Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

Nitric oxide is a versatile and critical effector molecule that can modulate many cellular functions. Although recognized as a regulator of infections, the inhibitory mechanism of nitric oxide against human cytomegalovirus (HCMV) replication remains elusive. We demonstrate that nitric oxide attenuates viral replication by interfering with HCMV-mediated modulation of several cellular processes. Nitric oxide exposure reduced HCMV genome synthesis and infectious viral progeny with cell-type-dependent differences observed. Mitochondrial respiration was severely reduced in both uninfected and HCMV-infected cells during exposure with little impact on ATP levels indicating changes in cellular metabolism. Metabolomics identified significantly altered small molecules in multiple pathways during nitric oxide exposure including nucleotide biosynthesis, tricarboxylic acid (TCA) cycle, and glutamine metabolism. Glutathione metabolites were increased coinciding with a reduction in the glutathione precursor glutamine. This shift was accompanied by increased antioxidant enzymes. Glutamine deprivation mimicked defects in HCMV replication and mitochondrial respiration observed during nitric oxide exposure. These data suggest that nitric oxide limits glutaminolysis by shuttling glutamine to glutathione synthesis. In addition, lipid intermediates were severely altered, which likely contributes to the observed increase in defective viral particles. Nitric oxide disrupts multiple cellular processes, and we had limited success in rescuing replication defects by supplementing with metabolic intermediates. Our studies indicate that nitric oxide attenuation of HCMV is multifactorial with interference in viral manipulation of cellular metabolism playing a central role.IMPORTANCE Human cytomegalovirus is a prevalent pathogen that can cause serious disease in patients with compromised immune systems, including transplant patients and during congenital infection. HCMV lytic replication likely occurs in localized sites of infection with immune cells infiltrating and releasing nitric oxide with other effector molecules. This nonspecific immune response results in both uninfected and infected cells exposed to high levels of nitric oxide. The absence of nitric oxide synthase has been associated with lethal HCMV infection. We demonstrate that nitric oxide inhibition of HCMV replication is multifactorial and cell type dependent. Our results indicate that nitric oxide controls replication by interfering with viral modulation of cellular metabolism while also affecting proliferation and mitochondrial respiration of neighboring uninfected cells. These studies identify the mechanism and contribution of nitric oxide during immune control of HCMV infection and provide insight into its role in other viral infections.

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Human cytomegalovirus (HCMV) is the leading cause of virus-mediated congenital birth defects. Congenitally infected infants can have a variety of symptoms manifesting within the central nervous system.

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