Efficient Antiviral State Research Articles

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes.

The first line of defense against viral infections is the innate immune response. Viruses are recognized by pathogen recognition receptors, such as the RIG-I like receptor family, that activate a signaling cascade that induces IFN production. In the present study, we visualized, for the first time in cells, both in overexpression and endogenous levels, complexes formed among key proteins involved in this innate immune signaling pathway. Through different techniques we were able to analyze how these proteins are distributed and reorganized spatially within the cell in order to transmit the signal, leading to an efficient antiviral state. In addition, this work presents a new means by how, when, and where viral proteins can target these pathways and act against the host immune system in order to counteract the activation of the immune response.

β-Catenin Upregulates the Constitutive and Virus-Induced Transcriptional Capacity of the Interferon Beta Promoter through T-Cell Factor Binding Sites.

Rapid upregulation of interferon beta (IFN-β) expression following virus infection is essential to set up an efficient innate antiviral response. Biological roles related to the antiviral and immune response have also been associated with the constitutive production of IFN-β in naive cells. However, the mechanisms capable of modulating constitutive IFN-β expression in the absence of infection remain largely unknown. In this work, we demonstrate that inhibition of the kinase glycogen synthase kinase 3 (GSK-3) leads to the upregulation of the constitutive level of IFN-β expression in noninfected cells, provided that GSK-3 inhibition is correlated with the binding of β-catenin to the IFN-β promoter. Under these conditions, IFN-β expression occurred through the T-cell factor (TCF) binding sites present on the IFN-β promoter independently of interferon regulatory factor 3 (IRF3). Enhancement of the constitutive level of IFN-β per se was able to confer an efficient antiviral state to naive cells and acted in synergy with virus infection to stimulate virus-induced IFN-β expression. Further emphasizing the role of β-catenin in the innate antiviral response, we show here that highly pathogenic Rift Valley fever virus (RVFV) targets the Wnt/β-catenin pathway and the formation of active TCF/β-catenin complexes at the transcriptional and protein level in RVFV-infected cells and mice.

ID: 108: Spatial insights in the interaction between the RIG-I like receptor pathway and viral proteins

The innate immune response relies on a set of Pathogen Recognition Receptors (PRRs) that sensor pathogen patterns (PAMPs). RIG-I is a cytosolic PRR that detects 5’-triphosphate double-stranded RNAs produced during infection. Once activated, the pathway leads to the induction of type I IFN and proinflammatory cytokines, leading to a cellular antiviral state. Upon recognition of the RNA, RIG-I hydrolyzes ATP and changes its conformation to an active state. The CARD domains are then exposed and become K63-linked polyubiquitinated by E3-ligases, such as TRIM25. The activation of this pathway is complex and well characterized, but most of the spatio-temporal events, and the subcellular localization where the essential proteins interact, are still under interrogation. Through different techniques, we analyzed how these proteins form complexes that are distributed and reorganized spatially within the cell in order to create an efficient antiviral state. RIG-I is the main sensor for recognition of viruses such as Paramyxoviruses, Flaviviruses, Rhabdoviruses and Orthomyxoviruses. Many of them have developed numerous and different strategies to overcome the activation of the RLR pathway. We will discuss and show new insights on how, where, and when, viral proteins can counteract the activation of the RLR pathway. NS1 of Influenza A virus, or NS3/4A of Hepatitis C Virus, as an example, are IFN antagonistic viral proteins that interact with specific complexes in very well defined areas in the host cell in order to inhibit the antiviral state in an infected cell.

Inhibition of Borna disease virus multiplication by interferon: cell line differences in susceptibility.

It has previously been reported that de novo infection of primary rabbit brain cells with Borna disease virus (BDV) can be blocked with interferon-alpha/beta (IFN), whereas this cytokine has no inhibitory effect on BDV in persistently infected rat lung cells [v. Rheinbaben et al., J. Gen. Virol. (1985) 66: 2,777-2,780]. It remained unclear, however, whether these results indicated that IFN exclusively targets early steps of the BDV replication cycle or whether they simply reflected cell line differences. We now show that BDV replication was effectively inhibited by IFN in both acutely and persistently infected monkey Vero cells. By contrast, IFN had no clear protective effect on either de novo or persistent BDV infections of rat C6 glioblastoma cells. IFN protected C6 cells from the cytopathic effects of vesicular stomatitis virus, excluding the possibility that these cells are devoid of a functional IFN system. In primary rat fibroblasts and in a human oligodendroglial cell line, IFN induced an efficient antiviral state against BDV. These results indicate that BDV is highly susceptible to the antiviral effect of IFN in some cell lines, while others seem to lack undefined components of the IFN system which mediate protection against BDV.

Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins.

In mouse Mx+ cells, interferon alpha/beta induces the synthesis of the nuclear Mx protein, whose accumulation is correlated with specific inhibition of influenza viral protein synthesis. When Mx+ mouse cells are microinjected with the monoclonal anti-Mx antibody 2C12, interferon alpha/beta still induces Mx protein, but no longer inhibits efficiently the expression of influenza viral proteins as visualized by immunofluorescent labeling. However, interferon inhibition of an unrelated control virus, vesicular stomatitis virus, remains unchanged. Proteins with homology to mouse Mx protein are found in interferon-treated cells of a variety of mammalian species. In rat cells, for instance, rat interferon alpha/beta induces three Mx proteins which all cross-react with antibody 2C12 but differ in mol. wt and intracellular location, and it protects these cells well against influenza viruses. However, when rat cells are microinjected with antibody 2C12, interferon alpha/beta cannot induce an efficient antiviral state against influenza virus infection, whereas protection against vesicular stomatitis virus is not altered. These results show that both mouse and rat cells require functional Mx proteins for efficient protection against influenza virus. They further demonstrate that microinjection of antibodies is a promising way of elucidating the role of particular interferon-induced proteins in the intact cell.

