Biomedical subjects

Georg Kochs

Publications and source records attributed to Georg Kochs.

At least 19 recordsLinked to original sources

PubMed2026

Autoantibodies against type I interferons in patients with zoonotic H7N9 influenza: an observational case-control study.

BACKGROUND: The determinants of the species barrier preventing human infections with avian influenza A viruses (IAV) are incompletely understood. We previously identified loss-of-function variants of the interferon-regulated antiviral factor MxA as a genetic factor for increased susceptibility to infections with the H7N9 subtype. Given the central role of type I IFNs (IFN-I) in antiviral defence, we hypothesised that IFN-I-neutralising autoantibodies may similarly predispose to zoonotic H7N9 infection. METHODS: In this observational case-control study, serum samples collected between 2013 and 2017 from 199 Chinese patients with laboratory-confirmed H7N9 infection and 531 healthy, uninfected controls (269 poultry workers, 262 close contacts) were screened for IgG autoantibodies binding IFNα2, IFNβ1b, or IFNω using a multiplex bead-based assay. Positive samples were tested for IFN-neutralising activity in a luciferase-based reporter assay. To confirm their ability to block IFNα2-mediated antiviral activity, selected samples (n = 19) were analysed in IAV infection experiments. Associations between age, sex, H7N9 case status, case fatality, and the presence of neutralising autoantibodies were evaluated by logistic regression. Available whole-genome sequencing data from 26 individuals with neutralising autoantibodies were screened for variants in genes linked to IFN-I autoimmunity. FINDINGS: Neutralising autoantibodies against at least one IFN-I were detected in 19.1% (38/199) of patients but in only 1.1% (6/531) of controls, consistent with published general population data. Most patient sera targeted IFNα2 and/or IFNω (35/199), and 18.1% (36/199) neutralised even high IFN-I concentrations of 1-10 ng/ml. The presence of neutralising autoantibodies was associated with 8.2- to 25.3-fold higher odds of H7N9 infection (p < 0.0001), depending on antibody specificity and reference group. Autoantibody prevalence increased significantly with age in patients (44.8% ≥70 years; OR = 1.05; 95% CI 1.02-1.07; p = 0.0001), but was not associated with sex (OR for males vs. females = 0.52; 95% CI 0.23-1.14; p = 0.106). All selected sera containing neutralising autoantibodies blocked IFNα2-induced antiviral activity in cell culture. No known genetic predisposition for IFN-I autoimmunity was identified. INTERPRETATION: Our findings suggest that IFN-I-targeting autoimmunity is associated with susceptibility to zoonotic IAV infection with the H7N9 subtype, and possibly also other subtypes, including panzootic H5N1. Given the ease of implementation, screening for anti-IFN-I autoantibodies could be readily integrated into surveillance or targeted testing. This could be relevant in environments with increased exposure to zoonotic IAVs. FUNDING: Shenzhen Medical Research Fund, National Natural Science Foundation of China, Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, Guangdong Provincial Science and Technology Program, Program for Youzuzhikeyan of Shenzhen University, German Research Foundation, Swiss National Science Foundation.

Humans↗

PubMed2007

The intracellular sites of early replication and budding of SARS-coronavirus.

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.

Animals↗

PubMed2006

Protective role of beta interferon in host defense against influenza A virus.

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.

Animals↗

PubMed2006

The interferon response circuit: induction and suppression by pathogenic viruses.

Type I interferons (IFN-alpha/beta) are potent antiviral cytokines and modulators of the adaptive immune system. They are induced by viral infection or by double-stranded RNA (dsRNA), a by-product of viral replication, and lead to the production of a broad range of antiviral proteins and immunoactive cytokines. Viruses, in turn, have evolved multiple strategies to counter the IFN system which would otherwise stop virus growth early in infection. Here we discuss the current view on the balancing act between virus-induced IFN responses and the viral counterplayers.

Animals↗

PubMed2006

Rapid and simple detection of IFN-neutralizing antibodies in chronic hepatitis C non-responsive to IFN-alpha.

