Argininosuccinate synthase 1 (ASS1) orchestrates arginine metabolism and ornithine production to modulate CHIKV infection.

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 1 (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. IMPORTANCE: Metabolic reprogramming of the host is crucial for the virus to establish itself within the cell, and in this process, the virus hijacks several host metabolic pathways. We examined the role of an important arginine metabolizing enzyme, human argininosuccinate synthase (ASS1), during CHIKV infection in liver cells through silencing and overexpressing ASS1 and by L-arginine supplementation. We demonstrate that ASS1 favors CHIKV replication and also plays important roles in several downstream cellular processes during virus infection. This study further deepens our understanding of the significance of the crucial metabolites involved in the arginine metabolism pathway during CHIKV infection and how CHIKV exploits the specific pathway to enhance its replication.