Nitric Oxide Attenuates Human Cytomegalovirus Infection yet Disrupts Neural Cell Differentiation and Tissue Organization

Abstract

Human cytomegalovirus (HCMV) is a prevalent betaherpesvirus that is asymptomatic in healthy individuals but can cause serious disease in immunocompromised patients. HCMV is also the leading cause of viral-mediated birth defects. Many of these defects manifest within the central nervous system and include microcephaly, sensorineural hearing loss, and cognitive developmental delays. Nitric oxide is a critical effector molecule produced as a component of the innate immune response during infection. Using a 3-dimensional cortical organoid model, we demonstrate that nitric oxide inhibits HCMV spread and simultaneously disrupts neural rosette structures resulting in tissue disorganization. Nitric oxide also attenuates HCMV replication in 2-dimensional cultures of neural progenitor cells (NPCs), a prominent cell type in cortical organoids that differentiate into neurons and glial cells. The multipotency factor SOX2 was decreased during nitric oxide exposure, suggesting early neural differentiation is affected. Maximal mitochondrial respiration was also reduced in both uninfected and infected NPCs. We determined this reduction likely influences neural differentiation as neurons (Tuj1+GFAP-Nestin-) and glial populations (Tuj1-GFAP+Nestin-) were reduced following differentiation. We also observed changes in calcium signaling during exposure to nitric oxide with increased cellular response to ATP (purinergic receptors) and KCl (voltage gated calcium channels). Importantly, nitric oxide could not rescue HCMV-mediated defects in calcium response. Our studies indicate a prominent, immunopathogenic role of nitric oxide in promoting developmental defects within the brain despite its antiviral activity during congenital HCMV infection. Author summaryHuman cytomegalovirus (HCMV) infection can result in serious disease to immunocompromised individuals. HCMV is also the leading cause of viral-mediated congenital birth defects. Congenitally-infected infants can have a variety of symptoms, including microcephaly, sensorineural hearing loss, and developmental delays. The use of 3-dimensional (3-D) cortical organoids to model infection of the fetal brain has advanced the current understanding of developmental defects and allowed a broader investigation of the mechanisms behind disease. Here, we investigate the effect of nitric oxide, a critical effector molecule, on cortical development and HCMV infection. We demonstrate that nitric oxide plays an antiviral role during infection yet results in significant disorganization to cortical tissue. Despite inhibiting viral replication in neural progenitor cells, nitric oxide contributes to differentiation defects of these cells and does not rescue functional consequences of HCMV infection on calcium signaling. Our results indicate that immunopathogenic consequences of nitric oxide during congenital infection promote developmental defects that undermine its antiviral activity.