Different mechanisms have been proposed for the failure of interferon (IFN) therapy in patients with chronic hepatitis C and multiple sclerosis, for example, the presence of IFN-neutralizing antibodies. In this study, a novel assay system based on the IFN-inducible Mx-promoter was used to detect IFN-neutralizing antibodies in sera of patients with chronic hepatitis C. To monitor IFN bioactivity in IFN-treated patients, a real-time RT-PCR for MxA gene expression in PBMCs was established. Using these two methods, patients with chronic hepatitis C virus (HCV) infection receiving IFN therapy and patients with treatment induced HCV clearance were monitored. Importantly, neutralizing anti-IFN antibodies were detected in the sera of 3 of 38 chronically HCV-infected patients who failed to respond to therapy but none in sera of patients who cleared HCV after IFN therapy. Interestingly, the presence of these antibodies correlated with the lack of MxA induction in PBMCs after initiation of IFN-alpha therapy. Retrospective analysis of one patient's sera revealed that the anti-IFN-alpha antibodies had already developed after the first of four unsuccessful IFN therapies, suggesting that neutralizing antibodies may have contributed to the failure of previous IFN treatments. In summary, a novel screening assay was established that may be helpful for testing IFN non-responders for the presence of clinically relevant anti-IFN-alpha antibodies and for selecting alternative IFN preparations not neutralized by these antibodies.

Adult↗

PubMed2006

Interferon-induced, antiviral human MxA protein localizes to a distinct subcompartment of the smooth endoplasmic reticulum.

Human MxA protein belongs to the superfamily of dynamin-like large GTPases that are involved in intracellular membrane trafficking. MxA is induced by interferons-alpha/beta (IFN-alpha/beta) and is a key component of the antiviral response against RNA viruses. Here, we show that MxA localizes to membranes that are positive for specific markers of the smooth endoplasmic reticulum, such as Syntaxin17, but is excluded from other membrane compartments. Overexpression of MxA leads to a characteristic reorganization of the associated membranes. Interestingly, Hook3, mannose-6-phosphate receptor, and Lamp-1, which normally accumulate in cis- Golgi, endosomes, and lysosomes, respectively, also colocalized with MxA, indicating that these markers were redistributed to the MxA-positive compartment. Functional assays, however, did not show any effect of MxA on endocytosis or the secretory pathway. The present results demonstrate that MxA is an IFN-induced antiviral effector protein that resembles the constitutively expressed large GTPase family members in its capacity to localize to and reorganize intracellular membranes.

Animals↗

PubMed2005

Thogoto virus ML protein suppresses IRF3 function.

The Thogoto virus (THOV) is a member of the family Orthomyxoviridae. It prevents induction of alpha/beta interferons (IFN) in cell culture and in vivo via the action of the viral ML protein. Phenotypically, the effect of THOV ML resembles that of the NS1 protein of influenza A virus (FLUAV) in that it blocks the expression of IFN genes. IFN expression depends on IFN regulatory factor 3 (IRF3). Upon activation, IRF3 forms homodimers and accumulates in the nucleus where it binds the transcriptional coactivator CREB-binding protein (CBP). Here, we show that expression of ML blocked the transcriptional activity of IRF3 after stimulation by virus infection. Further biochemical analysis revealed that ML acts by blocking IRF3 dimerization and association with CBP. Surprisingly, however, ML did not interfere with the nuclear transport of IRF3. Thus, the action of ML differs strikingly from that of FLUAV NS1 that prevents IFN induction by retaining IRF3 in the cytoplasm.

Animals↗

PubMed2005

Assay and functional analysis of dynamin-like Mx proteins.

Mx proteins are interferon-induced large guanosine triphosphatases (GTPases) that share structural and functional properties with dynamin and dynamin-like proteins, such as self-assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a range of RNA viruses, including influenza viruses and members of the bunyavirus family. These viruses are inhibited at an early stage in their life cycle, soon after host cell entry and before genome amplification. The association of the human MxA GTPase with membranes of the endoplasmic reticulum seems to support its antiviral function by providing an interaction platform that facilitates viral target recognition, MxA oligomerization, and missorting of the resulting multiprotein complex into large intracellular aggregates.

