Virus-host interactions of emerging respiratory pathogens

Respiratory virus infections are a major cause of morbidity and mortality worldwide. Decades of research have yielded many breakthroughs in our understanding of virus-host interactions, but many aspects of viral pathogenesis still remain unresolved. Vaccines and antiviral treatments have been developed, but they are imperfect or completely lacking for many viral agents. Novel emerging viruses form an additional challenge, which can only be overcome by proper preparation and quick response against these unexpected infectious threats. Understanding virus-host interactions is critical for elucidating aspects of viral pathogenesis and devising better treatment strategies against viral pathogens. Our research has focused on the virus-host interactions of two major respiratory pathogens with a recent history of outbreaks by a novel viral agent, the influenza A virus and coronavirus. Influenza A virus has plagued humankind throughout human history and continues to cause annual epidemics and occasional pandemics associated with significant mortality. NS1 protein is one of the major virulence factors of influenza A virus. It has a multitude of different interactions with host cell components that either aid viral replication or hamper the antiviral response exhibited by the host cell. These interactions are located both in the host cell nucleus and the cytoplasm, and three signals regulating the intracellular localization of NS1 protein have been identified. While the critical impact of nuclear localization signal 1 is well known, the other two localization signals have remained poorly characterized. In this work we provide a detailed description of the NS1 nuclear export signal (NES), showing that the NES region is well conserved within different influenza A strains and that certain mutations within the region cause attenuation of virus replication. Through the use of different mutant viruses, we show that the attenuated phenotype is not due to impaired localization alone, but rather involves defects in the functions of NS1. We also show that NS1 is not exported through the classical CRM1-dependent pathway and we establish the nucleolar proteins that bind NS1 and thus guide its nucleolar localization. Human coronaviruses are a major cause of the common cold. For a long time coronaviruses were thought to cause only mild upper respiratory tract infections in humans. However, this view changed with the emergence of the highly lethal SARS coronavirus in 2002 and the identification of MERS coronavirus in 2012. While the SARS outbreak was efficiently contained, MERS coronavirus continues to circulate in camels and causes repeated introductions into the human population in the Middle East. Our MERS coronavirus research concentrated on characterizing MERS infection of human macrophages and dendritic cells, two important cell types of the innate immune system. We show that MERS coronavirus does not replicate productively in these leucocytes, but a significant innate immune response is generated. Altogether this work identifies important aspects of virus-host interaction of two important respiratory pathogens. We provide new information on the mechanisms and impact of influenza A virus NS1 intracellular localization and we characterize MERS coronavirus infection in primary human leucocytes as well as highlight important differences in the host cell responses between MERS and SARS coronaviruses.Hengitystievirusten aiheuttamat infektiot ovat maailmanlaajuisesti merkittävä sairastavuutta ja kuolleisuutta aiheuttava haaste kansanterveydelle. Vuosikymmenten tutkimustyö on tuottanut lukuisia läpimurtoja hengitystievirusinfektioiden mekanismien tuntemuksessa, mutta monet virusten taudinaiheutuskyvyn piirteet ovat yhä epäselviä. Rokotusten ja antiviraaliyhdisteiden kehitykseen suunnatut ponnistelut ovat tuottaneet lukuisia rokotteita ja lääkkeitä hengitystieviruksia vastaan, mutta ne ovat usein suojausteholtaan puutteellisia tai puuttuvat kokonaan tiettyjä virusryhmiä vastaan. Erilaisista eläinperäisistä lähteistä ilmaantuvat uudenlaiset virukset muodostavat erityisen uhkan, johon varautuminen edellyttää valmistautumista sekä valmiutta nopeisiin vastatoimiin. Viruksen ja isäntäsolun välisten vuorovaikutusten ymmärtäminen on keskeisessä asemassa kehitettäessä uusia parempia hoitomenetelmiä sekä valmistauduttaessa uusiin virusuhkiin. Viruksen ja isäntäsolun vuorovaikutuksiin keskittyvä tutkimuksemme käsittelee kahta merkittävää hengitystievirusta, influenssa A -virusta sekä MERS-koronavirusta. Jo antiikin ajoista lähtien ihmiskunnan vitsauksena ollut influenssa A -virus aiheuttaa vuosittaisia talviaikaan sijoittuvia epidemioita sekä ajoittaisia maailmanlaajuisia pandemioita. NS1-proteiini on merkittävä influenssa A -viruksen virulenssitekijä, jolla on lukuisia infektiota edistäviä tai isäntäsolun immuunivastetta estäviä vuorovaikutuksia isäntäsolun proteiinien kanssa. Nämä vuorovaikutukset sijoittuvat sekä tumaan että solulimaan. NS1-proteiinista on tunnistettu kolme solunsisäistä paikantumista ohjaavaa signaalia, joista yhden tiedetään olevan tärkeä virusinfektiolle, mutta kahden muun merkitys on jäänyt epäselväksi. Tässä työssä luomme seikkaperäisen kuvauksen NS1-proteiinin tumasta solulimaan kulkeutumisen välittävän signaalin toiminnasta. Näytämme että kyseinen paikantumissignaali on hyvin säilynyt eri viruskantojen välillä ja osoitamme, että tietyt signaaliin kohdistuvat mutaatiot aiheuttavat viruksen replikaatiokyvyn huomattavan heikentymisen. Osoitamme erilaisten mutanttivirusten avulla, että heikentynyt fenotyyppi ei johdu ainoastaan NS1-proteiinin virheellisestä paikantumisesta, vaan aiheutuu NS1-proteiinin toiminnallisista vioista. Osoitamme myös, että NS1-proteiinin kulkeutuminen tumasta solulimaan ei ole CRM1-välitteinen, sekä tunnistamme NS1-proteiinin kanssa vuorovaikuttavat tumajyväsproteiinit. Koronavirukset tunnetaan merkittävinä flunssan aiheuttajina ja niitä pidettiin pitkään lähinnä lievien hengitystieinfektioiden aiheuttajina ihmisissä, mutta tämä näkemys on muuttunut merkittävää kuolleisuutta aiheuttaneiden SARS- ja MERS-epidemioiden myötä. SARS-koronavirus ilmaantui vuonna 2002 ja saatiin nopeasti taltutettua, mutta vuonna 2012 havaittu MERS-koronavirus kiertää yhä kameleissa aiheuttaen toistuvia ihmisinfektioita Lähi-Idässä. MERS-koronavirusta käsittelevä osa työstämme keskittyi analysoimaan MERS-koronaviruksen vuorovaikutusta ihmisen makrofagien ja dendriittisolujen kanssa. Osoitamme, että MERS-koronavirusinfektio ei johda merkittävään virusreplikaatioon näissä soluissa, mutta huomattava immuunivaste syntyy siitä huolimatta. Kaiken kaikkiaan työmme tuo ilmi tärkeitä piirteitä näiden merkittävien hengitystievirusten vuorovaikutussuhteista isäntäsolujen kanssa. Tuotamme uutta tietoa influenssa A -viruksen NS1-proteiinin solunsisäisen paikantumisen mekanismeista ja merkityksestä sekä analysoimme MERS-koronavirusinfektion piirteet ihmisen valkosoluissa korostaen samalla MERS- ja SARS-koronavirusten välisiä eroja

