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The role of the type I interferons and viperin during neurotropic flavivirus infection
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. (Anna Överby)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Flaviviruses are globally distributed pathogens that cause millions of human infections annually. One of the most detrimental outcomes of flavivirus infection is encephalitis, which is caused by neurotropic flaviviruses such as West Nile virus (WNV), Japanese encephalitis virus (JEV), and Tick-borne encephalitis virus (TBEV). The type I interferons (IFNs) are powerful cytokines, and they are known as the first line of defense against viral infection. IFNs are expressed at low or undetectable levels at the basal state, but recognition of invading pathogens triggers a robust IFN response. After synthesis, IFN is secreted and acts in an autocrine or paracrine manner by binding to the interferon-α/β receptor (IFNAR) receptor, which is expressed on the surface of all nucleated cells. Binding to IFNAR mediates a downstream cascade that triggers expression of hundreds of interferon-stimulated genes (ISGs). Some ISGs express signaling molecules to amplify the response while others are potent antiviral proteins that can efficiently limit viral infection. The impact of the type I IFN response in tick-borne flavivirus infection was not previously known. We found that the type I IFN response was crucial for protection of mice against neurotropic infection with tick-borne flaviviruses such as TBEV and Langat virus (LGTV). The response was needed both in the periphery as well as in the central nervous system (CNS), as transgenic mice lacking either peripherally or CNS-located IFNAR both succumbed to LGTV infection. Although we found that the local IFN response within the CNS is essential for protection against lethal LGTV infection, the cells responsible for the local IFN production were not known.

Astrocytes are one of the most abundant cell types within the CNS, but their role in neurotropic flavivirus infection was not fully characterized. In other viral infections, astrocytes are potent IFN producers, thus we were interested in characterizing the role of the type I IFN response in astrocytes during neurotropic flavivirus infection and its contribution to flavivirus pathogenesis. We found that upon flavivirus infection, astrocytes mount a strong type I IFN response that protects neighboring astrocytes from TBEV, JEV, WNV, and ZIKV infection. Furthermore, IFN signaling was found to protect astrocytes from TBEV-induced cytopathic effects. However, the ISGs that mediated these effects were not known.

In vitro studies of viperin, which was discovered in 2001 as an ISG with broad antiviral activity, has shown strong antiviral activity against TBEV, but its role in vivo and mode of action in flavivirus infection was not known. Using mice deficient in viperin, we wanted to determine the role of viperin in flavivirus infection. We found that viperin plays a region-specific role in the brain by controlling LGTV replication in the olfactory bulb and cerebrum. Remarkably, viperin was able to inhibit TBEV replication in primary cortical neurons isolated from the cerebrum but not in granule cell neurons isolated from the cerebellum. Furthermore, IFN treatment failed to compensate for loss of viperin in cortical neurons, indicating that viperin might be the most important ISG against TBEV in cortical neurons. Interestingly, we also found that viperin is needed for the IFN-mediated antiviral response against WNV and ZIKV in cortical neurons. Thus, viperin showed broad but region-specific antiviral mechanisms against different flaviviruses.

Although viperin has been shown to inhibit many viruses, the molecular antiviral mechanism is not clear and appears to differ between viruses. We performed a co-immunoprecipitation (CoIP) screen to identify TBEV proteins that could interact with viperin, and prM, E, NS2A, NS2B, and NS3 were identified. Interaction of viperin with NS3 resulted in degradation of the viral protein. We screened NS3 of JEV, yellow fever virus (YFV), ZIKV, and TBEV. Interestingly, although all NS3 proteins tested interacted with viperin, only those of ZIKV, and TBEV were significantly degraded by viperin. The degradation of NS3 correlated well with the antiviral activity of viperin, as only TBEV and ZIKV were inhibited.

In summary, this work revealed the importance of the local type I IFN response in the brain during neurotropic infections by flaviviruses. We identified astrocytes to be an important IFN producer within the CNS during neurotropic flavivirus infection. Astrocytes release type I IFN quickly after viral infection, and this interferon protects neighboring neurons and astrocytes from infection. Furthermore, viperin, a very potent antiviral ISG, is highly expressed in astrocytes and it is essential for controlling viral replication and mediating viral clearance in both neurons and astrocytes of the cerebrum. We also found that viperin specifically targeted the NS3 proteins of TBEV and ZIKV for degradation.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2017. , 66 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1921
Keyword [en]
TBEV, Flavivirus, Interferon, Neurotropic, Viperin
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-140290ISBN: 978-91-7601-780-7 (print)OAI: oai:DiVA.org:umu-140290DiVA: diva2:1146863
Public defence
2017-10-27, Hörsal D Unod T9, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-10-06 Created: 2017-10-04 Last updated: 2017-10-06Bibliographically approved
List of papers
1. Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses
Open this publication in new window or tab >>Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses
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2014 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 89, no 21, 12202-12212 p.Article in journal (Refereed) Published
Abstract [en]

Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.

Place, publisher, year, edition, pages
American Society for Microbiology, 2014
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-96495 (URN)10.1128/JVI.01215-14 (DOI)000343314900004 ()
Available from: 2014-12-01 Created: 2014-11-21 Last updated: 2017-10-04Bibliographically approved
2. Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects
Open this publication in new window or tab >>Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects
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2016 (English)In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 13, 277Article in journal (Refereed) Published
Abstract [en]

Background: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. Methods: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR(-/-)) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. Results: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. Conclusions: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.

Keyword
Astrocytes, Interferon, TBEV, Flavivirus, Viperin
National Category
Immunology Neurosciences
Identifiers
urn:nbn:se:umu:diva-127599 (URN)10.1186/s12974-016-0748-7 (DOI)000385979600001 ()27776548 (PubMedID)
Available from: 2016-12-09 Created: 2016-11-16 Last updated: 2017-10-04Bibliographically approved
3. Brain region- and cell type-specific role of viperin in neurotropic flavivirus infection
Open this publication in new window or tab >>Brain region- and cell type-specific role of viperin in neurotropic flavivirus infection
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-140287 (URN)
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-04
4. Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation
Open this publication in new window or tab >>Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation
(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-140289 (URN)
Available from: 2017-10-04 Created: 2017-10-04 Last updated: 2017-10-04

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