The 2–5a system in control and interferon treated k/balb cells infected with encephalomyocarditis virus

Summary Murine K/BALB cells have constitutively high levels of 2–5A synthetase, but are not resistant to infection by viruses; interferon treatment is necessary for the induction of an antiviral state in these cells; therefore we studied the 2–5A system (2–5A synthetase, 2–5A-dependent endonuclease, in vivo synthesis of 2–5A and activation of the endonuclease) in these K/BALB cells in parallel with murine L-929 cells, which have been used previously to illustrate the 2–5A system. The synthesis of 2-5A and the activation of the 2–5A-dependent nuclease occur in both control and interferon-treated K/BALB cells upon infection with encephalomyocarditis virus (EMCV). However, the level and the activity of the nuclease in control cells are very low compared to interferon-treated cells. In interferon-treated, EMCV-infected K/BALB or L-929 cells, comparable amounts of 2–5A were detectable, whereas the endonuclease activity observed in vivo was much lower in K/BALB cells. Accordingly, inhibition of EMCV growth by interferon was found to be 100 times less efficient in K/BALB cells than in L-929 cells. These results suggest that the level and the activity of the 2–5A dependent endonuclease might be crucial for the establishment of an efficient antiviral state in K/BALB cells. It should be noted, however, that the double-stranded RNA-dependent protein kinase activity is enhanced in K/BALB cells treated with interferon, and thus might also be responsible, at least in part, for the antiviral action.

Inhibition of influenza viral mRNA synthesis in cells expressing the interferon-induced Mx gene product

Interferons alpha and beta induce an efficient antiviral state against influenza virus in mouse cells that possess the Mx gene, but not in mouse cells that lack this gene. In Mx-containing cells treated with interferon the amount of viral mRNA synthesized as a result of primary transcription is drastically reduced. Only two viral mRNAs could be detected by Northern analysis and by translating the poly(A)+ RNA from infected cells in wheat germ extracts: a reduced amount of the mRNA for nonstructural protein 1 and an even lower amount of the mRNA for the matrix protein. The other viral mRNAs were not made in detectable amounts. In addition, the rate of viral mRNA synthesis catalyzed by the inoculum transcriptase, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, interferon treatment of cells lacking the Mx gene had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. These results indicate that the interferon-induced Mx gene product, a 75,000-molecular-weight protein that accumulates in the nucleus, inhibits influenza viral mRNA synthesis which occurs in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed.

Interferon-induced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses

In mouse cells carrying the dominant influenza resistance allele M x + (but not in M x − cells) interferon-α/β (IFN) induces an efficient antiviral state against influenza viruses and, concomitantly, the synthesis of a 75,000-Da protein (protein Mx). Here, indirect immunofluorescence using monoclonal antibodies was used to demonstrate that protein Mx accumulates in the nucleus of IFN-treated M x + cells, suggesting a nuclear site of action. Protein Mx is present in the nucleus of untreated influenza virus-resistant macrophages freshly explanted from the peritoneal cavity of M x + mice but is lost with time in culture when peritoneal macrophages become permissive for influenza virus.

Mx-dependent resistance to influenza viruses is induced by mouse interferons α and β but not γ

In cells derived from congenic BALB.A2G- Mx mice carrying the resistance gene Mx, but not in cells from BALB/c mice lacking Mx, mouse interferons α and β induced the synthesis of a unique cellular protein that was associated with an efficient antiviral state with selectivity for influenza viruses. In contrast, native or recombinant mouse interferon γ failed to efficiently protect Mx-bearing cells against influenza viruses and did not noticeably induce the synthesis of the Mx-associated protein, although interferon γ was as effective as interferons α and β in protecting BALB.A2G- Mx and BALB/c cells against the rhabdovirus VSV. These results demonstrate that different types of interferons differentially regulate the expression of the Mx gene and thereby induce distinct antiviral states.

Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus.

Mouse cells carrying the dominant resistance gene Mx develop a more efficient antiviral state toward influenza viruses in response to interferon than do Mx-negative cells. We have identified an Mx gene-associated product by labeling cultured peritoneal macrophages and embryonic cells with [35S]methionine in the presence or absence of interferon. The radioactive proteins from unfractionated cytoplasmic extracts were separated electrophoretically in two dimensions and were revealed by fluorography. A protein with a Mr of 72,500 and an isoelectric point of 6.3 was induced by mouse interferon type I (a mixture of alpha and beta interferons) in cells carrying the gene Mx but not in cells lacking Mx. The induction of this protein could be blocked by actinomycin D. The maximal rate of synthesis was reached in embryonic cells 4-5 hr after treatment with 10(3) reference units of interferon per ml. When the allele Mx (present in the inbred mouse strain A2G) was repeatedly backcrossed on different genetic backgrounds (BALB/c, C57BL/6, A/J), a clear correlation between the inducibility by interferon of this protein and the presence of the allele Mx was observed. The results suggest that this protein induced by the interaction of interferon with Mx plays a role in the selective antiviral state against influenza viruses that is observed in interferon-treated Mx-bearing cells.