Ole J. Hamming

Type III Interferon (IFN) Induces a Type I IFN-Like Response in a Restricted Subset of Cells through Signaling Pathways Involving both the Jak-STAT Pathway and the Mitogen-Activated Protein Kinases

ABSTRACT Type III interferon (IFN) is a novel member of the interferon family. Type III IFN utilizes a receptor complex different from that of type I IFN, but both types of IFN induce STAT1, STAT2, and STAT3 activation. Here we describe a detailed comparison of signal transduction initiated by type I and type III IFN. Gene expression array analysis showed that IFN types I and III induced a similar subset of genes. In particular, no genes were induced uniquely by type III IFN. Next, we used chromatin immunoprecipitation (ChIP) analysis to investigate the promoter activation by types I and III IFN. The ChIP assays demonstrated that stimulation of cells with both type I and type III IFN resulted in the recruitment of ISGF3 transcription factor components to the promoter region of responsive genes and in an increase of polymerase II loading and histone acetylation. Whereas IFN type I signaling was observed for a broad spectrum of cell lines, type III IFN signaling was more restricted. The lack of IFN type III signaling was correlated with a low expression of the IL28Ra component of the IFN type III receptor, and IL28Ra overexpression was sufficient to restore IFN type III signaling. We also tested the activation of mitogen-activated protein (MAP) kinases by type III IFN and found that type III IFN relies strongly upon both p38 and JNK MAP kinases for gene induction.

Interferon-λ Is Functionally an Interferon but Structurally Related to the Interleukin-10 Family

Interferon-λ (IFN-λ) is an antiviral cytokine that signals through a distinct receptor complex, composed of the IFN-λR1 and interleukin-10R2 (IL-10R2) receptor chains. We have determined the crystal structure of human IFN-λ3 and characterized the interaction with its receptor complex through structure-based site-directed mutagenesis. The ability of IFN-λ3 mutants to signal was determined by measuring the antiviral activity and induced STAT2 phosphorylation. In conclusion, our data show that, although IFN-λ is functionally an interferon, it is clearly structurally related to members of the IL-10 family. In particular, we found an interesting similarity between IFN-λ and IL-22, and we suggest that IFN-λ and IL-22 possess parallel functions, protecting epithelial tissue against viral and bacterial infections, respectively.

The Two Groups of Zebrafish Virus-Induced Interferons Signal via Distinct Receptors with Specific and Shared Chains

Because the availability of fish genomic data, the number of reported sequences for fish type II helical cytokines is rapidly growing, featuring different IFNs including virus-induced IFNs (IFNφ) and IFN-γ, and IL-10 with its related cytokines (IL-20, IL-22, and IL-26). Many candidate receptors exist for these cytokines and various authors have postulated which receptor chain would be involved in which functional receptor in fish. To date, only the receptor for zebrafish IFNφ1 has been identified functionally. Three genes encoding virus-induced IFNφs have been reported in zebrafish. In addition to these genes clustered on chromosome 3, we have identified a fourth IFNφ gene on chromosome 12. All these genes possess the intron-exon organization of mammalian λ IFNs. In the zebrafish larva, all induce the expression of reporter antiviral genes; protection in a viral challenge assay was observed for IFNφ1 and IFNφ2. Using a combination of gain- and loss-of-function experiments, we also show that all zebrafish IFNφs do not bind to the same receptor. Two subgroups of fish virus-induced IFNs have been defined based on conserved cysteines, and we find that this subdivision correlates with receptor usage. Both receptor complexes include a common short chain receptor (CRFB5) and a specific long chain receptor (CRFB1 or CRFB2).

Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons

ABSTRACT In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.

Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential

ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections. A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses

The IFNL4 gene is a recently discovered type III interferon, which in a significant fraction of the human population harbours a frameshift mutation abolishing the IFNλ4 ORF. The expression of IFNλ4 is correlated with both poor spontaneous clearance of hepatitis C virus (HCV) and poor response to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL‐10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion of IFNλ4 is impaired compared to that of IFNλ3, and this impairment is not due to a weak signal peptide, which was previously believed. We found that IFNλ4 gets N‐linked glycosylated and that this glycosylation is required for secretion. Nevertheless, this glycosylation is not required for activity. Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection.

Crystal Structure of Zebrafish Interferons I and II Reveals Conservation of Type I Interferon Structure in Vertebrates

ABSTRACT Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFNφ1 and IFNφ2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-α] and beta interferon [IFN-β]) and type III IFNs (lambda interferon [IFN-λ]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFNφ1 and IFNφ2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.

Crystal Structure of Interleukin-21 Receptor (IL-21R) Bound to IL-21 Reveals That Sugar Chain Interacting with WSXWS Motif Is Integral Part of IL-21R

Background: The class I cytokine IL-21 exerts pleiotropic effects on innate and adaptive immunity. Results: We obtained the crystal structure of the partially glycosylated IL-21 receptor (IL-21R) bound to IL-21. Conclusion: A sugar chain is an integral part of IL-21R. Significance: This structure offers an insight into the putative role of the class I cytokine receptor signature motif. IL-21 is a class I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. It signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common γ-chain. A hallmark of the class I cytokine receptors is the class I cytokine receptor signature motif (WSXWS). The exact role of this motif has not been determined yet; however, it has been implicated in diverse functions, including ligand binding, receptor internalization, proper folding, and export, as well as signal transduction. Furthermore, the WXXW motif is known to be a consensus sequence for C-mannosylation. Here, we present the crystal structure of IL-21 bound to IL-21R and reveal that the WSXWS motif of IL-21R is C-mannosylated at the first tryptophan. We furthermore demonstrate that a sugar chain bridges the two fibronectin domains that constitute the extracellular domain of IL-21R and anchors at the WSXWS motif through an extensive hydrogen bonding network, including mannosylation. The glycan thus transforms the V-shaped receptor into an A-frame. This finding offers a novel structural explanation of the role of the class I cytokine signature motif.

The Structure of Human Interferon Lambda and What It Has Taught Us

Type III interferon (IFN) or IFN-lambda is a novel family of class II cytokines that induces antiviral activities both in vitro and in vivo through its own distinctive receptor complex. The recent crystal structure of human IFN-lambda3 revealed a cytokine with structural similarity to the interleukin-10 family, despite functionally being an IFN. Here, we review the structure of IFN-lambda and its relation to the other members of the class II cytokines. Further, we analyze the structural differences between the tree human isoforms of IFN-lambda and relate this to the observed differences in potency.

Lambda Interferons: New Cytokines with Old Functions

Interferon lambda (IFN-λ) is a member of the class II cytokine family, and like the other members of this family, they are small helical proteins. Since their discovery significant efforts have been made to determine their role in innate and adaptive immunity. Their strong antiviral activity, both in vitro and in vivo, has firmly established their interferon status. However, in contrast to type I interferon, only a very limited subset of cells/tissues responds to interferon lambda. In addition to inducing an antiviral state in responsive cells, recent data suggest that IFN-λ plays a role in shaping the adaptive immune response. However, the data is not in complete agreement regarding the effect of IFN-λ on the adaptive immune system. Recently IFN-λ has entered clinical trials against hepatitis C Virus and IFN-λ is a promising future therapeutic, against different viruses replicating in responsive tissues, like that of the airway epithelia. In this review we describe the knowledge acquired during the past six years about the structure and function of interferon lambda.