Argininosuccinate synthetase 1 depletion produces a metabolic state conducive to herpes simplex virus 1 infection

Abstract

Herpes simplex virus 1 (HSV-1) infection triggers specific metabolic changes in its host cell. To explore the interactions between cellular metabolism and HSV-1 infection, we performed an siRNA screen of cellular metabolic genes, measuring their effect on viral replication. The screen identified multiple enzymes predicted to influence HSV-1 replication, including argininosuccinate synthetase 1 (AS1), which consumes aspartate as part of de novo arginine synthesis. Knockdown of AS1 robustly enhanced viral genome replication and the production of infectious virus. Using high-resolution liquid chromatography-mass spectrometry, we found that the metabolic phenotype induced by knockdown of AS1 in human fibroblasts mimicked multiple aspects of the metabolic program observed during HSV-1 infection, including an increase in multiple nucleotides and their precursors. Together with the observation that AS1 protein and mRNA levels decrease during wild-type infection, this work suggests that reduced AS1 activity is partially responsible for the metabolic program induced by infection.metabolomics | herpesviruses M any viruses, including herpes simplex virus 1 (HSV-1), human cytomegalovirus (HCMV), influenza, Dengue, hepatitis C, and hepatitis E (1-11), have been shown to significantly perturb metabolic homeostasis over the course of infection. HSV-1 is an intriguing case study, because the virus encodes several of its own metabolic enzymes, including a dUTPase, uracil-DNA glycosylase, ribonucleotide reductase, and thymidine kinase (12). Infection of host cells by multiple strains of HSV-1 induces alterations to central carbon metabolism, such as increased flux through upper glycolysis, and the anapleurotic feeding of carbons into the citric acid (TCA) cycle through the activity of pyruvate carboxylase (1). Infection also induces nucleotide synthesis, manifested by the increased flux of carbons through aspartate to nucleotides, as well as the increased pool levels of multiple nucleotides themselves (1).Although the metabolic reprogramming that occurs during HSV-1 infection has been described, the importance of specific host cell metabolic enzymes to infection is relatively unknown. Here we sought to further resolve the relationship between infection and metabolism by screening for cellular metabolic enzymes that modulate the efficiency of viral infection in primary human fibroblasts. The screen identified several dozen enzymes that modulate HSV-1 growth, including argininosuccinate synthetase (AS1), which antagonized viral replication.AS1 acts as a homotetramer to catalyze the synthesis of argininosuccinate from aspartate and citrulline (13,14). The enzyme plays a role in the production of urea in the kidney and liver, but functions primarily as the limiting step in the citrulline-nitric oxide (NO) cycle in other cell types (15). As a constituent of the citrulline-NO cycle, AS1 participates in the de novo production of the nonessential amino acid arginine as well as the soluble factor NO.We found that AS1 knockdown by siRNA in...

Chemical Methods for Probing Virus–Host Proteomic Interactions

“…The HSV-1 and HCMV infection metabolomes were also studied in the context of siRNA knockdown screening of cellular enzymes by Grady et al, 118 who identified the argininosuccinate synthase 1 (AS1) as a single key metabolic enzyme whose down-regulation mimics an HSV-1 infection state. Contrarily to HSV-1 infection, AS1 knockdown cells decreased HCMV infection, emphasizing the disparity between the modes of action of these two viruses on host cell metabolism.…”

Activity-based proteomic and metabolomic approaches for understanding metabolism

“…HCMV infected cells showed enhanced glycolytic flux and TCA cycle to fuel fatty acid biosynthesis (68). Furthermore, reducing the expression of a single metabolic enzyme, argininosuccinate synthetase (AS1), was sufficient to mimic these HSV-1 induced metabolomic changes to improve viral replication (69). These metabolomic approaches can point to potential new sites for antiviral therapy (70).…”

Exploring Metabolic Pathways and Regulation through Functional Chemoproteomic and Metabolomic Platforms

“…Grady et al used an siRNA screen to show that argininosuccinate synthase 1 (ASS1) knockdown increased virus yield and subsequently used metabolomic profiling to show that ASS1 inactivation resulted in a metabolic signature that closely resembled HSV-1 infection, in which levels of aspartate, carbamoyl-aspartate, one of the first committed metabolites on the pathway to nucleotide synthesis, and nucleotides and their precursors were reduced (Grady et al, 2013). …”