Replication efficiencies of human cytomegalovirus-infected epithelial cells are dependent on source of virus production.

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 (TB40Fb) or epithelial cells (TB40Epi), 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 TB40Fb than TB40Epi. In contrast, pp28 accumulation was higher in TB40Epi which coincided with a significant increase in titer for TB40Epi over TB40Fb. These differences were most evident during live-cell imaging, which revealed syncytia-like formation during infection by TB40Epi. Simulations of the late lytic replication cycle yielded a larger synthesis constant for pp28 in TB40Epi 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.

BioModME for building and simulating dynamic computational models of complex biological systems.

Summary: Molecular mechanisms of biological functions and disease processes are exceptionally complex, and our ability to interrogate and understand relationships is becoming increasingly dependent on the use of computational modeling. We have developed "BioModME," a standalone R-based web application package, providing an intuitive and comprehensive graphical user interface to help investigators build, solve, visualize, and analyze computational models of complex biological systems. Some important features of the application package include multi-region system modeling, custom reaction rate laws and equations, unit conversion, model parameter estimation utilizing experimental data, and import and export of model information in the Systems Biology Matkup Language format. The users can also export models to MATLAB, R, and Python languages and the equations to LaTeX and Mathematical Markup Language formats. Other important features include an online model development platform, multi-modality visualization tool, and efficient numerical solvers for differential-algebraic equations and optimization. Availability and implementation: All relevant software information including documentation and tutorials can be found at https://mcw.marquette.edu/biomedical-engineering/computational-systems-biology-lab/biomodme.php. Deployed software can be accessed at https://biomodme.ctsi.mcw.edu/. Source code is freely available for download at https://github.com/MCWComputationalBiologyLab/BioModME.

Simulating the Evolution of Signaling Signatures During CART-Cell and Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.Clinical Relevance- Analysis of CART-cell signaling signatures can inform future CAR receptor design and combination therapy approaches aimed at improving therapy response.

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

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.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Human cytomegalovirus UL138 interaction with USP1 activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquitinating complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated in infection and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection.

Human Cytomegalovirus Induces Significant Structural and Functional Changes in Terminally Differentiated Human Cortical Neurons.

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.

Human Cytomegalovirus UL138 Interaction with USP1 Activates STAT1 in infection.

Innate immune responses are crucial for limiting virus infection. However, viruses often hijack our best defenses for viral objectives. Human Cytomegalovirus (HCMV) is a beta herpesvirus which establishes a life-long latent infection. Defining the virus-host interactions controlling latency and reactivation is vital to the control of viral disease risk posed by virus reactivation. We defined an interaction between UL138, a pro-latency HCMV gene, and the host deubiquintase complex, UAF1-USP1. UAF1 is a scaffold protein pivotal for the activity of ubiquitin specific peptidases (USP), including USP1. UAF1-USP1 sustains an innate immune response through the phosphorylation and activation of signal transducer and activator of transcription-1 (pSTAT1), as well as regulates the DNA damage response. After the onset of viral DNA synthesis, pSTAT1 levels are elevated and this depends upon UL138 and USP1. pSTAT1 localizes to viral centers of replication, binds to the viral genome, and influences UL138 expression. Inhibition of USP1 results in a failure to establish latency, marked by increased viral genome replication and production of viral progeny. Inhibition of Jak-STAT signaling also results in increased viral genome synthesis in hematopoietic cells, consistent with a role for USP1-mediated regulation of STAT1 signaling in the establishment of latency. These findings demonstrate the importance of the UL138-UAF1-USP1 virus-host interaction in regulating HCMV latency establishment through the control of innate immune signaling. It will be important going forward to distinguish roles of UAF1-USP1 in regulating pSTAT1 relative to its role in the DNA damage response in HCMV infection. Importance: Human cytomegalovirus (HCMV) is one of nine herpesviruses that infect humans. Following a primary infection, HCMV establishes a life-long latent infection that is marked by sporadic, and likely frequent reactivation events. While these reactivation events are asymptomatic in the immune competent host, they pose important disease risks for the immune compromised, including solid organ or stem cell transplant recipients. Its complex interactions with host biology and deep coding capacity make it an excellent model for defining mechanisms important for viral latency and reactivation. Here we define an interaction with host proteins that commandeer typically antiviral innate immune signaling for the establishment of latency.

Simulating the Evolution of Signaling Signatures during CART-Cell - Tumor Cell Interactions.

Immunotherapies have been proven to have significant therapeutic efficacy in the treatment of cancer. The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for in silico based prediction of CART-cell functionality prior to experimentation.

Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in OriLyt.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.

Computational modeling of protracted HCMV replication using genome substrates and protein temporal profiles.

