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Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made toward identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and IFN-stimulated gene (ISG) response is another antiviral strategy that has been known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the proinflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, has been shown to be less proinflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV-infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to that of IFN-α-treated cells. Similarly, mice treated with two doses of IFN-λ were better protected than mice that received a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from a lethal Middle East respiratory syndrome CoV (MERS-CoV) infection. A two-dose IFN-λ regimen significantly reduced lung viral titers and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identified an effective regimen for IFN-λ use and demonstrated the protective efficacy of IFN-λ in MERS-CoV-infected mice. IMPORTANCE Effective antiviral agents are urgently required to prevent and treat individuals infected with SARS-CoV-2 and other emerging viral infections. The COVID-19 pandemic has catapulted our efforts to identify, develop, and evaluate several antiviral agents. However, a narrow therapeutic window has limited the protective efficacy of several broad-spectrum and CoV-specific antivirals. IFN-λ is an antiviral agent of interest due to its ability to induce a robust endogenous antiviral state and low levels of inflammation. Here, we evaluated the protective efficacy and effective treatment regimen of IFN-λ in mice infected with a lethal dose of MERS-CoV. We show that while prophylactic and early therapeutic IFN-λ administration is protective, delayed treatment is detrimental. Notably, a combination of prophylactic and delayed therapeutic administration of IFN-λ protected mice from severe MERS. Our results highlight the prophylactic and therapeutic use of IFN-λ against lethal hCoV and likely other viral lung infections.

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the devastating COVID-19 lung disease pandemic. Here, we tested the inhibitory activities of the antiviral interferons of type I (IFN-α) and type III (IFN-λ) against SARS-CoV-2 and compared them with those against SARS-CoV-1, which emerged in 2003. Using two mammalian epithelial cell lines (human Calu-3 and simian Vero E6), we found that both IFNs dose-dependently inhibit SARS-CoV-2. In contrast, SARS-CoV-1 was restricted only by IFN-α in these cell lines. SARS-CoV-2 generally exhibited a broader IFN sensitivity than SARS-CoV-1. Moreover, ruxolitinib, an inhibitor of IFN-triggered Janus kinase/signal transducer and activator of transcription signaling, boosted SARS-CoV-2 replication in the IFN-competent Calu-3 cells. We conclude that SARS-CoV-2 is sensitive to exogenously added IFNs. This finding suggests that type I and especially the less adverse effect-prone type III IFN are good candidates for the management of COVID-19., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Felgenhauer et al.)

Host factors restricting the transmission of respiratory viruses are poorly characterized. We analyzed the contribution of type I and type III interferon (IFN) using a mouse model in which the virus is selectively administered to the upper airways, mimicking a natural respiratory virus infection. Mice lacking functional IFN-λ receptors ( Ifnlr1 -/- ) no longer restricted virus dissemination from the upper airways to the lungs. Ifnlr1 -/- mice shed significantly more infectious virus particles via the nostrils and transmitted the virus much more efficiently to naïve contacts compared with wild-type mice or mice lacking functional type I IFN receptors. Prophylactic treatment with IFN-α or IFN-λ inhibited initial virus replication in all parts of the respiratory tract, but only IFN-λ conferred long-lasting antiviral protection in the upper airways and blocked virus transmission. Thus, IFN-λ has a decisive and non-redundant function in the upper airways that greatly limits transmission of respiratory viruses to naïve contacts., Competing Interests: JK, DS, LY, MS, HG, RH, DG, TM, PS No competing interests declared, (© 2018, Klinkhammer et al.)

The type III interferon (IFN-λ) family includes 4 IFN-λ subtypes in man. In the mouse, only the genes coding for IFN-λ2 and -λ3 are present. Unlike mouse and human type I IFNs (IFN-α/β), which exhibit strong species specificity, type III IFNs were reported to act in a cross-specific manner. We reexamined the cross-specificity and observed that mouse and human IFN-λ exhibit some species specificity, although much less than type I IFNs. Mouse IFN-λ3 displayed clear species specificity, being 25-fold less active in human cells than the closely related mouse IFN-λ2. This specificity likely depends on amino acids in α helices A and F that diverged from other IFN-λ sequences. Human IFN-λ4, in contrast, retained high activity in mouse cells. We next developed a firefly luciferase-based reporter cell line, named Fawa-λ-luc, to detect IFN-λ in biological fluids with high specificity and sensitivity. Fawa-λ-luc cells, derived from mouse epithelial cells that are responsive to IFN-λ, were made nonresponsive to type I IFNs by inactivation of the Ifnar2 gene and strongly responsive to IFN-λ by overexpression of the mouse IFNLR1. This bioassay was as sensitive as a commercially available enzyme-linked immunosorbent assay in detecting mouse IFN-λ in cell culture supernatant, as well as in serum and bronchoalveolar lavage samples of virus-infected mice. The assay also enabled the sensitive detection of human IFN-λ activity, including that of the divergent IFN-λ4 with a bias, however, due to variable activity of IFN-λ subtypes. [ABSTRACT FROM AUTHOR]

The members of the oligoadenylate synthetase (OAS) family of proteins are antiviral restriction factors that target a wide range of RNA and DNA viruses. They function as intracellular double-stranded RNA (dsRNA) sensors that, upon binding to dsRNA, undergo a conformational change and are activated to synthesize 2'-5'-linked oligoadenylates (2-5As). 2-5As of sufficient length act as second messengers to activate RNase L and thereby restrict viral replication. We expressed human OAS3 using the baculovirus system and purified it to homogeneity. We show that recombinant OAS3 is activated at a substantially lower concentration of dsRNA than OAS1, making it a potent in vivo sensor of dsRNA. Moreover, we find that OAS3 synthesizes considerably longer 2-5As than previously reported, and that OAS3 can activate RNase L intracellularly. The combined high affinity for dsRNA and the capability to produce 2-5As of sufficient length to activate RNase L suggests that OAS3 is a potent activator of RNase L. In addition, we provide experimental evidence to support one active site of OAS3 located in the C-terminal OAS domain and generate a low-resolution structure of OAS3 using SAXS. [ABSTRACT FROM AUTHOR]

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-a and IFN-b) and III (IFN-l) interferons are host-produced antiviral cytokines. Prolonged IFN-a and IFN-b responses can lead to harmful proinflammatory effects, whereas IFN-l mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-l driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19). [ABSTRACT FROM AUTHOR]