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Modulation of Potassium Channels Inhibits Bunyavirus Infection*

Open AccessPublished:December 16, 2015DOI:https://doi.org/10.1074/jbc.M115.692673
      Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

      Introduction

      The Bunyaviridae family represents the largest taxonomic grouping of negative sense RNA− viruses, with over 350 named members (
      • Adams M.J.
      • Lefkowitz E.J.
      • King A.M.
      • Carstens E.B.
      Recently agreed changes to the International Code of Virus Classification and Nomenclature.
      ). The family is divided into five genera, Orthobunyavirus, Hantavirus, Tospovirus, Phlebovirus, and Nairovirus and takes its name from Bunyamwera virus (BUNV),
      The abbreviations used are: BUNV
      Bunyamwera virus
      ORF
      open reading frame
      RLU
      relative luminescence unit
      MQAE
      6-methoxy-quinolyl acetoethyl ester
      NPPB
      5-nitro-1–3-phenylpropylamino benzoic acid
      IAA
      indyanyloxyacetic acid
      TEA
      tetraethylammonium
      CPE
      cytopathic effect
      CME
      clathrin-mediated endocytosis
      SBV
      Schmallenberg virus.
      the prototype of the genus Orthobunyavirus.
      All bunyaviruses share common elements of virion structure being enveloped and containing an RNA genome that comprises three separate RNA segments named small (S), medium (M), and large (L). These three segments encode four structural proteins using an expression strategy that is conserved across all members of the family: the S segment encodes the nucleoprotein (N), the M segment encodes two glycoproteins (Gn and Gc), and the L segment encodes an RNA-dependent RNA polymerase (L protein). Most Bunyaviridae family members including BUNV also encode two non-structural proteins; NSs from the S segment, and NSm from the M segment (
      • Bridgen A.
      • Weber F.
      • Fazakerley J.K.
      • Elliott R.M.
      Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.
      ,
      • Elliott R.M.
      Orthobunyaviruses: recent genetic and structural insights.
      ).
      Bunyaviruses are predominantly arthropod-borne viruses capable of infecting a wide range of hosts including humans, plants, and animals. Four of the five bunyavirus genera include members that are associated with lethal hemorrhagic fevers in infected humans, and many are considered to be emerging pathogens due to a complex combination of factors including travel, animal trade, and climate change (
      • Elliott R.M.
      Bunyaviruses and climate change.
      ,
      • Maltezou H.C.
      • Papa A.
      Crimean-Congo hemorrhagic fever: risk for emergence of new endemic foci in Europe?.
      ,
      • Zhang Y.Z.
      • He Y.W.
      • Dai Y.A.
      • Xiong Y.
      • Zheng H.
      • Zhou D.J.
      • Li J.
      • Sun Q.
      • Luo X.L.
      • Cheng Y.L.
      • Qin X.C.
      • Tian J.H.
      • Chen X.P.
      • Yu B.
      • Jin D.
      • Guo W.P.
      • Li W.
      • Wang W.
      • Peng J.S.
      • Zhang G.B.
      • Zhang S.
      • Chen X.M.
      • Wang Y.
      • Li M.H.
      • Li Z.
      • Lu S.
      • Ye C.
      • de Jong M.D.
      • Xu J.
      Hemorrhagic fever caused by a novel Bunyavirus in China: pathogenesis and correlates of fatal outcome.
      ). Bunyavirus emergence is facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity (
      • Briese T.
      • Calisher C.H.
      • Higgs S.
      Viruses of the family Bunyaviridae: are all available isolates reassortants?.
      ). Schmallenberg (SBV) and Ngari (NGAV) viruses are orthobunyaviruses that exemplify this phenomenon; NGAV, which causes a highly fatal hemorrhagic fever in humans (
      • Bowen M.D.
      • Trappier S.G.
      • Sanchez A.J.
      • Meyer R.F.
      • Goldsmith C.S.
      • Zaki S.R.
      • Dunster L.M.
      • Peters C.J.
      • Ksiazek T.G.
      • Nichol S.T.
      • RVF Task Force
      A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.
      ) is a reassortant possessing S and L segments from BUNV and the M segment from the closely related Batai virus (BATV) (
      • Briese T.
      • Bird B.
      • Kapoor V.
      • Nichol S.T.
      • Lipkin W.I.
      Batai and Ngari viruses: M segment reassortment and association with severe febrile disease outbreaks in East Africa.
      ). Similarly, SBV, which is a pathogen responsible for teratogenic disease in sheep and cattle (
      • Hoffmann B.
      • Scheuch M.
      • Höper D.
      • Jungblut R.
      • Holsteg M.
      • Schirrmeier H.
      • Eschbaumer M.
      • Goller K.V.
      • Wernike K.
      • Fischer M.
      • Breithaupt A.
      • Mettenleiter T.C.
      • Beer M.
      Novel orthobunyavirus in Cattle, Europe, 2011.
      ), has a complex genetic background possessing segments shared with previously characterized Sathuperi and Shamonda viruses (
      • Goller K.V.
      • Höper D.
      • Schirrmeier H.
      • Mettenleiter T.C.
      • Beer M.
      Schmallenberg virus as possible ancestor of Shamonda virus.
