Biomedical subjects

Friedemann Weber

Publications and source records attributed to Friedemann Weber.

29 records · Page 2Linked to original sources

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

High-efficiency detection of severe acute respiratory syndrome virus genetic material.

A Taqman amplicon targeting the nucleocapsid gene of severe acute respiratory syndrome coronavirus (SARS-CoV) is 5 log(10) times more sensitive for SARS-CoV target RNA extracted from infected cells and 2.79 log(10) times more sensitive for RNA extracted from patient material of the index case in Frankfurt than an amplicon targeting the polymerase gene.

Bronchoalveolar Lavage Fluid↗

PubMed2004

NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription.

Rift Valley fever virus (RVFV) is an important cause of epizootics and epidemics in Africa and a potential agent of bioterrorism. A better understanding of the factors that govern RVFV virulence and pathogenicity is required, given the urgent need for antiviral therapies and safe vaccines. We have previously shown that RVFV strains with mutations in the NSs gene are excellent inducers of alpha/beta interferon (IFN-alpha/beta) and are highly attenuated in mice. Here, we demonstrate that NSs is sufficient to block IFN-beta gene expression at the transcriptional level. In cells transiently expressing NSs, IFN-beta transcripts were not inducible by viral infection or by transfection of poly(I:C). NSs with anti-IFN activity accumulated in the nucleus. In contrast, mutant forms of NSs that had lost their IFN-inhibiting activity remained in the cytoplasm, indicating that nuclear localization plays a role. IFN synthesis is regulated by specific transcription factors, including interferon regulatory factor (IRF-3), NF-kappaB, and AP-1. In the presence of NSs, IRF-3 was still activated and moved to the nucleus. Likewise, NF-kappaB and AP-1 were activated normally, as shown in electrophoretic mobility shift assays. Moreover, NSs was found to inhibit transcriptional activity of a constitutive promoter, in agreement with recent findings showing that NSs targets the basal cellular transcription factor TFIIH. The present results suggest that NSs, unlike other viral IFN antagonists, does not inhibit IFN-specific transcription factors but blocks IFN gene expression at a subsequent step.

Amino Acid Sequence↗

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

Bunyamwera virus nonstructural protein NSs counteracts interferon regulatory factor 3-mediated induction of early cell death.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.

Annexin A5↗

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↗

PubMed2003

Activation of PKR by Bunyamwera virus is independent of the viral interferon antagonist NSs.

Double-stranded RNA (dsRNA) is a by-product of viral RNA polymerase activity, and its recognition is one mechanism by which the innate immune system is activated. Cellular responses to dsRNA include induction of alpha/beta interferon (IFN) synthesis and activation of the enzyme PKR, which exerts its antiviral effect by phosphorylating the eukaryotic initiation factor eIF-2 alpha, thereby inhibiting translation. We have recently identified the nonstructural protein NSs of Bunyamwera virus (BUNV), the prototype of the family Bunyaviridae, as a virulence factor that blocks the induction of IFN by dsRNA. Here, we investigated the potential of NSs to inhibit PKR. We show that wild-type (wt) BUNV that expresses NSs triggered PKR-dependent phosphorylation of eIF-2 alpha to levels similar to those of a recombinant virus that does not express NSs (BUNdelNSs virus). Furthermore, the sensitivity of viruses in cell culture to IFN was independent of PKR and was not determined by NSs. PKR knockout mice, however, succumbed to infection approximately 1 day earlier than wt mice or mice deficient in expression of RNase L, another dsRNA-activated antiviral enzyme. Our data indicate that (i) bunyaviruses activate PKR, but are only marginally sensitive to its antiviral effect, and (ii) NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems.

Animals↗

PubMed2002

Antigenic drift, antigenic shift and interferon antagonists: how bunyaviruses counteract the immune system.

Members of the Bunyaviridae family are amongst the most widespread viruses in the world. They can be found on every inhabited continent at virtually every latitude, and are able to infect a wide range of arthropods, plants and mammals including humans. More than 300 named viruses are contained within the family Bunyaviridae (Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses (2000) 599), and several members cause significant disease in humans or domestic animals. Despite being recognised as an emerging threat, relatively little is known about their virulence mechanisms. Here, we try to summarise the current state of knowledge about how the viruses of the Bunyaviridae succeed in establishing infection in the face of a powerful immune system.

Animals↗

PubMed2002

Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Animals↗

PubMed2000

Formation of virus-like particles from cloned cDNAs of Thogoto virus.

Thogoto virus (THOV) is the type species of tick-transmitted orthomyxoviruses. Here, we describe the generation of virus-like particles (VLP) of THOV from cloned cDNAs. To synthesize the six structural proteins of THOV in mammalian cells, we used T7-controlled expression plasmids and a recombinant vaccinia virus producing T7 RNA polymerase. A minireplicon encoding a reporter gene flanked by THOV promoter sequences was expressed by the cellular RNA polymerase I. The recombinant proteins were functional in encapsidation, amplification and transcription of the minireplicon RNA. Furthermore, the artificial nucleocapsids were packaged into THO-VLPs that transferred the minireplicon to indicator cells. This system should be helpful in generating recombinant THOV entirely from cloned cDNAs.

Animals↗