FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2913398081> ?p ?o ?g. }

• W2913398081 endingPage "1098.e4" @default.
• W2913398081 startingPage "1089" @default.
• W2913398081 abstract "•Zika protease NS2B3 is a heterodimer that cleaves host proteins in hNPCs•NS2B3 expression causes protease-dependent cytokinesis defects and apoptosis•NS2B3 associates with the septin cytoskeleton and cleaves Septin-2•Non-cleavable Septin-2 partially restores cytokinesis in NS2B3-expressing cells Zika virus (ZIKV) targets neural progenitor cells in the brain, attenuates cell proliferation, and leads to cell death. Here, we describe a role for the ZIKV protease NS2B-NS3 heterodimer in mediating neurotoxicity through cleavage of a host protein required for neurogenesis. Similar to ZIKV infection, NS2B-NS3 expression led to cytokinesis defects and cell death in a protease activity-dependent fashion. Among binding partners, NS2B-NS3 cleaved Septin-2, a cytoskeletal factor involved in cytokinesis. Cleavage of Septin-2 occurred at residue 306 and forced expression of a non-cleavable Septin-2 restored cytokinesis, suggesting a direct mechanism of ZIKV-induced neural toxicity.Video AbstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3ZjczOTg4ODVlYzI1MDhjZTA2N2Y2NGI5MWI2MjkyZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NzE2ODIwfQ.LVPrc_FDBwkBKyNFMcfuDWUjkE5Ztbe7TyBQ44jaGukzM-R7VGtlvSGzPOW5NOZ8901uZJth84j-d8-2ldu4PAqbN7hMyIflHxKw_TBPSnshkJBaOmz58GeiZDBK8qiqU0MjDW_QEFFWqllscav5pzoA1Lmmg_22vW53tYtOuj-Gq4CFI8A91sfPkKk9g2sgYwDSki48gssg5lWLlWlzF9JpI_B8grTxByvycs5eUBzRLPd-44g7zGNVIau518J38bUnIVo_fKsu_QJyzfg0pcluxo0KVPDveDFon_UzE5kUqPkl71U5jegaa7zyH8DxuqJ6HbDZYTxX-07p3cktww(mp4, (35.69 MB) Download video Zika virus (ZIKV) targets neural progenitor cells in the brain, attenuates cell proliferation, and leads to cell death. Here, we describe a role for the ZIKV protease NS2B-NS3 heterodimer in mediating neurotoxicity through cleavage of a host protein required for neurogenesis. Similar to ZIKV infection, NS2B-NS3 expression led to cytokinesis defects and cell death in a protease activity-dependent fashion. Among binding partners, NS2B-NS3 cleaved Septin-2, a cytoskeletal factor involved in cytokinesis. Cleavage of Septin-2 occurred at residue 306 and forced expression of a non-cleavable Septin-2 restored cytokinesis, suggesting a direct mechanism of ZIKV-induced neural toxicity. Zika virus (ZIKV) represents a new threat to global health, due to the unexpected association with congenital neurodevelopmental birth defects observed in the fetuses of infected women during critical periods of pregnancy. During the peak of the epidemic in Brazil in 2016, the reported incidence of microcephaly increased 26-fold over baseline (Butler, 2016Butler D. Zika virus: Brazil’s surge in small-headed babies questioned by report.Nature. 2016; 530: 13-14Crossref PubMed Scopus (47) Google Scholar). ZIKV is capable of direct infection of neural progenitor cells in the fetal brain, leading to delayed mitosis, activation of p53, and apoptotic cell death (Ghouzzi et al., 2017Ghouzzi V.E. Bianchi F.T. Molineris I. Mounce B.C. Berto G.E. Rak M. Lebon S. Aubry L. Tocco C. Gai M. et al.ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53.Cell Death Dis. 2017; 8: e2567Crossref PubMed Scopus (10) Google Scholar, Li et al., 2016cLi H. Saucedo-Cuevas L. Shresta S. Gleeson J.G. The neurobiology of Zika virus.Neuron. 2016; 92: 949-958Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, Ming et al., 2016Ming G.L. Tang H. Song H. Advances in Zika virus research: Stem cell models, challenges, and opportunities.Cell Stem Cell. 2016; 19: 690-702Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Zhang et al., 2016Zhang F. Hammack C. Ogden S.C. Cheng Y. Lee E.M. Wen Z. Qian X. Nguyen H.N. Li Y. Yao B. et al.Molecular signatures associated with ZIKV exposure in human cortical neural progenitors.Nucleic Acids Res. 2016; 44: 8610-8620Crossref PubMed Scopus (119) Google Scholar). ZIKV contains a positive single-stranded 11-kb RNA genome encoding three structural (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), providing functional diversity required for its life cycle. Individual ZIKV proteins tested to date, when introduced into human neural precursor cells (hNPCs) or fetal murine brain, can impact neurogenesis but are insufficient to mediate cell death (Liang et al., 2016Liang Q. Luo Z. Zeng J. Chen W. Foo S.S. Lee S.A. Ge J. Wang S. Goldman S.A. Zlokovic B.V. et al.Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy.Cell Stem Cell. 2016; 19: 663-671Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, Yoon et al., 2017Yoon K.J. Song G. Qian X. Pan J. Xu D. Rho H.S. Kim N.S. Habela C. Zheng L. Jacob F. et al.Zika-virus-encoded NS2A disrupts mammalian cortical neurogenesis by degrading adherens junction proteins.Cell Stem Cell. 2017; 21: 349-358.e6Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Some ZIKV proteins can form heteromultimeric complexes, including prM and E, which assemble during viral packaging (Lorenz et al., 2002Lorenz I.C. Allison S.L. Heinz F.X. Helenius A. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum.J. Virol. 2002; 76: 5480-5491Crossref PubMed Scopus (188) Google Scholar), and NS2B and NS3, which form a functional heterodimeric protease (Zuo et al., 2009Zuo Z. Liew O.W. Chen G. Chong P.C. Lee S.H. Chen K. Jiang H. Puah C.M. Zhu W. Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays.J. Virol. 2009; 83: 1060-1070Crossref PubMed Scopus (43) Google Scholar). NS2B is a co-factor of NS3, which enhances its proteolytic activity and stabilizes its folding. NS3 is a multifunctional protein common to the flavivirus genus, with an N-terminal serine protease domain and a C-terminal nucleoside triphosphatase (NTPase) and RNA helicase domains (Wengler and Wengler, 1991Wengler G. Wengler G. The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase.Virology. 1991; 184: 707-715Crossref PubMed Scopus (161) Google Scholar). Cytokinesis is the final stage of cell division whereby the two daughter cells physically separate, which requires contribution from the septin cytoskeleton. During cytokinesis, the contractile ring forms beneath the cell’s equatorial surface to form the cleavage furrow, and then ingression of the furrow results in the formation of an intercellular bridge called the midbody (D’Avino et al., 2015D’Avino P.P. Giansanti M.G. Petronczki M. Cytokinesis in animal cells.Cold Spring Harb. Perspect. Biol. 2015; 7: a015834PubMed Google Scholar) and then finally abscission as the two daughter cells separate. Cytokinesis failure results in binucleated and multi-centrosome-containing cells, with resultant aneuploidy, genotoxic stress, and cell death (Hayashi and Karlseder, 2013Hayashi M.T. Karlseder J. DNA damage associated with mitosis and cytokinesis failure.Oncogene. 2013; 32: 4593-4601Crossref PubMed Scopus (99) Google Scholar). Septins are highly conserved guanosine triphosphate (GTP)-binding proteins that hetero-oligomerize (Mostowy and Cossart, 2012Mostowy S. Cossart P. Septins: the fourth component of the cytoskeleton.Nat. Rev. Mol. Cell Biol. 2012; 13: 183-194Crossref PubMed Scopus (480) Google Scholar) and are crucial for midbody formation during cytokinesis (Shindo and Wallingford, 2014Shindo A. Wallingford J.B. PCP and septins compartmentalize cortical actomyosin to direct collective cell movement.Science. 2014; 343: 649-652Crossref PubMed Scopus (127) Google Scholar, Spiliotis et al., 2005Spiliotis E.T. Kinoshita M. Nelson W.J. A mitotic septin scaffold required for Mammalian chromosome congression and segregation.Science. 2005; 307: 1781-1785Crossref PubMed Scopus (196) Google Scholar) as well as membrane trafficking, spermatogenesis, and dendrite branching (Beites et al., 1999Beites C.L. Xie H. Bowser R. Trimble W.S. The septin CDCrel-1 binds syntaxin and inhibits exocytosis.Nat. Neurosci. 1999; 2: 434-439Crossref PubMed Scopus (309) Google Scholar, Ihara et al., 2005Ihara M. Kinoshita A. Yamada S. Tanaka H. Tanigaki A. Kitano A. Goto M. Okubo K. Nishiyama H. Ogawa O. et al.Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa.Dev. Cell. 2005; 8: 343-352Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, Xie et al., 2007Xie Y. Vessey J.P. Konecna A. Dahm R. Macchi P. Kiebler M.A. The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology.Curr. Biol. 2007; 17: 1746-1751Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Of the 14 septin paralogs in humans, Septin 2, 6, and 7 bind in a heterotrimeric linear fashion (Weirich et al., 2008Weirich C.S. Erzberger J.P. Barral Y. The septin family of GTPases: architecture and dynamics.Nat. Rev. Mol. Cell Biol. 2008; 9: 478-489Crossref PubMed Scopus (256) Google Scholar), which then binds other trimers to produce filaments and rings (Sirajuddin et al., 2007Sirajuddin M. Farkasovsky M. Hauer F. Kühlmann D. Macara I.G. Weyand M. Stark H. Wittinghofer A. Structural insight into filament formation by mammalian septins.Nature. 2007; 449: 311-315Crossref PubMed Scopus (334) Google Scholar). Congenital microcephaly refers to birth head circumference <2 SDs below mean, which can have genetic or environmental/viral origins (Volpe et al., 2018Volpe J. Inder T. Darras B. de Vries L.S. du Plessis A. Neil J. Perlman J. Volpe’s Neurology of the Newborn. Elsevier, 2018Google Scholar), and ZIKV-related microcephaly shares many radiographic and pathological features with genetic forms of disease (Besnard et al., 2016Besnard M. Eyrolle-Guignot D. Guillemette-Artur P. Lastère S. Bost-Bezeaud F. Marcelis L. Abadie V. Garel C. Moutard M.L. Jouannic J.M. et al.Congenital cerebral malformations and dysfunction in fetuses and newborns following the 2013 to 2014 Zika virus epidemic in French Polynesia.Euro Surveill. 2016; 21: 13Crossref Scopus (148) Google Scholar, Chimelli et al., 2017Chimelli L. Melo A.S.O. Avvad-Portari E. Wiley C.A. Camacho A.H.S. Lopes V.S. Machado H.N. Andrade C.V. Dock D.C.A. Moreira M.E. et al.The spectrum of neuropathological changes associated with congenital Zika virus infection.Acta Neuropathol. 2017; 133: 983-999Crossref PubMed Scopus (122) Google Scholar, de Fatima Vasco Aragao et al., 2016de Fatima Vasco Aragao M. van der Linden V. Brainer-Lima A.M. Coeli R.R. Rocha M.A. Sobral da Silva P. Durce Costa Gomes de Carvalho M. van der Linden A. Cesario de Holanda A. Valenca M.M. Clinical features and neuroimaging (CT and MRI) findings in presumed Zika virus related congenital infection and microcephaly: retrospective case series study.BMJ. 2016; 353: i1901Crossref PubMed Scopus (281) Google Scholar). Two genes mutated in recessive microcephaly in particular, KIF14 (kinesin 14) and CIT (citron kinase), encode proteins that localize to the mitotic cleavage furrow. Loss of either demonstrates delayed mitosis due to failed abscission, supernumerary centrosomes, and multinucleated cells (LoTurco et al., 2003LoTurco J.J. Sarkisian M.R. Cosker L. Bai J. Citron kinase is a regulator of mitosis and neurogenic cytokinesis in the neocortical ventricular zone.Cereb. Cortex. 2003; 13: 588-591Crossref PubMed Scopus (28) Google Scholar, Madaule et al., 1998Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Role of citron kinase as a target of the small GTPase Rho in cytokinesis.Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar, Moawia et al., 2017Moawia A. Shaheen R. Rasool S. Waseem S.S. Ewida N. Budde B. Kawalia A. Motameny S. Khan K. Fatima A. et al.Mutations of KIF14 cause primary microcephaly by impairing cytokinesis.Ann. Neurol. 2017; 82: 562-577Crossref PubMed Scopus (47) Google Scholar), resulting in chromosomal instability and p53-sensitive cell death (Bianchi et al., 2017Bianchi F.T. Tocco C. Pallavicini G. Liu Y. Vernì F. Merigliano C. Bonaccorsi S. El-Assawy N. Priano L. Gai M. et al.Citron kinase deficiency leads to chromosomal instability and TP53-sensitive microcephaly.Cell Rep. 2017; 18: 1674-1686Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Although the ZIKV-related and KIF14/CIT-related microcephaly differ in that ZIKV cases show frequent brain calcification (de Fatima Vasco Aragao et al., 2016de Fatima Vasco Aragao M. van der Linden V. Brainer-Lima A.M. Coeli R.R. Rocha M.A. Sobral da Silva P. Durce Costa Gomes de Carvalho M. van der Linden A. Cesario de Holanda A. Valenca M.M. Clinical features and neuroimaging (CT and MRI) findings in presumed Zika virus related congenital infection and microcephaly: retrospective case series study.BMJ. 2016; 353: i1901Crossref PubMed Scopus (281) Google Scholar, Zare Mehrjardi et al., 2017Zare Mehrjardi M. Poretti A. Huisman T.A. Werner H. Keshavarz E. Araujo Júnior E. Neuroimaging findings of congenital Zika virus infection: a pictorial essay.Jpn. J. Radiol. 