Influenza A virus-host interactions and their control by viral non-structural protein NS1

Viruses infect all domains of life. They establish complex interactions with their host cells to subvert and hijack multiple cellular processes and warrant their own replication. Understanding virus-host interactions is critical to control spread of pathogenic viruses, develop vaccines and search for antivirals. Besides that, understanding virus-host interactions allows deciphering complex cellular processes and provides useful tools for biotechnology. My research is dedicated to influenza A virus, an important pathogen that infects humans worldwide, represents a constant health care threat and elicits continuous efforts to control the human spread of the disease. Influenza A expresses a non-structural protein NS1 that is a key regulator of viral interactions with the host cell and an important virulence factor. Versatile functions of NS1 modulate multiple cellular functions to secure viral replication. This work addresses several aspects of NS1-mediated modulation of core cellular processes. We discovered that NS1 binds to dsDNA and inhibits transcription of cellular genes, thus limiting antiviral responses. We found that NS1 secures general protein synthesis and mapped several residues within NS1 that are essential for this function. Further, we showed that the length of C-terminal ``tail'' of NS1 is essential for control of cellular antiviral responses and virus pathogenicity. The presented results increase the understanding of influenza A virus-host interactions and can be further utilized in the search for antivirals and vaccine development. In addition, this work provides a biotechnological application of influenza A NS1 protein for improvement of cell-free translation system.Virukset ovat vuorovaikutuksessa isäntäsolujensa kanssa. Ne manipuloivat solun sisäisiä prosesseja ja turvaavat oman lisääntymisensä. Virus-soluvuorovaikutusten ymmärtäminen on erittäin tärkeää jotta voidaan estää patogeenisten virusten leviäminen, kehittää rokotteita ja etsiä uusia viruslääkkeitä. Tämän lisäksi virus-soluvuorovaikutusten ymmärtäminen auttaa selvittämään monimutkaisia solun prosesseja ja tarjoaa siten käyttökelpoisia työkaluja bioteknologian alalle. Tutkimukseni kohteena on tärkeä, ihmisiä maailmanlaajuisesti infektoiva influenssa A -virus. Tämä virus on jatkuva uhka terveydenhuollolle ja viruksen ihmisvälitteisen leviämisen estoon pyritään löytämään jatkuvasti keinoja. Influenssa A -virus tuottaa NS1-proteiinia joka ei ole osa viruksen rakennetta. NS1-proteiini on avainasemassa virusinfektion aikana, se säätelee viruksen vuorovaikutuksia solun kanssa ja vaikuttaa viruksen virulenssiin. NS1-proteiinin moninaiset toiminnot muokkaavat useita solun toimintoja ja varmistavat viruksen lisääntymisen. Tässä tutkimuksessa selvitetään millä eri tavoilla NS1-proteiini vaikuttaa solun prosesseihin. Havaitsimme että NS1 sitoutuu kaksijuosteiseen DNA:han ja estää siten solun geenien ilmentämisen estäen solun antiviraalisia vasteita. Havaitsimme myös että NS1 turvaa proteiinien tuotannon ja määritimme NS1:n alueet jotka ovat välttämättömiä tällä toiminnolle. Lisäksi osoitimme että NS1-proteiinin C-terminaalisen "hännän" pituus on tärkeä solun antiviraalisten vasteiden kontolloimisessa ja viruksen patogeenisuudessa. Tutkimuksessa saadut tulokset auttavat ymmärtämään influenssa A -viruksen ja solun vuorovaikutuksia ja tuloksia voi hyödyntää viruslääkkeiden ja rokotteiden kehittämisessä. Lisäksi nämä tulokset mahdollistavat influenssa A -viruksen NS1-proteiinin bioteknologisen hyödyntämisen, proteiinin avulla voidaan parantaa soluvapaita proteiinituottosysteemejä

Molecular Organisation of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions

Molecular Organisation of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions

SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies

Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019–2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments

Human picornaviruses associated with neurological diseases and their neutralization by antibodies

Picornaviruses are the most commonly encountered infectious agents in mankind. They typically cause mild infections of the gastrointestinal or respiratory tract, but sometimes also invade the central nervous system. There, they can cause severe diseases with long-term sequelae and even be lethal. The most infamous picornavirus is poliovirus, for which significant epidemics of poliomyelitis were reported from the end of the nineteenth century. A successful vaccination campaign has brought poliovirus close to eradication, but neurological diseases caused by other picornaviruses have increasingly been reported since the late 1990s. In this review we focus on enterovirus 71, coxsackievirus A16, enterovirus 68 and human parechovirus 3, which have recently drawn attention because of their links to severe neurological diseases. We discuss the clinical relevance of these viruses and the primary role of humoral immunity in controlling them, and summarize current knowledge on the neutralization of such viruses by antibodies.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

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

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.</p

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.Peer reviewe