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Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
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2014 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 16, no 6, 834-848 p.Article in journal (Refereed) Published
Abstract [en]

Viperin is an interferon-induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S-adenosyl-l-methionine (SAM) domain which has been shown in vitro to hold a [4Fe-4S] cluster. We identified tick-borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of Fe-55, indicative for the presence of an Fe-S cluster. Mutation of the cysteine residues ligating the Fe-S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of Fe-55. Mutants lacking the extreme C-terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe-4S] cluster assembly, and acts through an enzymatic, Fe-S cluster- and SAM-dependent mechanism to inhibit viral RNA synthesis.

Place, publisher, year, edition, pages
2014. Vol. 16, no 6, 834-848 p.
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-90413DOI: 10.1111/cmi.12241ISI: 000335930200004OAI: oai:DiVA.org:umu-90413DiVA: diva2:733561
Available from: 2014-07-10 Created: 2014-06-23 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Viperin vs. tick-borne encephalitis virus: mechanism of a potent antiviral protein
Open this publication in new window or tab >>Viperin vs. tick-borne encephalitis virus: mechanism of a potent antiviral protein
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tick-borne encephalitis virus (TBEV) is a very important virus medically, causing mild or severe encephalitis often with long-lasting sequelae. Treatment of tick-borne encephalitis is limited to supportive care, and antiviral drugs are much needed.

The type-I interferon (IFN) system is the first line of host defense against many viruses. Infected cells secrete type-I IFN to alert neighboring cells. These cells in turn upregulate the expression of antiviral proteins to protect themselves from the virus.

In this work, we found that the interferon-induced host protein viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) has a pronounced antiviral effect against TBEV.

 

Viperin is an evolutionarily conserved protein with three domains: the N-terminus, the radical S-adenosyl methionine (SAM) domain, and the C-terminus. Viperin shows antiviral activity against a broad spectrum of different viruses. However, its mode of action appears to be virus-specific.

We therefore concentrated on determining the antiviral mechanism of viperin against TBEV. The specific questions addressed in this thesis are: (1) which steps of the TBEV infectious cycle are targeted by viperin?, (2) which domains of viperin are responsible for its antiviral activity?, and (3) which interaction partners does viperin need in order to have an antiviral effect against TBEV?

First, we investigated which step(s) of the TBEV life cycle viperin targets by using several assays to examine the effects of viperin on virus binding, entry, genome replication, assembly, and release.

We found that viperin inhibited the replication of positive-sense genomic RNA and also targeted particle release, selectively enhancing the release of membrane-associated capsid particles.

For inhibition of genome replication, viperin was dependent on the host cellular protein CIAO1 (cytosolic iron-sulfur assembly component 1). CIAO1 interacted with the C-terminus of viperin and was necessary for the maturation and stability of viperin, and also for loading of an iron-sulfur cluster onto the SAM domain. The SAM domain required this iron-sulfur cluster to perform its function as a radical SAM enzyme, which was required for the inhibition of TBEV genome replication. In addition to the SAM domain and the C-terminus, viperin needed its N-terminus in order to be fully antivirally active during late replication, since the N-terminus directed viperin to the endoplasmic reticulum, where genome replication takes place.

Furthermore, viperin targeted GBF1 (Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1), a host protein known to be involved in the secretory pathway. Interaction between the N-terminus of viperin and GBF1 appeared to induce an enhance release of capsid particles independently of the later steps of the classical secretory pathway. The enhanced secretion of capsid particles by viperin occurred at the expense of whole, infectious virions and is therefore a completely novel antiviral mechanism.

 

In summary, this work identified viperin as a very strong inhibitor of TBEV, and its antiviral mechanism was characterized in detail. Viperin was found to target multiple steps in the TBEV infectious cycle by both inhibiting viral RNA replication and inducing secretion of capsid particles. These findings provide new insights into the interplay between TBEV and viperin, and offer new approaches to our understanding of the molecular and cellular mechanisms of TBEV infection, which may contribute to the development of a treatment for TBEV.

Abstract [sv]

Fästingburen encefalit (TBE) är en sjukdom som orsakas av TBEvirus och överförs till människor genom fästingbett. Hur allvarlig sjukdomen blir varierar från person till person. Några blir inte ens sjuka, andra får milda influensaliknande symptom, medan vissa får hjärnhinneinflammation (meningit) och/ eller hjärninflammation (encefalit), med symptom såsom huvudvärk, kräkningar, medvetanderubbningar och förlamning. Symptomen kan vara i flera månader och i 30-50 % av fallen kvarstår neurologiska besvär. Det finns bra vaccination mot TBE, men om man blir sjuk finns idag ingen behandling som gör en frisk, utan det handlar istället om att lindra symptomen.