2017; 35: 89-94Crossref PubMed Scopus (36) Google Scholar), recent evidence suggests that ZIKV-infected cells strikingly share several of these mitotic defects with KIF14 or CIT deficiency (Carleton et al., 2006Carleton M. Mao M. Biery M. Warrener P. Kim S. Buser C. Marshall C.G. Fernandes C. Annis J. Linsley P.S. RNA interference-mediated silencing of mitotic kinesin KIF14 disrupts cell cycle progression and induces cytokinesis failure.Mol. Cell. Biol. 2006; 26: 3853-3863Crossref PubMed Scopus (91) Google Scholar, Li et al., 2016bLi H. Bielas S.L. Zaki M.S. Ismail S. Farfara D. Um K. Rosti R.O. Scott E.C. Tu S. Chi N.C. et al.Biallelic mutations in citron kinase link mitotic cytokinesis to human primary microcephaly.Am. J. Hum. Genet. 2016; 99: 501-510Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Moawia et al., 2017Moawia A. Shaheen R. Rasool S. Waseem S.S. Ewida N. Budde B. Kawalia A. Motameny S. Khan K. Fatima A. et al.Mutations of KIF14 cause primary microcephaly by impairing cytokinesis.Ann. Neurol. 2017; 82: 562-577Crossref PubMed Scopus (47) Google Scholar, Onorati et al., 2016Onorati M. Li Z. Liu F. Sousa A.M.M. Nakagawa N. Li M. Dell’Anno M.T. Gulden F.O. Pochareddy S. Tebbenkamp A.T.N. et al.Zika virus disrupts phospho-TBK1 localization and mitosis in human neuroepithelial stem cells and radial glia.Cell Rep. 2016; 16: 2576-2592Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, Souza et al., 2016Souza B.S. Sampaio G.L. Pereira C.S. Campos G.S. Sardi S.I. Freitas L.A. Figueira C.P. Paredes B.D. Nonaka C.K. Azevedo C.M. et al.Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells.Sci. Rep. 2016; 6: 39775Crossref PubMed Scopus (136) Google Scholar, Wolf et al., 2017Wolf B. Diop F. Ferraris P. Wichit S. Busso C. Missé D. Gönczy P. Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning.Open Biol. 2017; 7: 160231Crossref PubMed Scopus (26) Google Scholar). Due to the similarities with these forms of microcephaly, and because ZIKV targets the radial glial neural stem cells of the brain (Garcez et al., 2016Garcez P.P. Loiola E.C. Madeiro da Costa R. Higa L.M. Trindade P. Delvecchio R. Nascimento J.M. Brindeiro R. Tanuri A. Rehen S.K. Zika virus impairs growth in human neurospheres and brain organoids.Science. 2016; 352: 816-818Crossref PubMed Scopus (815) Google Scholar, Wu et al., 2016Wu K.Y. Zuo G.L. Li X.F. Ye Q. Deng Y.Q. Huang X.Y. Cao W.C. Qin C.F. Luo Z.G. Vertical transmission of Zika virus targeting the radial glial cells affects cortex development of offspring mice.Cell Res. 2016; 26: 645-654Crossref PubMed Scopus (220) Google Scholar), we reasoned that ZIKV proteins might inadvertently impact the function of one or more proteins involved in neuronal cell division. Here, we show that the active NS2B3 ZIKV protease is capable of mediating cytotoxic effects of ZIKV, including delayed cytokinesis and failed mitotic abscission. We traced this to off-target direct cleavage of the host Septin 2 protein at residue 306, which leads to alterations of Septin cytoskeletal components. Introduction of a non-cleavable Septin 2 partially restores cytokinesis in cells expressing NS2B3. We thus isolate a ZIKV toxic component mediating cytokinesis defects. These results suggest a potential target for drugs to reduce the burden of disease. To test for potential toxic effects of the ZIKV protease, we utilized just the N-terminal NS3 protease domain fused with the NS2B protein, termed NS2B3, derived from the American strain, which fully reconstitutes protease activity (Lei et al., 2016Lei J. Hansen G. Nitsche C. Klein C.D. Zhang L. Hilgenfeld R. