Abstract
Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production.
Highlights
Respiratory syncytial virus (RSV) is a leading etiological agent of lower respiratory tract infections in young children and immunocompromised individuals [1,2,3]
Colocalization of RSV nonstructural protein 1 (NS1) with mitochondria of infected cells In an effort to track the very early events of RSV infection, we used 4-D live cell imaging (Perkin Elmer UltraVIEWHVoX 3D Live Cell Imaging System) of A549 cells stained with live mitochondrial stain CMXRos and infected with rgRSV at 1 multiplicity of infection (MOI)
The viral RNA is detected by RIG-I, which interacts with mitochondrial antiviral signaling protein (MAVS) leading to the induction of NFkB-responsive genes and type-I IFN production [30,31]
Summary
Respiratory syncytial virus (RSV) is a leading etiological agent of lower respiratory tract infections in young children and immunocompromised individuals [1,2,3]. RSV infection causes an estimated 64 million cases of respiratory disease and 166,000 deaths annually worldwide and RSV-induced bronchiolitis and pneumonia result in over 2000 deaths and 100,000 hospitalizations annually in the USA [4]. RSV infections recur throughout life since immunity to natural RSV infections fails to generate protective memory responses [5]. Previous efforts to generate a vaccine to RSV using formalin-inactivated virus led to enhanced respiratory disease and the deaths of two vaccinated children after later RSV infection [6,7]. It was found that formalinRSV vaccination failed to adequately stimulate receptors of innate immunity, such as Toll-like receptors (TLRs) [8]. New approaches for RSV antivirals involve targeting the RSV proteins that enhance infection and viral survival through RNA interference with the nucleocapsid [9,10,11] and nonstructural protein 1
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