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

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 virus-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. Congenitally infected fetal brains show regions of brain damage, including necrotic foci with infiltrating macrophages and microglia, cell types that produce nitric oxide 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 that early neural differentiation is affected. Nitric oxide also reduced maximal mitochondrial respiration in both uninfected and infected NPCs. We determined that this reduction likely influences neural differentiation, as neurons (Tuj1+ GFAP- Nestin-) and glial populations (Tuj1- GFAP+ Nestin-) were reduced following differentiation. 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. IMPORTANCE 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. The use of 3-dimensional (3-D) cortical organoids to model infection of the fetal brain has advanced the current understanding of development and allowed broader investigation of the mechanisms behind disease. However, the impact of the innate immune molecule nitric oxide during HCMV infection has not been explored in neural cells or cortical 3-D models. Here, we investigated the effect of nitric oxide on cortical development during HCMV infection. We demonstrate that nitric oxide plays an antiviral role during infection yet results in disorganized cortical tissue. Nitric oxide contributes to differentiation defects of neuron and glial cells from neural progenitor cells despite inhibiting viral replication. Our results indicate that immunopathogenic consequences of nitric oxide during congenital infection promote developmental defects that undermine its antiviral activity.

Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus.

Human cytomegalovirus (HCMV) is a betaherpesvirus that can cause severe birth defects including vision and hearing loss, microcephaly, and seizures. Currently, no approved treatment options exist for in utero infections. Here, we aimed to determine the impact of HCMV infection on the transcriptome of developing neurons in an organoid model system. Cell populations isolated from organoids based on a marker for infection and transcriptomes were defined. We uncovered downregulation in key cortical, neurodevelopmental, and functional gene pathways which occurred regardless of the degree of infection. To test the contributions of specific HCMV immediate early proteins known to disrupt neural differentiation, we infected NPCs using a recombinant virus harboring a destabilization domain. Despite suppressing their expression, HCMV-mediated transcriptional downregulation still occurred. Together, our studies have revealed that HCMV infection causes a profound downregulation of neurodevelopmental genes and suggest a role for other viral factors in this process.

Human cytomegalovirus lytic infection inhibits replication-dependent histone synthesis and requires stem loop binding protein function.

Replication-dependent (RD) histones are deposited onto human cytomegalovirus (HCMV) genomes at the start of infection. We examined how HCMV affects the de novo production of RD histones and found that viral infection blocked the accumulation of RD histone mRNAs that normally occurs during the S phase. Furthermore, RD histone mRNAs present in HCMV-infected cells did not undergo the unique 3' processing required for their normal nuclear export and translation. The protein that orchestrates processing in the nucleus, stem loop­binding protein (SLBP), was found predominantly in the cytoplasm, and RD histone proteins were not de novo synthesized in HCMV-infected cells. Intriguingly, however, we found that SLBP was required for the efficient synthesis and assembly of infectious progeny virions. We conclude that HCMV infection attenuates RD histone mRNA accumulation and processing and the de novo protein synthesis of the RD histones, while utilizing SLBP for an alternative purpose to support infectious virion production.

Crosstalk between Plk1, p53, cell cycle, and G2/M DNA damage checkpoint regulation in cancer: computational modeling and analysis.

Different cancer cell lines can have varying responses to the same perturbations or stressful conditions. Cancer cells that have DNA damage checkpoint-related mutations are often more sensitive to gene perturbations including altered Plk1 and p53 activities than cancer cells without these mutations. The perturbations often induce a cell cycle arrest in the former cancer, whereas they only delay the cell cycle progression in the latter cancer. To study crosstalk between Plk1, p53, and G2/M DNA damage checkpoint leading to differential cell cycle regulations, we developed a computational model by extending our recently developed model of mitotic cell cycle and including these key interactions. We have used the model to analyze the cancer cell cycle progression under various gene perturbations including Plk1-depletion conditions. We also analyzed mutations and perturbations in approximately 1800 different cell lines available in the Cancer Dependency Map and grouped lines by genes that are represented in our model. Our model successfully explained phenotypes of various cancer cell lines under different gene perturbations. Several sensitivity analysis approaches were used to identify the range of key parameter values that lead to the cell cycle arrest in cancer cells. Our resulting model can be used to predict the effect of potential treatments targeting key mitotic and DNA damage checkpoint regulators on cell cycle progression of different types of cancer cells.

Implications of a 'Third Signal' in NK Cells.

Innate and adaptive immune systems are evolutionarily divergent. Primary signaling in T and B cells depends on somatically rearranged clonotypic receptors. In contrast, NK cells use germline-encoded non-clonotypic receptors such as NCRs, NKG2D, and Ly49H. Proliferation and effector functions of T and B cells are dictated by unique peptide epitopes presented on MHC or soluble humoral antigens. However, in NK cells, the primary signals are mediated by self or viral proteins. Secondary signaling mediated by various cytokines is involved in metabolic reprogramming, proliferation, terminal maturation, or memory formation in both innate and adaptive lymphocytes. The family of common gamma (γc) cytokine receptors, including IL-2Rα/ß/γ, IL-7Rα/γ, IL-15Rα/ß/γ, and IL-21Rα/γ are the prime examples of these secondary signals. A distinct set of cytokine receptors mediate a 'third' set of signaling. These include IL-12Rß1/ß2, IL-18Rα/ß, IL-23R, IL-27R (WSX-1/gp130), IL-35R (IL-12Rß2/gp130), and IL-39R (IL-23Rα/gp130) that can prime, activate, and mediate effector functions in lymphocytes. The existence of the 'third' signal is known in both innate and adaptive lymphocytes. However, the necessity, context, and functional relevance of this 'third signal' in NK cells are elusive. Here, we define the current paradigm of the 'third' signal in NK cells and enumerate its clinical implications.

MyD88 is an essential regulator of NK cell-mediated clearance of MCMV infection.

The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.

Nitric Oxide Circumvents Virus-Mediated Metabolic Regulation during Human Cytomegalovirus 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.

Network mechanisms and dysfunction within an integrated computational model of progression through mitosis in the human cell cycle.

The cellular protein-protein interaction network that governs cellular proliferation (cell cycle) is highly complex. Here, we have developed a novel computational model of human mitotic cell cycle, integrating diverse cellular mechanisms, for the purpose of generating new hypotheses and predicting new experiments designed to help understand complex diseases. The pathogenic state investigated is infection by a human herpesvirus. The model starts at mitotic entry initiated by the activities of Cyclin-dependent kinase 1 (CDK1) and Polo-like kinase 1 (PLK1), transitions through Anaphase-promoting complex (APC/C) bound to Cell division cycle protein 20 (CDC20), and ends upon mitotic exit mediated by APC/C bound to CDC20 homolog 1 (CDH1). It includes syntheses and multiple mechanisms of degradations of the mitotic proteins. Prior to this work, no such comprehensive model of the human mitotic cell cycle existed. The new model is based on a hybrid framework combining Michaelis-Menten and mass action kinetics for the mitotic interacting reactions. It simulates temporal changes in 12 different mitotic proteins and associated protein complexes in multiple states using 15 interacting reactions and 26 ordinary differential equations. We have defined model parameter values using both quantitative and qualitative data and using parameter values from relevant published models, and we have tested the model to reproduce the cardinal features of human mitosis determined experimentally by numerous laboratories. Like cancer, viruses create dysfunction to support infection. By simulating infection of the human herpesvirus, cytomegalovirus, we hypothesize that virus-mediated disruption of APC/C is necessary to establish a unique mitotic collapse with sustained CDK1 activity, consistent with known mechanisms of virus egress. With the rapid discovery of cellular protein-protein interaction networks and regulatory mechanisms, we anticipate that this model will be highly valuable in helping us to understand the network dynamics and identify potential points of therapeutic interventions.

Human Cytomegalovirus Disruption of Calcium Signaling in Neural Progenitor Cells and Organoids.

The herpesvirus human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Infection can result in infants born with a variety of symptoms, including hepatosplenomegaly, microcephaly, and developmental disabilities. Microcephaly is associated with disruptions in the neural progenitor cell (NPC) population. Here, we defined the impact of HCMV infection on neural tissue development and calcium regulation, a critical activity in neural development. Regulation of intracellular calcium involves purinergic receptors and voltage-gated calcium channels (VGCC). HCMV infection compromised the ability of both pathways in NPCs as well as fibroblasts to respond to stimulation. We observed significant drops in basal calcium levels in infected NPCs which were accompanied by loss in VGCC activity and purinergic receptor responses. However, uninfected cells in the population retained responsiveness. Addition of the HCMV inhibitor maribavir reduced viral spread but failed to restore activity in infected cells. To study neural development, we infected three-dimensional cortical organoids with HCMV. Infection spread to a subset of cells over time and disrupted organoid structure, with alterations in developmental and neural layering markers. Organoid-derived infected neurons and astrocytes were unable to respond to stimulation whereas uninfected cells retained nearly normal responses. Maribavir partially restored structural features, including neural rosette formation, and dampened the impact of infection on neural cellular function. Using a tissue model system, we have demonstrated that HCMV alters cortical neural layering and disrupts calcium regulation in infected cells.IMPORTANCE Human cytomegalovirus (HCMV) replicates in several cell types throughout the body, causing disease in the absence of an effective immune response. Studies on HCMV require cultured human cells and tissues due to species specificity. In these studies, we investigated the impact of infection on developing three-dimensional cortical organoid tissues, with specific emphasis on cell-type-dependent calcium signaling. Calcium signaling is an essential function during neural differentiation and cortical development. We observed that HCMV infects and spreads within these tissues, ultimately disrupting cortical structure. Infected cells exhibited depleted calcium stores and loss of ATP- and KCl-stimulated calcium signaling while uninfected cells in the population maintained nearly normal responses. Some protection was provided by the viral inhibitor maribavir. Overall, our studies provide new insights into the impact of HCMV on cortical tissue development and function.