INTRODUCTION
Respiratory syncytial virus (RSV) is a leading cause of viral pneumonia in infants, the elderly, and immunocompromised patients. For the year 2015, approximately 3.2 million hospital admissions due to acute low respiratory tract infection in children younger than 5 years were attributed to RSV worldwide ( 1 ). Clinically approved and effective therapeutic options are scarce, with prophylactic monoclonal antibodies (i.e., palivizumab) ( 2 ) and antiviral compounds such as ribavirin showing variable clinical outcomes ( 3 ). No licensed vaccine is currently available. Therefore, understanding host-virus interactions and mechanisms governing the ability of RSV to alter the immune response is essential for the development of effective therapeutic and prophylactic interventions.
An emerging branch of evidence shows that innate immune responses are regulated by a process known as autophagy. Autophagy is at the center of cellular homeostasis, as this process degrades damaged proteins and organelles in specialized enclosed compartments. The by-products of such degradation are then recycled by the cell, a feature useful during stress and nutrient starvation under which autophagy preserves cell viability (reviewed in reference 4 ). The autophagy pathway comprises a network of interacting protein complexes that drive the formation of membranous compartments known as autophagosomes ( 5 ). The autophagy core complex consists of VPS15-VPS34-ATG14-Beclin1, which reorganizes membranes derived from cytoplasmic organelles, converting them into the phagophore and autophagosome that ultimately drive autophagy. Autophagy can be regulated by posttranslational modifications in its core complex proteins. For instance, recent work indicates that the proautophagy mediator Beclin1 is subject to ISGylation, resulting in inhibition of autophagy ( 6 ). ISGylation entails the covalent conjugation of interferon-stimulated gene 15 (ISG15) to target proteins ( 7 8 ) as a means to regulate a wide spectrum of cellular activities, including autophagy. To date, there are no reports of autophagy regulation by viral proteins through modulation of ISGylation. In the current study, we show that RSV NS2 protein deregulates ISGylation of Beclin1 to promote autophagy during infection.
Autophagy participates in immune responses against virus infections by degrading viral proteins and blocking virus spread ( 9 ), activating and signaling Toll-like receptors (TLRs) ( 10 ), and mediating secretion of immune modulators ( 11 ) and cytokines ( 10 ), among numerous others. RSV infection induces autophagy in cultured lung epithelial cells, mouse airway cells, dendritic cells, and macrophages ( 10 14 – 12 ). RSV-induced autophagy contributes to maturation and cytokine expression in dendritic cells ( 12 ) and activation of the TGF-β/SMAD signaling pathway in macrophages ( 14 ). Despite the various roles of autophagy during RSV infection, the mechanisms responsible for triggering RSV-induced autophagy remain unknown. Specifically, the role of any viral component in inducing autophagy during RSV infection has not yet been identified. Here, we identify RSV nonstructural protein 2 (NS2) as a key regulator of autophagy during infection.
Single-stranded negative-sense RNA viruses encode several nonstructural proteins. Orthomyxoviruses (e.g., influenza viruses) encode NS1, while paramyxoviruses encode nonstructural proteins such as NS1 and NS2 (RSV), C protein (human parainfluenza 3, measles, Nipah, and Hendra viruses), V protein (human parainfluenza 3, mumps, measles, Nipah, and Hendra viruses), and W protein (measles, Nipah, and Hendra viruses). These proteins are not packaged in the virus particle and are expressed only in infected cells. Nonstructural proteins of single-stranded negative-sense RNA viruses antagonize the innate immune response by interfering with type I interferon signaling and inflammasome-mediated interleukin-1beta (IL-1β) production. To date, the role of RSV nonstructural proteins in autophagy remains unexplored.
Our current study highlights a new role of nonstructural proteins by showing that RSV NS2 mediates RSV-induced autophagy. Our data indicate that NS2 induces autophagy by interacting with the proautophagy mediator Beclin1, preventing its degradation and leading to enhanced Beclin1 protein levels and functional autophagy. Surprisingly, NS2 also hinders ISGylation of Beclin1, partially relieving the antiautophagy effect of ISGylated Beclin1 and enabling Beclin1 to proceed with autophagy. Studies with NS2-deficient RSV have revealed that NS2 contributes to RSV-mediated autophagy during infection. Thus, our current study identifies RSV NS2 as a proautophagic viral factor and provides a mechanistic model for RSV-induced autophagy in which NS2 employs a dual approach: increase Beclin1 protein levels by preventing its degradation and relieve it from ISGylation to trigger autophagy.