För att hitta en möjlig behandling är det viktigt att förstå vad som händer i de individuella cellerna i kroppen när man blir sjuk och vad cellerna gör för att bekämpa viruset. Eftersom virus använder cellens byggstenar för att föröka sig och sprida sig till andra celler har cellerna utvecklat mekanismer för att känna igen och försvara sig mot virus. En central komponent i cellens tidiga försvar mot virusinfektioner är interferon, som skickas ut som en varningssignal till närliggande celler. Dessa celler kan då producera proteiner som skyddar dem mot virusangrepp.

I denna avhandling identifierades och undersöktes ett protein, viperin, som är aktivt i försvaret mot TBE-virus. Vi fann att viperin kunde förhindra virusökning inne i cellerna, samt begränsa mängden av funktionella virus som lämnar cellerna. Viperin arbetar inte ensamt, utan behöver andra proteiner från cellen för att vara aktivt. Viperin binder till och är beroende av CIAO1 för att vara fullt fungerande och förhindra tillökning av virus arvsmassa i cellen. Ett annat protein som viperin binder till är GBF1. GBF1 är viktig för transport av nyproducerade proteiner ut ur cellen. Denna transportled används också av TBE-virus för att lämna cellen.Viperin påverkar GBF1 så att bildandet av nya viruspartiklar störs, vilket leder till att färre hela, fungerande virus kommer ut ur cellen. Istället utsöndras många partiklar som endast består av den inre delen av viruset, den så kallade kapsiden. Kapsidpartiklarna kan vara enbart en biprodukt av störd virusproduktion eller så kan de kanske ha ytterligare funktioner som att aktivera immunsystemets celler, men det återstår att utforska.

Viperin påverkar TBE-virus på två olika sätt och kan därmed bromsa infektionen mycket effektivt. Det här arbetet hjälper oss att i mer detalj förstå hur det antivirala proteinet viperin motverkar TBE-virus. I och med det, har vi identifierat nya svagheter hos viruset som förhoppningsvis kan utnyttjas i framtiden för att utveckla läkemedel mot TBE.

Abstract [de]

Frühsommer-Meningoenzephalitis (FSME) ist eine Erkrankung, die vom FSME Virus ausgelöst und durch Zeckenbiss auf den Menschen übertragen wird. Nach Ansteckung kann es zu sehr unterschiedlich ausgeprägtem Krankheitsverlauf kommen. Während viele Menschen gar nicht erkranken und andere nur leichte grippeähnliche Symptome zeigen, bekommen andere eine Hirnhautentzündung (Meningitis) oder/und Hirnentzündung (Enzephalitis), die sich unter anderem in Kopfschmerzen, Erbrechen bis hin zu Bewusstseinsstörungen und Lähmungen äußern. Die Symptome können mehrere Monate anhalten und in einigen Fällen heilt die Krankheit nie vollständig aus. Es gibt zwar eine gute Impfung gegen FSME, aber einmal erkrankt gibt es keine Behandlungsmöglichkeit zur Heilung, lediglich die Symptome können gelindert werden.

Um eine mögliche Behandlung zu finden ist es wichtig, dass man versteht, was genau in den einzelnen Körperzellen bei einer Erkrankung geschieht und welche Mechanismen dort zur Bekämpfung des Virus ablaufen. Da Viren die Bausteine der Zellen nutzen um sich zu vermehren, haben die Zellen im Gegenzug Möglichkeiten entwickelt um die Eindringlinge zu erkennen, sich gegen diese zu verteidigen und benachbarte Körperzellen vor den Angreifern zu warnen. Die Nachbarzellen können daraufhin Proteine (Eiweiße) produzieren, die sie gegen das Virus schützen.

