Human cytomegalovirus induces significant structural and functional changes in terminally differentiated human cortical neurons

Abstract

ABSTRACT 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 and 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 and neurodegenerative conditions. Using a model system representative of the human forebrain, we demonstrate that induced pluripotent stem cell-derived excitatory glutamatergic and inhibitory GABAergic neurons are fully permissive to HCMV with 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 proliferative cell nuclear antigen suggesting alterations in cell cycle machinery. These findings are consistent with previous HCMV-mediated changes in various cell types and implicate the ability of viral proteins to alter proliferative pathways to promote virion production. Infection also induces significant structural changes in forebrain neurons, such as the formation of syncytia and the 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 central nervous system, which has significant implications for overall brain health and function. IMPORTANCE Human cytomegalovirus (HCMV) is a highly prevalent viral pathogen that can cause serious neurological deficits in infants experiencing an in utero infection. Also, as a life-long infection, HCMV has been associated with several diseases in the adult brain. HCMV is known to infect early neural progenitor cells, but whether it also infects terminally differentiated neurons is still debated. Here, we differentiated human-induced pluripotent stem cells into neurons for 84–120 days to test the ability of HCMV to infect terminally differentiated neurons and assess the downstream functional consequences. We discovered that mature human neurons are highly permissive to HCMV infection, exhibited late replication hallmarks, and produced infectious virus. Moreover, infection in terminally differentiated neurons essentially eliminated neuron function. These results demonstrate that terminally differentiated human neurons are permissive to HCMV infection, which can significantly alter both structural and functional features of this mature neuron population.