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Viperin, a multifunctional radical SAM enzyme: biogenesis and antiviral mechanisms
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Anna Överby)
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: urn:nbn:se:umu:diva-128754ISBN: 978-91-7601-627-5 (print)OAI: oai:DiVA.org:umu-128754DiVA: diva2:1056165
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
List of papers
1. Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity
Open this publication in new window or tab >>Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity
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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.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-90413 (URN)10.1111/cmi.12241 (DOI)000335930200004 ()
External cooperation:
Available from: 2014-07-10 Created: 2014-06-23 Last updated: 2016-12-14Bibliographically approved
2. Unusual iron-sulfur cluster maturation of the antiviral radical SAM protein viperin
Open this publication in new window or tab >>Unusual iron-sulfur cluster maturation of the antiviral radical SAM protein viperin
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(English)Manuscript (preprint) (Other academic)
Keyword
Viperin, Iron-sulphur cluster, CIA1, CIA2A, CIA2B and MMS19
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-128726 (URN)
Available from: 2016-12-13 Created: 2016-12-13 Last updated: 2016-12-14
3. Viperin induces secretion of tick-borne encephalitis virus capsid particles by interacting with GBF1
Open this publication in new window or tab >>Viperin induces secretion of tick-borne encephalitis virus capsid particles by interacting with GBF1
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-125661 (URN)
Available from: 2016-09-14 Created: 2016-09-14 Last updated: 2016-12-16

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