3T3 Cells↗

PubMed2004

Inverse interference: how viruses fight the interferon system.

Viruses need to multiply extensively in the infected host in order to ensure transmission to new hosts and survival as a population. This is a formidable task, given the powerful innate and adaptive immune responses of the host. In particular, the interferon (IFN) system plays an important role in limiting virus spread at an early stage of infection. It has become increasingly clear that viruses have evolved multiple strategies to escape the IFN system. They either inhibit IFN synthesis, bind and inactivate secreted IFN molecules, block IFN-activated signaling, or disturb the action of IFN-induced antiviral proteins. The molecular mechanisms involved range from a broad shut-off of the host cell metabolism to fine-tuned elimination of key components of the IFN system. Type I (alpha/beta) IFNs are produced in direct response to virus infection and double-stranded RNA (dsRNA) molecules that are sensed as a danger signal by infected cells. IFNs induce the expression of a number of antiviral proteins, some of which are again activated by dsRNA. Therefore, many viruses produce dsRNA-binding proteins to sequester the danger signal or express virulence genes that target specific components of the IFN system, such as members of the IFN regulatory factor (IRF) family or components of the JAK-STAT signaling pathway. Finally, some viruses have adopted means to directly suppress the very antiviral effector proteins of the IFN-induced antiviral state directed against them. Evidently, viruses and their host's innate immune responses have coevolved, leading to a subtle balance between virus-promoting and virus-inhibiting factors. A better understanding of virus-host interactions is now emerging with great implications for vaccine development and drug design.

Gene Expression Regulation↗

PubMed2004

Functional comparison of the two gene products of Thogoto virus segment 6.

The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-beta by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins.

Animals↗

PubMed2004

Missorting of LaCrosse virus nucleocapsid protein by the interferon-induced MxA GTPase involves smooth ER membranes.

The interferon-induced human MxA protein belongs to the class of dynamin-like, large guanosine-5'-triphosphatases that are involved in intracellular vesicle trafficking and organelle homeostasis. MxA shares many properties with the other members of this protein superfamily, including the propensity to self-assemble and to associate with lipid membranes. However, MxA is unique in that it has antiviral activity and inhibits the replication of several RNA viruses. Here, we determined the role of membranes for the antiviral function of MxA using LaCrosse-bunyavirus (LACV). We show that MxA does not affect trafficking and sorting of viral glycoproteins but binds and mislocates the viral nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. We further demonstrate that MxA localizes to a subcompartment of the smooth endoplasmic reticulum where the viral N protein accumulates. In infected MxA-expressing cells, oligomeric MxA/N complexes are formed in close association with COP-I-positive vesicular-tubular membranes. Our results suggest that this membrane compartment is the preferred place where MxA and N interact, leading to efficient sequestration and missorting of an essential viral component.

Animals↗

PubMed2004

Thogoto virus lacking interferon-antagonistic protein ML is strongly attenuated in newborn Mx1-positive but not Mx1-negative mice.

The Thogoto virus ML protein suppresses interferon synthesis in infected cells. Nevertheless, a virus mutant lacking ML remained highly pathogenic in standard laboratory mice. It was strongly attenuated, however, in mice carrying the interferon-responsive Mx1 gene found in wild mice, demonstrating that enhanced interferon synthesis is protective only if appropriate antiviral effector molecules are present. Our study shows that the virulence-enhancing effects of some viral interferon antagonists may escape detection in conventional animal models.

Animals↗

PubMed2003

Viral evasion of the interferon system: old viruses, new tricks.

Interferon-alpha/beta (IFN-alpha/beta) plays an important role in antiviral defense, but many viruses have evolved mechanisms to evade this barrier. The emerging understanding of how viral proteins intercept the IFN response was the focus of a workshop held at the University of Freiburg, Germany, on September 27-28, 2002.

Animals↗

PubMed2003

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids.

La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.

Animals↗

PubMed2003

Novel gene product of Thogoto virus segment 6 codes for an interferon antagonist.

