Extracellular ISG15 triggers ISGylation via a type-I interferon independent mechanism to regulate host response during virus infection.

Type-I interferons (IFN) are key antiviral factors that induce several cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity. Apart from ISGylation, unconjugated ISG15 is also released from cells during immuno-stimulation and virus infection. The role of extracellular ISG15 during virus infection was unknown. We now show that extracellular ISG15 acts as a soluble antiviral factor to restrict virus infection in human epithelial cells via an IFN-independent mechanism. Additionally, we demonstrated that extracellular ISG15 induces expression of intracellular ISG15 and ISGylation in the absence of IFN. Mechanistic studies revealed that extracellular ISG15 interacts with cell surface integrin (α5ß1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified ISG15 protein as a new soluble factor that confers IFN-independent antiviral activity by inducing ISGylation. Additionally, we have unfolded an IFN-independent non-canonical ISGylation by extracellular ISG15 that operates via the integrin-FAK pathway.

Proinflammatory Responses in SARS-CoV-2 and Soluble Spike Glycoprotein S1 Subunit Activated Human Macrophages.

Critically ill COVID-19 patients display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We inoculated and treated human macrophage cell line THP-1 with SARS-CoV-2 and purified, glycosylated, soluble SARS-CoV-2 spike protein S1 subunit (S1) to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication or viral entry, virus exposure resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that extracellular soluble S1 protein is a key viral component inducing pro-inflammatory responses in macrophages, independent of virus replication. Thus, virus- or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.

Human Respiratory Syncytial Virus NS2 Protein Induces Autophagy by Modulating Beclin1 Protein Stabilization and ISGylation.

Paramyxoviruses such as respiratory syncytial virus (RSV) are the leading cause of pneumonia in infants, the elderly, and immunocompromised individuals. Understanding host-virus interactions is essential for the development of effective interventions. RSV induces autophagy to modulate the immune response. The viral factors and mechanisms underlying RSV-induced autophagy are unknown. Here, we identify the RSV nonstructural protein NS2 as the virus component mediating RSV-induced autophagy. We show that NS2 interacts and stabilizes the proautophagy mediator Beclin1 by preventing its degradation by the proteasome. NS2 further impairs interferon-stimulated gene 15 (ISG15)-mediated Beclin1 ISGylation and generates a pool of "hypo-ISGylated" active Beclin1 to engage in functional autophagy. Studies with NS2-deficient RSV revealed that NS2 contributes to RSV-mediated autophagy during infection. The present study is the first report to show direct activation of autophagy by a paramyxovirus nonstructural protein. We also report a new viral mechanism for autophagy induction wherein the viral protein NS2 promotes hypo-ISGylation of Beclin1 to ensure availability of active Beclin1 to engage in the autophagy process. IMPORTANCE Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. Here, we identify the RSV nonstructural protein 2 (NS2) as the virus component involved in autophagy induction. NS2 promotes autophagy by interaction with and stabilization of the proautophagy mediator Beclin1 and by impairing its ISGylation to overcome autophagy inhibition. To the best of our knowledge, this is the first report of a viral protein regulating the autophagy pathway by modulating ISGylation of autophagy mediators. Our studies highlight a direct role of a paramyxovirus nonstructural protein in activating autophagy by interacting with the autophagy mediator Beclin1. NS2-mediated regulation of the autophagy and ISGylation processes is a novel function of viral nonstructural proteins to control the host response against RSV.

Proinflammatory responses in SARS-CoV-2 infected and soluble spike glycoprotein S1 subunit activated human macrophages.

Critically ill COVID-19 patients infected with SARS-CoV-2 display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We used SARS-CoV-2 infected and glycosylated soluble SARS-CoV-2 Spike S1 subunit (S1) treated THP-1 human-derived macrophage-like cell line to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication, virus infection resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that S1 is a key viral component inducing inflammatory response in macrophages, independently of virus replication. Thus, virus-infected or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.