection can lead to a range of symptomatology from mononucleosis to sepsis in immunocompromised individuals. HCMV is also the leading viral cause of congenital birth defects. Lytic replication is supported by many cell types with different kinetics and efficiencies leading to a plethora of pathologies. The goal of these studies was to elucidate HCMV replication efficiencies for viruses produced on different cell types upon infection of epithelial cells by combining experimental approaches with data-driven computational modeling. HCMV was generated from a common genetic background of TB40-BAC4, propagated on fibroblasts (TB40 Fb ) or epithelial cells (TB40 Epi ), and used to infect epithelial cells. We quantified cell-associated viral genomes (vDNA), protein levels (pUL44, pp28), and cell-free titers over time for each virus at different multiplicities of infection. We combined experimental quantification with data-driven simulations and determined that parameters describing vDNA synthesis were similar between sources. We found that pUL44 accumulation was higher in TB40 Fb than TB40 Epi . In contrast, pp28 accumulation was higher in TB40 Epi which coincided with a significant increase in titer for TB40 Epi over TB40 Fb . These differences were most evident during live-cell imaging, which revealed syncytia-like formation during infection by TB40 Epi . Simulations of the late lytic replication cycle yielded a larger synthesis constant for pp28 in TB40 Epi along with increase in virus output despite similar rates of genome synthesis. By combining experimental and computational modeling approaches, our studies demonstrate that the cellular source of propagated virus impacts viral replication efficiency in target cell types., Competing Interests: Competing Interest Statement: The authors declare no competing interest.

Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that typically presents asymptomatically in healthy individuals despite lifelong latency. However, in 10-15% of congenital cases, this beta-herpesvirus demonstrates direct effects on the central nervous system, including microcephaly, cognitive/learning delays, and hearing deficits. HCMV has been widely shown to infect neural progenitor cells, but the permissiveness of fully differentiated neurons to HCMV is controversial and chronically understudied, despite potential associations between HCMV infection with neurodegenerative conditions. Using a model system representative of the human forebrain, we demonstrate that induced pluripotent stem cell (iPSC)-derived, excitatory glutamatergic and inhibitory GABAergic neurons are fully permissive to HCMV, demonstrating complete viral replication, competent virion production, and spread within the culture. Interestingly, while cell proliferation was not induced in these post-mitotic neurons, HCMV did increase expression of proliferative markers Ki67 and PCNA suggesting alterations in cell cycle machinery. These finding are consistent with previous HCMV-mediated changes in various cell types and implicate the virus' ability to alter proliferative pathways to promote virion production. HCMV also induces significant structural changes in forebrain neurons, such as the formation of syncytia and retraction of neurites. Finally, we demonstrate that HCMV disrupts calcium signaling and decreases neurotransmission, with action potential generation effectively silenced after 15 days post infection. Taken together, our data highlight the potential for forebrain neurons to be permissive to HCMV infection in the CNS, which could have significant implications on overall brain health and function.