      ). The threat of widespread arthropod borne transmission of these potentially lethal emerging viruses means the development of preventative and therapeutic strategies is urgently required.
      One strategy to identify new anti-viral therapies is to target virus host interactions that are essential for virus multiplication. Many examples of such interactions have been described for bunyaviruses (reviewed in Refs.
      • Walter C.T.
      • Bento D.F.
      • Alonso A.G.
      • Barr J.N.
      Amino acid changes within the Bunyamwera virus nucleocapsid protein differentially affect the mRNA transcription and RNA replication activities of assembled ribonucleoprotein templates.
      ,
      • Schoen A.
      • Weber F.
      Orthobunyaviruses and innate immunity induction: alieNSs vs. PredatoRRs.
      ) and other recent examples include those involved in virus entry (
      • Meier R.
      • Franceschini A.
      • Horvath P.
      • Tetard M.
      • Mancini R.
      • von Mering C.
      • Helenius A.
      • Lozach P.Y.
      Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetration.
      ), counteracting the antiviral response (
      • Barry G.
      • Varela M.
      • Ratinier M.
      • Blomström A.L.
      • Caporale M.
      • Seehusen F.
      • Hahn K.
      • Schnettler E.
      • Baumgärtner W.
      • Kohl A.
      • Palmarini M.
      NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery.
      ,
      • Cheng E.
      • Haque A.
      • Rimmer M.A.
      • Hussein I.T.
      • Sheema S.
      • Little A.
      • Mir M.A.
      Characterization of the Interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19).
      ,
      • Weber F.
      • Bridgen A.
      • Fazakerley J.K.
      • Streitenfeld H.
      • Kessler N.
      • Randall R.E.
      • Elliott R.M.
      Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of α/β interferon.
      ) and modulating host gene expression (
      • Cheng E.
      • Haque A.
      • Rimmer M.A.
      • Hussein I.T.
      • Sheema S.
      • Little A.
      • Mir M.A.
      Characterization of the Interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19).
      ,
      • Kainulainen M.
      • Habjan M.
      • Hubel P.
      • Busch L.
      • Lau S.
      • Colinge J.
      • Superti-Furga G.
      • Pichlmair A.
      • Weber F.
      Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.
      ). Another group of potential targets are host cell ion channels, which regulate ion homeostasis across all cellular membranes and are key players in a broad and extensive range of cellular processes including the cell cycle, gene expression, cell signaling, and innate immunity (
      • Becchetti A.
      • Munaron L.
      • Arcangeli A.
      The role of ion channels and transporters in cell proliferation and cancer.
      ,
      • Lang F.
      • Shumilina E.
      • Ritter M.
      • Gulbins E.
      • Vereninov A.
      • Huber S.M.
      Ion channels and cell volume in regulation of cell proliferation and apoptotic cell death.
      ,
      • Feske S.
      • Skolnik E.Y.
      • Prakriya M.
      Ion channels and transporters in lymphocyte function and immunity.
      ). Ion channels are emerging as key factors required during virus replicative cycles, and have been assigned critical roles in virus entry, survival, and release. Examples include roles for both potassium (K+) and chloride ion (Cl) channels during the hepatitis C virus lifecycle (
      • Igloi Z.
      • Mohl B.P.
      • Lippiat J.D.
      • Harris M.
      • Mankouri J.
      Requirement for chloride channel function during the hepatitis C virus life cycle.
      ,
      • Mankouri J.
      • Dallas M.L.
      • Hughes M.E.
      • Griffin S.D.
      • Macdonald A.
      • Peers C.
      • Harris M.
      Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence.
      ), the ability of human immunodeficiency virus proteins to modulate K+ channel activity (
      • Hsu K.
      • Seharaseyon J.
      • Dong P.
      • Bour S.
      • Marbán E.
      Mutual functional destruction of HIV-1 Vpu and host TASK-1 channel.
      ,
      • Irvine E.
      • Keblesh J.
      • Liu J.
      • Xiong H.
      Voltage-gated potassium channel modulation of neurotoxic activity in human immunodeficiency virus type-1(HIV-1)-infected macrophages.
      ) and the recently identified requirement of two-pore calcium (Ca2+) channels (TPCs) in endosomal membranes for successful entry of Ebola virus (
      • Sakurai Y.
      • Kolokoltsov A.A.
      • Chen C.C.
      • Tidwell M.W.
      • Bauta W.E.
      • Klugbauer N.
      • Grimm C.
      • Wahl-Schott C.
      • Biel M.
      • Davey R.A.
      Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.
      ). Here, we report that BUNV multiplication depends on the function of cellular K+ channels. Using the available ion channel pharmacological tools, a small family of two-pore potassium ion channels (K2P) was identified as the candidate K+ channel family critical for BUNV multiplication. We propose that targeting K+ channel function may represent a new, pharmacologically safe and broad ranging therapeutic strategy for this important family of pathogens.

