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Flaviviruses are globally distributed pathogens causing millions of human infections every year. Flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. Mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response.

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 in, Originally included in thesis in manuscript form.Errata: Panayiotou C, Lindqvist R, Kurhade C, Vonderstein K, Pasto J, Edlund K, Upadhyay AS, Överby AK. 2018. Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation. J Virol 92 (12): e00501-18. DOI:10.1128/JVI.00501-18.

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune-modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin-JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease.

Junín arenavirus infections are associated with high levels of interferons in both severe and fatal cases. Upon Junín virus (JUNV) infection a cell signaling cascade initiates, that ultimately attempts to limit viral replication and prevent infection progression through the expression of host antiviral proteins. The interferon stimulated gene (ISG) viperin has drawn our attention as it has been highlighted as an important antiviral protein against several viral infections. The studies of the mechanistic actions of viperin have described important functional domains relating its antiviral and immune‐modulating actions through cellular lipid structures. In line with this, through silencing and overexpression approaches, we have identified viperin as an antiviral ISG against JUNV. In addition, we found that lipid droplet structures are modulated during JUNV infection, suggesting its relevance for proper virus multiplication. Furthermore, our confocal microscopy images, bioinformatics and functional results also revealed viperin‐JUNV protein interactions that might be participating in this antiviral pathway at lipid droplet level. Altogether, these results will help to better understand the factors mediating innate immunity in arenavirus infection and may lead to the development of pharmacological agents that can boost their effectiveness thereby leading to new treatments for this viral disease., Supplemental Material : Abstracts: poster presentations Meeting abstract: P3550

Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and Fe-55-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. Fe-55-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for (55F)e/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.

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.