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- Panayiotou, Christakis, et al.
(författare)
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Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation
- 2018
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 92:7
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Tidskriftsartikel (refereegranskat)abstract
- Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possible availability of new drug targets that can be used for therapeutic intervention.IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV non-structural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention.
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| 2. |
- Upadhyay, Arunkumar S., et al.
(författare)
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Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity
- 2014
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Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 16:6, s. 834-848
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Vonderstein, Kirstin, 1986-, et al.
(författare)
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Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles
- 2018
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Ingår i: Journal of Virology. - : American Society of Microbiology. - 0022-538X .- 1098-5514. ; 92:1
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Tidskriftsartikel (refereegranskat)abstract
- Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention.Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.
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| 6. |
- Vonderstein, Kirstin, 1986-
(författare)
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Viperin vs. tick-borne encephalitis virus : mechanism of a potent antiviral protein
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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