Argininosuccinate Synthase 1 (ASS1) Orchestrates Arginine Metabolism and Ornithine Production to Modulate CHIKV Infection
Description
Abstract extracted from manuscript. Viruses reprogram the host metabolic machinery to ensure a continuous supply of macromolecules and energy for their own survival. Important cellular pathways are impacted during infection, resulting in changes in key metabolic precursors that influence infection outcomes. The present study was undertaken to evaluate the impact of L-arginine and argininosuccinate synthase (ASS1), an important upstream enzyme of the arginine metabolism pathway, during chikungunya virus (CHIKV) infection in the human liver-derived, Huh-7 cells. Using dose- dependent and time-course L-arginine supplementation experiments, we demonstrated that CHIKV exploits cellular arginine for enhanced viral replication. Loss-of-function and gain- of-function studies of ASS1, combined with nitric oxide donor treatments, revealed that arginine metabolism influences multiple downstream pathways, including ornithine synthesis, proline metabolism, and nitric oxide production during CHIKV infection. We further examined the relationship between ASS1 expression and STAT3 signaling in the context of viral infection. Our results demonstrate that exogenous L-arginine supplementation and ASS1 overexpression enhance CHIKV replication in Huh-7 cells. Conversely, ASS1 silencing resulted in 95% reduction in viral titers. Mechanistically, ASS1 modulated arginase 1 activity, affecting ornithine production and downstream metabolites, while also influencing the cellular nitroso-redox environment. Additionally, ASS1 expression affected STAT3 levels and its subcellular localization: ASS1 overexpression correlated with reduced nuclear STAT3 accumulation and increased viral replication, whereas ASS1 depletion promoted STAT3 nuclear translocation and restricted viral infection. These findings reveal a complex interplay between arginine metabolism, innate immune signaling, and CHIKV replication, identifying ASS1 as a potential regulatory node in CHIKV-host interactions.
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Methods extracted as it is from the manuscript. Metabolite extraction: Huh7 Cells were grown in cell culture plates to 80% confluency and harvested at 24 hpi. Post-harvesting, chilled methanol (80%, 800 μL) was added to quench the metabolic activities. To the cell extract, ribitol (1 μL, 0.5 mg/mL) was added as a spike in the standard, followed by vortexing the mixture at 900 RPM on the remixer (Eppendorf, USA) for 30 minutes at 4°C. These samples were further centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was transferred to a new tube and vacuum-dried at 40°C using a CentiVap vacuum concentrator (Labconco, USA). These dried metabolites were stored at -20°C till mass spectrometry analysis. Derivatization: To the dried samples, methoxylamine hydrochloride (Sigma, 30 μL) was added, and the mixture was briefly vortexed before incubation at 60°C and 900 RPM on a thermomixer for 1 hour. Then, the methoxylated samples were silylated using N-Methyl-N- (trimethylsilyl)trifluoroacetamide (MSTFA, 60 μL) with trimethylchlorosilane (TMCS, 1%) (Sigma, USA) by incubating at 60°C and 900 RPM for 1 hour. The reaction mixtures were centrifuged, and the supernatant was transferred into a glass insert containing GC vials for further GC-MS analysis. Commercial standards of the target molecules were also derivatized using the above protocol to confirm the identity of metabolites. GC-MS analysis: Metabolite profiling was performed on Leco GC-TOF (Leco, USA). An aliquot of the sample (1 μL) was injected onto the HP-5MS (Agilent) column in splitless mode using Helium as the carrier gas at a constant flow rate of 1 mL/min. The initial oven temperature was set at 50°C for 1 min, then raised to 150°C at a rate of 10°C/min and held for 3 min. Additionally, the temperature was ramped to 300°C at a rate of 7°C/min and held for 3 minutes. The transfer line and ion source temperatures were maintained at 260 °C, 230 °C, and 230°C, respectively. The energy was -70eV in electron impact mode. The acquisition rate and mass range were 50 spectra/second and 35-600 m/z, respectively. Data analysis: Chroma TOF software (4.50.8.0, Leco, USA) was used for peak picking, baseline correction, deconvolution, alignment, and integration from the raw files obtained from GC-TOF runs. The identities of the metabolic features were confirmed by matching their fragmentation patterns in the NIST library and their retention times with those from their commercial standards.
Institutions
- International Centre for Genetic Engineering and BiotechnologyDelhi, New Delhi