      Discussion

      Ion channels underpin an array of essential cellular processes and drugs acting on specific ion channels are the treatment of choice for many diseases (
      • Bagal S.K.
      • Brown A.D.
      • Cox P.J.
      • Omoto K.
      • Owen R.M.
      • Pryde D.C.
      • Sidders B.
      • Skerratt S.E.
      • Stevens E.B.
      • Storer R.I.
      • Swain N.A.
      Ion channels as therapeutic targets: a drug discovery perspective.
      ). Using the available pharmacological tools targeting various cellular ion channels, this study showed that K+ channel function is critical to the growth of BUNV (FIGURE 1, FIGURE 2), the prototypic bunyavirus in both vertebrate and invertebrate cells. K+ channel dependence was also observed with HAZV and SBV, revealing this as a key virus-host interaction across other bunyaviruses (Fig. 3), suggesting it may be a general property that applies to all family members.
      It is not clear why the BUNV replicative cycle is dependent upon manipulation of host cell K+ channel currents. Previous reliance of bunyavirus replication had been noted by Frugulhetti and Rebello (
      • Frugulhetti I.C.
      • Rebello M.A.
      Na+ and K+ concentration and regulation of protein synthesis in L-A9 and Aedes albopictus cells infected with Marituba virus (Bunyaviridae).
      ), who reported that changes in K+ ion concentrations disrupted the growth of the orthobunyavirus Marituba virus. The stage of the life cycle affected was found to be mRNA translation, although the mechanism that underpinned this dependence was not pursued, and the role of cellular ion channels in this process was not investigated. In the present study, K+ channel modulation had no discernible effect on mRNA transcription and translation in the context of the BUNV minireplicon system (Fig. 4C) and so we suggest that our findings of K+ channel reliance are likely unrelated to these early findings. To better understand the nature of the K+ channel requirement, time of addition assays were performed which indicated that the K+ sensitive steps in the BUNV life cycle occur within the initial 6 h of virus infection, but do not include virus binding/entry (Fig. 4, A and B). It is thus most likely that K+ channel modulation contributes to early events within the virus life cycle including virus uncoating and/or events prior to the formation of RNA replication factories, postulated to assemble around the Golgi (
      • Fontana J.
      • López-Montero N.
      • Elliott R.M.
      • Fernández J.J.
      • Risco C.
      The unique architecture of Bunyamwera virus factories around the Golgi complex.
      ,
      • Shi X.
      • Lappin D.F.
      • Elliott R.M.
      Mapping the Golgi targeting and retention signal of Bunyamwera virus glycoproteins.
      ,
      • Novoa R.R.
      • Calderita G.
      • Cabezas P.
      • Elliott R.M.
      • Risco C.
      Key Golgi factors for structural and functional maturation of bunyamwera virus.
      ).
      A key finding of this study is that K2P channels are the K+ family member required for BUNV infection. BUNV was inhibited by diverse pharmacological agents that modulate K2P channels (
      • Mathie A.
      • Al-Moubarak E.
      • Veale E.L.
      Gating of two pore domain potassium channels.
      ,
      • Musset B.
      • Meuth S.G.
      • Liu G.X.
      • Derst C.
      • Wegner S.
      • Pape H.C.
      • Budde T.
      • Preisig-Müller R.
      • Daut J.
      Effects of divalent cations and spermine on the K+ channel TASK-3 and on the outward current in thalamic neurons.
      ,
      • Enyedi P.
      • Czirják G.
      Molecular background of leak K+ currents: two-pore domain potassium channels.
      ,
      • Kudo S.
      • Ishizaki T.