Immuno-modulating properties of saliphenylhalamide, SNS-032, obatoclax, and gemcitabine

Influenza A viruses (IAVs) impact the public health and global economy by causing yearly epidemics and occasional pandemics. Several anti-IAV drugs are available and many are in development. However, the question remains which of these antiviral agents may allow activation of immune responses and protect patients against co- and re-infections. To answer to this question, we analysed immuno-modulating properties of the antivirals saliphenylhalamide (SaliPhe), SNS-032, obatoclax, and gemcitabine, and found that only gemcitabine did not impair immune responses in infected cells. It also allowed activation of innate immune responses in lipopolysaccharide (LPS)- and interferon alpha (IFN alpha)-stimulated macrophages. Moreover, immuno-mediators produced by gemcitabine-treated IAV-infected macrophages were able to prime immune responses in non-infected cells. Thus, we identified an antiviral agent which might be beneficial for treatment of patients with severe viral infections. (C) 2015 The Authors. Published by Elsevier B.V.Peer reviewe

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

No Serological Evidence of Influenza A H1N1pdm09 Virus Infection as a Contributing Factor in Childhood Narcolepsy after Pandemrix Vaccination Campaign in Finland

Peer reviewe

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

SARS-CoV-2 variant-specific differences in inhibiting the effects of the PKR-activated integrated stress response

The integrated stress response (ISR) is a eukaryotic cell pathway that triggers translational arrest and the formation of stress granules (SGs) in response to various stress signals, including those caused by viral infections. The SARS-CoV-2 nucleocapsid protein has been shown to disrupt SGs, but SARS-CoV-2 interactions with other components of the pathway remains poorly characterized. Here, we show that SARS-CoV-2 infection triggers the ISR through activation of the eIF2α-kinase PKR while inhibiting a variety of downstream effects. In line with previous studies, SG formation was efficiently inhibited and the induced eIF2α phosphorylation only minimally contributed to the translational arrest observed in infected cells. Despite ISR activation and translational arrest, expression of the stress-responsive transcription factors ATF4 and CHOP was not induced in SARS-CoV-2 infected cells. Finally, we found variant-specific differences in the activation of the ISR between ancestral SARS-CoV-2 and the Delta and Omicron BA.1 variants in that Delta infection induced weaker PKR activation while Omicron infection induced higher levels of p-eIF2α, and greatly increased SG formation compared to the other variants. Our results suggest that different SARS-CoV-2 variants can affect normal cell functions differently, which can have an impact on pathogenesis and treatment strategies

Mutations within the conserved NS1 nuclear export signal lead to inhibition of influenza A virus replication

BACKGROUND: The influenza A virus NS1 protein is a virulence factor and an antagonist of host cell innate immune responses. During virus infection NS1 protein has several functions both in the nucleus and in the cytoplasm and its intracellular localization is regulated by one or two nuclear localization signals (NLS) and a nuclear export signal (NES). METHODS: In order to investigate the role of NS1 NES in intracellular localization, virus life cycle and host interferon responses, we generated recombinant A/Udorn/72 viruses harboring point mutations in the NES sequence. RESULTS: NS1 NES was found to be inactivated by several of the mutations resulting in nuclear retention of NS1 at late stages of infection confirming that this sequence is a bona fide functional NES. Some of the mutant viruses showed reduced growth properties in cell culture, inability to antagonize host cell interferon production and increased p-IRF3 levels, but no clear correlation between these phenotypes and NS1 localization could be made. Impaired activation of Akt phosphorylation by the replication-deficient viruses indicates possible disruption of NS1-p85β interaction by mutations in the NES region. CONCLUSION: We conclude that mutations within the NS1 NES result in impairment of several NS1 functions which extends further from the NES site being only involved in regulating the nuclear-cytoplasmic trafficking of NS1

Novel avian influenza A (H7N9) virus induces impaired interferon responses in human dendritic cells.

In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced "cytokine storm" seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs