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

Abstract

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.

Hallmarks of Metabolic Reprogramming and Their Role in Viral Pathogenesis

“…In HCMV-infected primary HFF cells, and during lytic replication, lipid synthesis is induced by the influx of carbons from glycolysis that enter the fatty acid synthesis pathway [ 123 ]. Along with the increase in metabolites in the fatty acid synthesis pathway, HCMV also induces the expression of many enzymes involved in this pathway as well, such as ACC1 and SREBPs [ 151 , 168 , 169 , 170 ]. HCMV causes an induction of the carbohydrate-response binding element protein (ChREBP).…”

Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells

“…Due to the gaseous nature of nitric oxide, a well-characterized nitric oxide-releasing donor diethlenetriamine/nitric oxide (DETA/ NO) was used as an inducer. 28 The gene circuits were activated by NO released from DETA/NO in the range of 1.5−200 μM, and the minimum detection limit was 1.5 μM DETA/NO (Figure 2B).…”

“…The gene circuits were initially designed using a simple regulatory and open-loop format (Figure A), in which the P norV promoter was fused with a superfolder gfp (sfGFP), and norR cloned from EcN was under the control of constitutive promoters with different strengths (P roA , P roC , and P roD ) (Figure A). Due to the gaseous nature of nitric oxide, a well-characterized nitric oxide-releasing donor diethlenetriamine/nitric oxide (DETA/NO) was used as an inducer . The gene circuits were activated by NO released from DETA/NO in the range of 1.5–200 μM, and the minimum detection limit was 1.5 μM DETA/NO (Figure B).…”

Nitric oxide for the prevention and treatment of viral, bacterial, protozoal and fungal infections

“…• Limit glutaminolysis by shuttling glutamine to glutathione synthesis, as in cytomegalovirus. 43 • Interaction with sulfhydryl-containing constituents of the bacterial cell. 44 • Disrupt zinc homeostasis, as in S. enterica.…”