Hans Henrik Gad

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.

Human interferon-|[lambda]|3 is a potent member of the type III interferon family

Type III interferon (IFN) or IFN-λ is a recently discovered family of IFNs that signal through the same downstream transcription factors as type I IFN but use a separate receptor complex composed of the IL-10R2 and the unique IFN-λR1 receptor chains. We have established a simple and efficient expression system to produce highly pure and active IFN-λ of the three human IFN-λ isoforms (IFN-λ1, -λ2 and -λ3) and used this to compare the biological activity of the different IFN-λ subtypes. Surprisingly, we found IFN-λ3 to possess the highest specific activity of the human IFN-λ subtypes, exhibiting a twofold higher activity than IFN-λ1 and a 16-fold higher activity than IFN-λ2. Furthermore, in comparison with the commercially available preparations of IFN-λ1 and -λ2, we found our IFN-λ preparation to be superior in activity.

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

Functional IRF3 deficiency in a patient with herpes simplex encephalitis

Andersen et al. identify a novel genetic etiology of herpes encephalitis in an adult patient carrying a heterozygous loss-of-function mutation in the IRF3 gene. This mutation results in impaired INF production in response to viral infection

IFNλ is a potent anti‐influenza therapeutic without the inflammatory side effects of IFNα treatment

Influenza A virus (IAV)‐induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαβ) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV‐infected Mx1‐positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ‐treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non‐inflammatory and hence superior treatment option for human IAV infection.

Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2′-5′ linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.

The 2=-5=-Oligoadenylate Synthetase 3 Enzyme Potently Synthesizes the 2=-5=-Oligoadenylates Required for RNase L Activation

ABSTRACT 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. IMPORTANCE We are the first to purify the OAS3 enzyme to homogeneity, which allowed us to characterize the mechanism utilized by OAS3 and identify the active site. We provide compelling evidence that OAS3 can produce 2′-5′-oligoadenylates of sufficient length to activate RNase L. This is contrary to what is described in the current literature but agrees with recent in vivo data showing that OAS3 harbors an antiviral activity requiring RNase L. Thus, our work redefines our understanding of the biological role of OAS3. Furthermore, we used a combination of mutagenesis and small-angle X-ray scattering to describe the active site and low-resolution structure of OAS3.

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.

Selection of a Novel and Highly Specific Tumor Necrosis Factor α (TNFα) Antagonist INSIGHT FROM THE CRYSTAL STRUCTURE OF THE ANTAGONIST-TNFα COMPLEX

Inhibition of tumor necrosis factor α (TNFα) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFα inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFα as a test target to validate this new technology because of the extensive experience available with protein-based TNFα antagonists. Here, we present a novel and highly specific TNFα antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFα complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFα.Inhibition of tumor necrosis factor alpha (TNFalpha) is a favorable way of treating several important diseases such as rheumatoid arthritis, Crohn disease, and psoriasis. Therefore, an extensive range of TNFalpha inhibitory proteins, most of them based upon an antibody scaffold, has been developed and used with variable success as therapeutics. We have developed a novel technology platform using C-type lectins as a vehicle for the creation of novel trimeric therapeutic proteins with increased avidity and unique properties as compared with current protein therapeutics. We chose human TNFalpha as a test target to validate this new technology because of the extensive experience available with protein-based TNFalpha antagonists. Here, we present a novel and highly specific TNFalpha antagonist developed using this technology. Furthermore, we have solved the three-dimensional structure of the antagonist-TNFalpha complex by x-ray crystallography, and this structure is presented here. The structure has given us a unique insight into how the selection procedure works at a molecular level. Surprisingly little change is observed in the C-type lectin-like domain structure outside of the randomized regions, whereas a substantial change is observed within the randomized loops. Thus, the overall integrity of the C-type lectin-like domain is maintained, whereas specificity and binding affinity are changed by the introduction of a number of specific contacts with TNFalpha.