      Pharmacokinetics of haloperidol: an update.
      ,
      • Shin H.W.
      • Soh J.S.
      • Kim H.Z.
      • Hong J.
      • Woo D.H.
      • Heo J.Y.
      • Hwang E.M.
      • Park J.Y.
      • Lee C.J.
      The inhibitory effects of bupivacaine, levobupivacaine, and ropivacaine on K2P (two-pore domain potassium) channel TREK-1.
      ), but no effects of K+ channel modulators targeting Kv, BK, and Kir channels was observed. The family of K2P channels is small containing only ∼17 members, which regulate the membrane potential of both “excitable” and “non-excitable” cells (
      • Enyedi P.
      • Czirják G.
      Molecular background of leak K+ currents: two-pore domain potassium channels.
      ). K2P channels respond to a diverse array of stimuli including pH, temperature and membrane stretch and their pharmacology is highly characteristic, as a result of their distinct structural features (
      • Goldstein S.A.
      K2P potassium channels, mysterious and paradoxically exciting.
      ,
      • Dong Y.Y.
      • Pike A.C.
      • Mackenzie A.
      • McClenaghan C.
      • Aryal P.
      • Dong L.
      • Quigley A.
      • Grieben M.
      • Goubin S.
      • Mukhopadhyay S.
      • Ruda G.F.
      • Clausen M.V.
      • Cao L.
      • Brennan P.E.
      • Burgess-Brown N.A.
      • Sansom M.S.
      • Tucker S.J.
      • Carpenter E.P.
      K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac.
      ,
      • Miller A.N.
      • Long S.B.
      Crystal structure of the human two-pore domain potassium channel K2P1.
      ). Using a fluorescent membrane potential probe, hyperpolarization of the plasma membrane was observed in BUNV-infected cells 6 hpi (Fig. 4), a shift consistent with an enhancement of K2P activity at the stage of the virus lifecycle sensitive to K+ channel modulation. Many viruses encode their own viroporin (reviewed in Ref.
      • Nieva J.L.
      • Madan V.
      • Carrasco L.
      Viroporins: structure and biological functions.
      ), which are pore forming proteins that have been documented to participate in several viral functions, including the promotion of release of virus particles, modulation of cellular vesicles, glycoprotein trafficking, and membrane permeability. As BUNV has no known viroporin, it is possible that BUNV proteins have evolved to interfere and depend on cellular K2P channels to aid virus pathogenesis. While we do not understand the mechanism(s) of K2P activation, our findings implicate BUNV structural proteins present in the virion to mediate these effects since viral gene expression would not have occurred at these timepoints, and thus no new proteins would be generated. Orthobunyaviruses have been shown to enter cells via clathrin mediated endocytosis (
      • Hollidge B.S.
      • Nedelsky N.B.
      • Salzano M.V.
      • Fraser J.W.
      • González-Scarano F.
      • Soldan S.S.
      Orthobunyavirus entry into neurons and other mammalian cells occurs via clathrin-mediated endocytosis and requires trafficking into early endosomes.
      ), but events post-entry are less well understood, although virion trafficking of CCHFV has been shown to be ESCRT dependent (
      • Shtanko O.
      • Nikitina R.A.
      • Altuntas C.Z.
      • Chepurnov A.A.
      • Davey R.A.
      Crimean-Congo hemorrhagic fever virus entry into host cells occurs through the multivesicular body and requires ESCRT regulators.
      ). The modulation/reliance upon host cell ion K2P channels at this post-entry stage is consistent with recent observations for other negative sense enveloped viruses. Post-entry trafficking of Ebola virus, a single stranded negative sense RNA virus, is dependent upon endosomal calcium channels termed two-pore channels (TPCs) (
      • Sakurai Y.
      • Kolokoltsov A.A.