Thogoto virus (THOV) is a tick-transmitted orthomyxovirus with a genome of six negative-stranded RNA segments. The sixth segment encodes two different transcripts: a spliced transcript that is translated into the matrix protein (M) and an unspliced transcript. Here, we report that the unspliced transcript encodes an elongated form of M named ML. A THOV isolate deficient in ML expression was an efficient interferon inducer, whereas ML-expressing wild-type strains were poor interferon inducers. These results were confirmed with recombinant THOVs rescued from cDNAs. Expression of ML efficiently suppressed activation of the beta interferon promoter by double-stranded RNA. These results indicate that ML is an accessory protein that functions as a potent interferon antagonist by blocking transcriptional activation of alpha/beta interferons.

Animals↗

PubMed2002

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes.

Bunyaviruses replicate in the cytoplasm of infected cells. New viral particles are formed by budding of nucleocapsids into the Golgi apparatus. We have previously shown that the IFN-induced human MxA protein inhibits bunyavirus replication by an unknown mechanism. Here we demonstrate that MxA binds to the nucleocapsid protein of La Crosse virus (LACV) and colocalizes with the viral protein in cytoplasmic complexes. Electron microscopy revealed that these complexes accumulated in the perinuclear area and consisted of highly ordered fibrillary structures. A similar MxA-mediated redistribution of viral nucleocapsid proteins was detected with other bunyaviruses, such as Bunyamwera virus and Rift Valley fever virus. MxA(E645R), a carboxy-terminal mutant of MxA without antiviral activity against LACV, did not lead to complex formation. Wild-type MxA, but not MxA(E645R), was able to bind to LACV nucleocapsid protein in coimmunoprecipitation assays, demonstrating the importance of the carboxy-terminal effector domain of MxA. These results illustrate an efficient mechanism of IFN action whereby an essential virus component is trapped in cytoplasmic inclusions and becomes unavailable for the generation of new virus particles.

Animals↗

PubMed2002

Self-assembly of human MxA GTPase into highly ordered dynamin-like oligomers.

Human MxA protein is a member of the interferon-induced Mx protein family and an important component of the innate host defense against RNA viruses. The Mx family belongs to a superfamily of large GTPases that also includes the dynamins and the interferon-regulated guanylate-binding proteins. A common feature of these large GTPases is their ability to form high molecular weight oligomers. Here we determined the capacity of MxA to self-assemble into homo-oligomers in vitro. We show that recombinant MxA protein assembles into long filamentous structures with a diameter of about 20 nm at physiological salt concentration as demonstrated by sedimentation assays and electron microscopy. In the presence of guanosine nucleotides the filaments rearranged into rings and more compact helical arrays. Our data indicate that binding and hydrolysis of GTP induce conformational changes in MxA that may be essential for viral target recognition and antiviral activity.

Aluminum Compounds↗

PubMed2002

Interferon-induced mx proteins: dynamin-like GTPases with antiviral activity.

Mx proteins are interferon-induced GTPases that belong to the dynamin superfamily of large GTPases. Similarities include a high molecular weight, a propensity to self-assemble, a relatively low affinity for GTP, and a high intrinsic rate of GTP hydrolysis. A unique property of Mx GTPases is their antiviral activity against a wide range of RNA viruses, including bunya- and orthomyxoviruses. The human MxA GTPase accumulates in the cytoplasm of interferon-treated cells, partly associating with the endoplasmic reticulum. In the case of bunyaviruses, MxA interferes with transport of the viral nucleocapsid protein (N) to the Golgi compartment, the site of virus assembly. In the case of Thogoto virus (an orthomyxovirus), MxA prevents the incoming viral nucleocapsids from being transported into the nucleus, the site of viral transcription and replication. In both cases, the GTP-binding and carboxy-terminal effector functions of MxA are required for target recognition. In general, Mx GTPases appear to detect viral infection by sensing nucleocapsid-like structures. As a consequence, these viral components are trapped and sorted to locations where they become unavailable for the generation of new virus particles.

Antiviral Agents↗