In dieser Arbeit wurde ein solches antivirales Protein namens Viperin, das die Zellen gegen das FSME-Virus schützt, identifiziert und genauer untersucht. Wir haben herausgefunden, dass Viperin die Vermehrung des Virus in der Zelle verxi hindert und außerdem die Menge an funktionsfähigen, infekti- ösen Viren, welche die Zelle verlassen, begrenzt. Dabei arbeitet Viperin nicht alleine, sondern steht in Wechselwirkung mit anderen zellulären Proteinen. Wir konnten zeigen, dass Viperin auf die Hilfe des Proteins CIAO1 angewiesen ist um voll funktionsfähig zu sein und die Vermehrung des viralen Erbguts in der Zelle zu verhindern. Ein weiteres Protein mit dem Viperin in Wechselwirkung steht heißt GBF1. GBF1 ist darin involviert, dass die in der Zelle hergestellten Proteine die Zelle auf dem klassischen sekretorischen Transportweg verlassen. Auch das FSME Virus benutzt normalerweise diesen Weg um die Zelle zu verlassen. Viperin beeinflusst GBF1 so, dass nicht mehr so viele ganze, funktionsfähige Viren aus der Zelle kommen, sondern viele Partikel ausgeschieden werden, die nur aus dem inneren Teil des Virus, dem sogenannte Kapsid, bestehen. Ob diese Kapsidpartikel nur ausgeschieden werden damit weniger ganze, funktionsfähige Viruspartikel entstehen oder aber ob sie eine bestimmte Funktion im Körper erfüllen wie z.B. die Zellen des Immunsystems zu aktivieren, bleibt zu erforschen.

Viperin attackiert also das FSME Virus auf zwei verschiedenen Wegen und schafft es damit sehr wirksam die Infektion einzudämmen. Die in dieser Arbeit gewonnenen Erkenntnisse darüber, wie das antivirale Protein Viperin gegen das FSME Virus wirkt, können hoffentlich in Zukunft dazu beitragen, Medikament gegen FSME zu entwickeln.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 92 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1832
Keyword
Tick-borne encephalitis virus, TBEV, type-I interferon, IFN, Interferon-stimulated gene, ISG, viperin, GBF1, CIAO1, antiviral protein, genome replication, particle release, capsid
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-125659 (URN)978-91-7601-538-4 (ISBN)
Public defence
2016-10-07, E04, byggnad 6A, NUS, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-09-16 Created: 2016-09-13 Last updated: 2017-09-21Bibliographically approved
2. Viperin, a multifunctional radical SAM enzyme: biogenesis and antiviral mechanisms
Open this publication in new window or tab >>Viperin, a multifunctional radical SAM enzyme: biogenesis and antiviral mechanisms
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an interferon-induced antiviral protein. It has three distinct functional domains: the N-terminal domain, the radical SAM (S-adenosylmethionine) domain for binding iron-sulphur cluster, and the C-terminus domain. Viperin has a broad antiviral effect, and is also involved in the immune response signalling. However, the function and antiviral mechanism of viperin are still not well characterized. Thus the overall aim of the thesis was to investigate and better understand the function of viperin and its antiviral mechanism by identifying the cellular network of interaction partners. Affinity purification and mass spectrometry analysis were used to identify cellular proteins that interact with viperin.

CIA1 (also known as CIAO1), a factor involved in loading of iron-sulphur (Fe/S) cluster was identified and confirmed to interact at the C-terminus of viperin. It was also seen that the C-terminal domain and the functional SAM domain of viperin was essential for loading the Fe/S cluster onto viperin. On a closer look at the biogenesis of viperin, we identified and confirmed viperin interaction with CIA2A, CIA2B, (also known as FAM96A and FAM96B respectively) and MMS19, which are other factors involved in the transfer of Fe/S clusters onto cytosolic Fe/S apo-proteins. Surprisingly, MMS19, which has been shown to act as an adapter protein for other Fe/S proteins, only interacted indirectly and was not required for transferring the Fe/S cluster. Similarly, the interaction of viperin with both the isoforms of CIA2 was interesting, but the role they play in viperin biogenesis and antiviral function is still not clear and requires further investigation.

Study of the antiviral action of viperin against tick-borne encephalitis virus (TBEV) showed that the activity of the SAM domain is essential for the strong inhibition of genome replication of TBEV. Furthermore, viperin also affects the assembly and release of TBEV. Viperin interacts with GBF1 and downregulates its activity, thus preferentially inducing the secretion of viral capsid protein from the cell, and therefore disrupting the formation of infectious virus particles. The N-terminal domain of viperin is important for its effect on assembly and release.

In summary, this work contributes to our general understanding of viperin biogenesis in the cell regarding the loading of Fe/S cluster onto viperin. It also addresses the importance of the different domains for its antiviral function against TBEV. Finally, mass spectrometry and viperin interactome analysis implicate many other interesting cellular pathways or processes that might give us a better understanding of viperin’s function and antiviral mechanism. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 68 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1869
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-128754 (URN)978-91-7601-627-5 (ISBN)
Public defence
2017-01-20, Room E04_R1, Building 6A, University Hospital of Umeå, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-12-16 Created: 2016-12-14 Last updated: 2016-12-16Bibliographically approved

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