      • Chen C.C.
      • Tidwell M.W.
      • Bauta W.E.
      • Klugbauer N.
      • Grimm C.
      • Wahl-Schott C.
      • Biel M.
      • Davey R.A.
      Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.
      ). In addition the capsid (C) protein of dengue virus, a single-stranded positive sense RNA virus, forms an association with lipid membranes that is intrinsically dependent on the intracellular concentrations of K+ ions (
      • Carvalho F.A.
      • Carneiro F.A.
      • Martins I.C.
      • Assunção-Miranda I.
      • Faustino A.F.
      • Pereira R.M.
      • Bozza P.T.
      • Castanho M.A.
      • Mohana-Borges R.
      • Da Poian A.T.
      • Santos N.C.
      Dengue virus capsid protein binding to hepatic lipid droplets (LD) is potassium ion dependent and is mediated by LD surface proteins.
      ). The cellular mechanisms of these ion-channel dependent events remain to be elucidated.
      As HAZV represents a model for CCHFV (
      • Elliott R.M.
      • Wilkie M.L.
      Persistent infection of Aedes albopictus C6/36 cells by Bunyamwera virus.
      ), a cause of a severe, often fatal disease in humans, this study highlights the potential of K+ channel modulating drugs as a novel therapeutic intervention against more dangerous pathogens of this family. It is interesting to note that compounds such as haloperidol and fluoxetine possess highly desirable drug properties in a clinical setting; good oral bioavailability (60–70%), generally well tolerated with severe side effects uncommon and the ability to cross the blood brain barrier (
      • Kudo S.
      • Ishizaki T.
      Pharmacokinetics of haloperidol: an update.
      ,
      • Kaakkola S.
      • Lehtosalo J.
      • Laitinen L.A.
      Changes in blood-brain barrier permeability to drugs in decompressed rats.
      ). This may be pertinent for bunyavirus treatment since BUNV infection in both mouse and horses is largely neurotropic (
      • Tauro L.B.
      • Rivarola M.E.
      • Lucca E.
      • Mariño B.
      • Mazzini R.
      • Cardoso J.F.
      • Barrandeguy M.E.
      • Teixeira Nunes M.R.
      • Contigiani M.S.
      First isolation of Bunyamwera virus (Bunyaviridae family) from horses with neurological disease and an abortion in Argentina.
      ,
      • Murphy F.A.
      • Harrison A.K.
      • Tzianabos T.
      Electron microscopic observations of mouse brain infected with Bunyamwera group arboviruses.
      ). Indeed BUNV infection was permissive in U87-MG cells; a human primary glioblastoma cell line, and could be inhibited by K+ channel modulation (Fig. 2).
      In conclusion, despite encompassing a large number of viruses capable of causing outbreaks that would impact public health, national economies and food security; little is known regarding key stages in the life cycle of bunyaviruses. This study has demonstrated the importance of K+ channel activity during the BUNV lifecycle and contributes to the growing field of viral ion channel interactions. Targeting these K+ channels in patients infected with severe Bunyaviridae infections may therefore represent a new prospect for future anti-viral drug development.

      Author Contributions

      S. H., B. K., B. H., E. L., and H. T. performed the experiments. S. H., B. K., A. K., J. N. B., J. M. conceived the experiments. J. D., M. D., C. P., E. S., C. M., A. K. provided reagents and expertise. B. K., J. N. B., and J. M. wrote the manuscript.

      Acknowledgments

      We acknowledge the late Richard M. Elliott for contribution of reagents, support, and advice during this and previous studies. The confocal microscope used for imaging was funded by a Royal Society equipment grant (Grant Number RG110306).

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