Nitric oxide production is downregulated during respiratory syncytial virus persistence by constitutive expression of arginase 1
Viral persistence alters cellular antiviral activities. Nitric oxide (NO), a highly reactive free radical and a potent antiviral molecule, can inhibit replication of RNA and DNA viruses, but its production and effect during viral persistence are largely unknown. NO synthesis is stimulated in epithelial cells during acute infection with respiratory syncytial virus (RSV) and interferes with viral replication. In this study, we compared the levels of production of NO and expression of its regulatory enzymes, inducible nitric oxide synthase (NOS II) and arginase 1 (Arg-1), during acute and persistent RSV infection in a macrophage cell line to investigate their role in the control and maintenance of viral infection. We observed that NO and NOS II mRNA were induced at higher levels in acutely infected macrophages than in persistently infected macrophages, while the kinetics of NOS II protein expression were similar in both types of infected cultures, except that its disappearance was delayed during acute infection. Thus, NOS II was inducible and expressed at high levels during persistent infection, but production of NO was low relative to acute infection. This was not associated with a lack of enzymatic activity but instead was due to constitutive expression of the Arg-1 enzyme at the mRNA and protein levels, suggesting that arginase restricts availability of L-arginine as a substrate for NOS II to synthesize NO. This hypothesis was supported by showing that arginase enzymatic activity was inhibited in persistently RSV-infected cells by Nω-hydroxy-nor-L-arginine, increasing L-arginine availability in conditioned medium and producing increased levels of nitrites, concurrently with a significant reduction in virus genome replication, implying that Arg-1 overexpression contributes to the maintenance of the RSV genome in the host in persistent infection.
The authors thank Jorge Gaona Bernal and Juan Miranda Ríos for helpful discussions and constructive comments during the course of this work. Also, thanks to Arturo A. Wilkins Rodríguez and Laila Gutiérrez Kobeh of the Research Unit for Translational Medicine, UNAM, for technical assistance with arginase activity determinations, and Isabel Ibarra-González and Marcela Vela-Amieva of the Laboratory of Inborn Errors of Metabolism and Screening of the National Institute of Pediatrics for technical assistance in L-arginine determinations, and finally, to Ana Flisser for English editing and the facilities granted for the culmination of the paper. This paper is part of the fulfillment of the requirements for the PhD degree of CSO within the Posgrado en Ciencias Biológicas of Universidad Nacional Autónoma de México.
This research was supported by grants from the Consejo Nacional de Ciencia y Tecnología, México (Grant 179838), by the Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (Grant PAPIIT IN218916), and by the School of Medicine, UNAM.
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Conflict of interest
The authors declare that they have not conflict of interest.
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