A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than parental. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern

The Influenza Virus Protein PB1-F2 Inhibits the Induction of Type I Interferon at the Level of the MAVS Adaptor Protein

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses

Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors

Wirkmechanismus und Induktion neutralisierender Antikörper gegen das transmembrane Hüllprotein gp41 von HIV-1

Die Entwicklung eines effektiven, subtypübergreifend wirksamen HIV-1-Impfstoff stellt auch mehr als 20 Jahre nach der Erstbeschreibung von AIDS eine der größten Herausforderungen der moderenen Virologie und Immunologie dar. Konventionelle Impfstoffansätze zur Induktion einer humoralen Immunantwort unter Verwendung von attenuierten oder abgetöteten Viren, gereinigten, viralen Proteine oder rekombinanten Untereinheitenvakzinen scheitern entweder an Sicherheitsbedenken oder induzierten lediglich bindende Antikörper. In den letzten Jahren sind die Ursachen für dieses Scheitern durch immer detailliertere Strukturuntersuchungen des HI-Virus aufgedeckt worden. Seit einigen Jahren wird das Augenmerk vermehrt auf maskierte Strukturen gelegt, die erst während des Infektionsprozesses und der daraus resultierenden Konformationsänderungen im Oberflächen- Transmembran-Komplex offengelegt werden. Monoklonale Antikörper, die aus HIVpositiven Patienten isoliert wurden und gegen den Oberflächen-Transmembran-Komplex gerichtet sind, erkennen solche Strukturen und zeigen teilweise eine Neutralisationsspektrum, das praktisch alle Subtypen der Gruppe M umfasst. Der monoklonale Antikörper 2F5 gehört zur kleinen Gruppe dieser subtypübergreifend virusneutralisierenden Antikörper und wurde bereits erfolgreich in passiven Immunisierungsstudien im Primatenmodell und im Menschen getestet. Bisher ist es nicht gelungen, trotz Kenntnis des linearen 2F5-Epitops (ELDKWA) im membranproximalen Bereich der Ektodomäne von gp41, den Wirkmechnismus von 2F5 vollständig zu verstehen und Antigene zu konstruieren, die in vivo virusneutralisierende Antiseren induzieren, die ein ähnlich breites Neutralisationsspektrum aufweisen wie 2F5. Hier konnte erstmals gezeigt werden, dass 2F5 in vitro Selektionsdruck auf den membranproximalen Anteil der Ektodomäne von gp41 ausübt und so die Bildung von neutralisationsresistenten Fluchtmutanten durch Aminosäureaustausche im 2F5-Kernepitop (LDKW) induziert. Durch Bindungsstudien wurde hier erstmals eine Sequenz aus dem N-terminalen Bereich der Ektodomäne von gp41 identifiziert (E1-Sequenz), die die Bindung von 2F5 an die Sequenz ELDKWA deutlich steigert ohne selbst durch den Antikörper gebunden zu werden. Dieser Effekt konnte sowohl im ELISA, als auch im in vitro Neutralisationstest beobachtet werden. Es konnte gezeigt werden, dass die Bindung von 2F5 an ELDKWA bereits vor der Anlagerung des Virus an die Zielzelle erfolgt und eine Vorinkubation von Virus und 2F5 folglich zu einer gesteigerten Hemmung der Virusreplikation in vitro führt. Auf Basis dieser Daten wurde ein neues, zweistufiges Bindungs- und Neutralisationsmodell erstellt, das sowohl die hier gewonnen Erkenntnisse über den Wirkmechanismus von 2F5, als auch die bisher veröffentlichten Strukturanalysen vereinigt. Ausgehend von diesem neuen Bindungs- und Neutralisationsmodell wurden rekombinante Antigene konstruiert, die Anteile aus dem membranproximalen Bereich der Ektodomäne von gp41 und die E1-Sequenz in unterschiedlichen Trägermolekülen beinhalten. Zusätzlich wurden Plasmide generiert, die nach DNA-Immunisierung zur Präsentation der codierten gp41-abgeleiteten Antigene auf der Zelloberfläche führen. Eines der hier entwickelten Antigene (Hybrid II) induzierte HIV-spezifische neutralisierende Seren im Rattenmodell und bildet möglichweise die Basis für die Entwicklung eines HIVVakzins

Induction of Antibodies Specific for Gp41 of HIV-1 by Gene Gun DNA Vaccination

All attempts to induce broadly neutralising antibodies such as mAb 2F5 and mAb 4E10 targeting conserved epitopes in the membrane proximal external region (MPER) of the transmembrane envelope (TM) protein gp41 of HIV-1 failed so far. In contrast, in previous studies, immunising with the ectodomain of the TM protein p15E of different gammaretroviruses, we successfully induced neutralising antibodies. These antibodies recognised epitopes located in the fusion peptide proximal region (FPPR) and in the MPER of p15E. The epitope in the MPER of p15E corresponds to that of the mAb 4E10 in gp41 in terms of location within the protein and partial sequence homology. In order to present the MPER of gp41 (containing the 2F5 and 4E10 epitopes) membrane-associated, rats were immunised with DNA constructs corresponding (i) to the entire gp41, (ii) to the C-terminal helix of gp41 and (iii) to hybrid proteins composed of a backbone derived from p15E of a gammaretrovirus with inserted FPPR and MPER from gp41 of HIV-1. After transfection in vitro these proteins were found expressed at the cell surface and the accessibility of the 2F5 epitope was demonstrated by flow cytometry. However, DNA vaccination in rats resulted only in low titres of antibodies specific for the MPER of HIV-1, and none of the sera was neutralising