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.Science. 2016; 353: 503-505Crossref PubMed Scopus (244) Google Scholar). We generated stable human 293T cell lines expressing NS2B3 and examined cell proliferation rates. We found that cells expressing NS2B3 exhibited a 33% reduction in EdU labeling as well as a 36% reduction in phospho-histone H3 (pH3) immunoreactivity (Figures 1A–1D), suggesting reduced cell division. Correlated with this was evidence of profound cell death, evidenced by extensive cleaved caspase-3 positivity (Figures 1E and 1F). There was a dramatic 580% increase in the percent of cells with multipolar spindles and supernumerary centrosomes following NS2B3 expression (Figures 1G and 1H), similar to effects observed in ZIKV-infected cells (Figures S1A–S1C; Onorati et al., 2016Onorati M. Li Z. Liu F. Sousa A.M.M. Nakagawa N. Li M. Dell’Anno M.T. Gulden F.O. Pochareddy S. Tebbenkamp A.T.N. et al.Zika virus disrupts phospho-TBK1 localization and mitosis in human neuroepithelial stem cells and radial glia.Cell Rep. 2016; 16: 2576-2592Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, Souza et al., 2016Souza B.S. Sampaio G.L. Pereira C.S. Campos G.S. Sardi S.I. Freitas L.A. Figueira C.P. Paredes B.D. Nonaka C.K. Azevedo C.M. et al.Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells.Sci. Rep. 2016; 6: 39775Crossref PubMed Scopus (136) Google Scholar, Wolf et al., 2017Wolf B. Diop F. Ferraris P. Wichit S. Busso C. Missé D. Gönczy P. Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning.Open Biol. 2017; 7: 160231Crossref PubMed Scopus (26) Google Scholar). None of these cellular phenotypes were observed in cells expressing proteolytically inactive NS2B3, containing a S135A (SA) mutation at the catalytic site (Zhou et al., 2006Zhou H. Singh N.J. Kim K.S. Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor.Proteins. 2006; 65: 692-701Crossref PubMed Scopus (20) Google Scholar). Taken together, these results suggest that NS2B3 is sufficient to alter cell proliferation and survival, in a fashion similar to ZIKV infection, and may act to cleave one or more host proteins. To identify putative ZIKV protease targets, we performed co-affinity purification using recombinant glutathione S-transferase (GST)-tagged NS2B3 (Figure 2A) incubated with hNPC lysates, followed by mass spectrometry analysis. We utilized the S135A mutant, reasoning that this might prolong interactions with binding partners due to its inability to cleave substrates. The co-affinity purification identified a series of proteins interacting with NS2B3 (Figure 2B; Table S1; Student’s t test; p < 0.05), which we termed the ZIKV proteaseome (ZPO). Strikingly, these proteins clustered into seven specific interaction modules (Figure 2C): cytoskeleton; protein folding; proteasome; RNA binding and processing; chromosome maintenance; metabolism; and ribosome, suggesting one or more proteins within each module might be a protease target. The cytoskeleton module within the ZPO had three hits in subunits of the septin cytoskeleton, Septin-2 (SEPT2), Septin-7 (SEPT7), and Septin-9 (SEPT9). We confirmed the interaction between ZIKV NS2B3 and SEPT2/SEPT7 by western blot (Figure S1A). We found that native ZIKV NS3 interacts with SEPT2 directly, and the interaction was enhanced by the presence of NS2B (Figure S1B), excluding the possibility of an artificial interaction mediated by nonnative NS2B3 fusion protein. Septins form a key component of the contractile ring and midbody (Estey et al., 2010Estey M.P. Di Ciano-Oliveira C. Froese C.D. Bejide M.T. Trimble W.S. Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission.J. Cell Biol. 2010; 191: 741-749Crossref PubMed Scopus (163) Google Scholar), which we confirmed in cultured cells (Figure S2C), and function with KIF14 and CIT in cytokinesis (El Amine et al., 2013El Amine N. Kechad A. Jananji S. Hickson G.R. Opposing actions of septins and Sticky on Anillin promote the transition from contractile to midbody ring.J. Cell Biol. 2013; 203: 487-504Crossref PubMed Scopus (55) Google Scholar, Kremer et al., 2005Kremer B.E. Haystead T. Macara I.G. Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4.Mol. Biol. Cell. 2005; 16: 4648-4659Crossref PubMed Scopus (158) Google Scholar), encoded by two genes recently implicated in congenital microcephaly. The septin ring is critical for telophase abscission, during which the two daughter cells separate. This made septins attractive candidates in the pathogenesis of ZIKV-induced microcephaly. We next tested whether SEPT2 or SEPT7 was a substrate of the protease by transient transfection of ZIKV NS2B3 in 293T cells. Cells transfected with NS2B3 wild-type (WT), but not S135A, showed a down-shifted band of SEPT2 when probed with an anti-SEPT2 antibody (Figure 3A), suggesting cleavage by NS2B3. We compared SEPT2 levels between cells with and without NS2B3 expression in the same field of view by immunocytochemistry using an antibody against C-terminal SEPT2 and found NS2B3 WT, but not S135A, led to reduction of SEPT2 immunoreactivity (Figures S3A and S3B), suggesting loss of C-terminal SEPT2 from NS2B3-mediated cleavage. Because transient transfection would leave some cells untransfected, which could blunt the observed effects, we next studied SEPT2 levels in 293T cells stably expressing NS2B3. We observed a reduction in endogenous SEPT2 level, as well as presence of the cleaved SEPT2, which was notably more intense than following transient transfection (Figure S3C). SEPT7 did not show a cleavage product but exhibited a reduced level (Figure 3B), consistent with previous observations that stoichiometric levels are required for stability of the reciprocal septins (Kinoshita et al., 2002Kinoshita M. Field C.M. Coughlin M.L. Straight A.F. Mitchison T.J. Self- and actin-templated assembly of Mammalian septins.Dev. Cell. 2002; 3: 791-802Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). SEPT9 level remained unchanged upon NS2B3 expression (Figure S3D). Next, we confirmed the presence of the down-shifted SEPT2 band and reduced SEPT7 in hNPCs infected with ZIKV comparing to cells with mock infection (Figures 3C and 3D), suggesting that the natural heterodimer produced by ZIKV infection is also capable of cleaving SEPT2. To exclude the possibility that SEPT2 was cleaved indirectly by a downstream protease, we repeated the experiment in vitro with recombinant proteins. ZIKV NS2B3 WT showed a dosage-dependent cleavage of human SEPT2, with molecular weight reduced by ∼9 kDa from the C terminus, using anti-N- and C-terminal SEPT2 antibodies (Figure 3E). The sizes were consistent with the cleavage products observed following cellular transfection and suggest a single cleavage within the C-terminal domain of SEPT2 near the guanine nucleotide binding (G) domain, likely inactivating its function (de Almeida Marques et al., 2012de Almeida Marques I. Valadares N.F. Garcia W. Damalio J.C. Macedo J.N. de Araújo A.P. Botello C.A. Andreu J.M. Garratt R.C. Septin C-terminal domain interactions: implications for filament stability and assembly.Cell Biochem. Biophys. 2012; 62: 317-328Crossref PubMed Scopus (28) Google Scholar, Meseroll et al., 2013Meseroll R.A. Occhipinti P. Gladfelter A.S. Septin phosphorylation and coiled-coil domains function in cell and septin ring morphology in the filamentous fungus Ashbya gossypii.Eukaryot. Cell. 2013; 12: 182-193Crossref PubMed Scopus (27) Google Scholar, Sirajuddin et al., 2007Sirajuddin M. Farkasovsky M. Hauer F. Kühlmann D. Macara I.G. Weyand M. Stark H. Wittinghofer A. Structural insight into filament formation by mammalian septins.Nature. 2007; 449: 311-315Crossref PubMed Scopus (334) Google Scholar). Typical flavivirus proteases prefer substrates with basic residues (Arg or Lys) at the P1 and P2 sites and a short side chain (Gly, Ser, or Ala) at the P1′ site (Chambers et al., 1990Chambers T.J. Hahn C.S. Galler R. Rice C.M. Flavivirus genome organization, expression, and replication.Annu. Rev. Microbiol. 1990; 44: 649-688Crossref PubMed Scopus (1589) Google Scholar, Yamshchikov and Compans, 1995Yamshchikov V.F. Compans R.W. Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.J. Virol. 1995; 69: 1995-2003PubMed Google Scholar). The C-terminal region of human SEPT2 revealed three such potential cleavage sites: R306; K310; and R341 (Figure 3F). To identify candidate cleavage sites, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) on gel-extracted SEPT2 C-terminal cleavage product, together with a control using full-length SEPT2. We used ASP-N and trypsin digestion combined with propionyl labeling of primary amines to distinguish between ZIKV protease and ASP-N and trypsin protease cleavages (Yamshchikov and Compans, 1995Yamshchikov V.F. Compans R.W. Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.J. Virol. 1995; 69: 1995-2003PubMed Google Scholar; Figures 3G and S3E). The recovered peptide fragments from all three digest patterns suggested that C-terminal ZIKV protease cleavage occurred before residue 330, ruling out potential cleavage site R341. We also identified peptide K310pVENEDMNK (Figure S3F; where p represents propionate) from propionyl-trypsin digestion, which argued against K310 cleavage. We identified the N-terminal labeled peptide G307GRKVENE314 (Figure 3H) from propionyl-ASP-N digestion, which led to cleavage after glutamic acid (E), suggesting cleavage by NS2B3 at R306. We confirmed these results by introducing an alanine substitution at R306 of SEPT2 (SEPT2R306A), which was impervious to ZIKV NS2B3 in our in vitro protease assay (Figure 3I). To examine the impact of R306 cleavage on SEPT2 function, we transfected constructs expressing EGFP-fused full-length SEPT2 or with a C-terminal truncation at residue 306 (ΔC). EGFP-SEPT2 concentrated at the cleavage furrow during telophase as expected; however, EGFP-SEPT2ΔC was mislocalized throughout the cell cortex (Figure S3G). In addition, SEPT2 in cells engineered to stably express NS2B3 failed to localize at the midbody during telophase, consistent with mislocalization as a result of NS2B3 cleavage (Figure S3H). We also found failed localization of SEPT2 and SEPT7 at the midbody in ZIKV-infected hNPCs (Figures S3I and S3J), suggesting disrupted septin complex upon ZIKV infection. Although septins are required for cytokinesis, their roles during neurogenesis are not known. We thus tested the roles of SEPT2 and SEPT7 during neurogenesis in cultured hNPCs (Figure S4A). We found that knockdown of SEPT2 or SEPT7 led to a 33% or 27% reduction in EdU-marked cells or 38% or 47% reduction in pH3-marked cells, respectively (Figures 4A, 4B, S4B, and S4C). The changes following SEPT2 or SEPT7 knockdown in hNPCs correlated with reduced survival and increased activated caspase-3 (Cas3), compared with control cells (Figure 4C), similar to what we found upon NS2B3 forced expression or published results following ZIKV infection. Considering the well-established role of septins in cytokinesis, we performed time-lapse recordings and observed ∼6% of cells with SEPT2 knockdown completely failed abscission, and the remainder managed to complete cytokinesis but took 200% longer time than control. Knockdown of SEPT7 led to less severe cytokinesis delay, with a 123% increase in length of cytokinesis as comparing to control (Figures 4D and 4E). Similarly, a 160% increase in cytokinesis length was observed in 293T cells stably expressing NS2B3, but not S135A NS2B3 (Figures 4F and 4G). We also observed a lengthening of cytokinesis by 248% in hNPCs with NS2B3 expression (Figures S4D and S4E) and by 200% by ZIKV infection (Figures S4F–S4H). Given that SEPT2 R306A was impervious to ZIKV NS2B3 cleavage, we attempted to rescue the NS2B3-induced phenotype by co-expression of SEPT2-R306A. First, we confirmed that SEPT2 R306A produced a stable and properly localized protein in cells (Figure S4I). We found that cells expressing both ZIKV NS2B3 and SEPT2-R306A showed a partial rescue of cytokinesis delay (Figures 3H and 3I) and formation of multipolar spindle cells (Figures S4J and S4K) compared with ZIKV NS2B3 alone. In addition, co-expression of SEPT2-R306A restored SEPT7 level at the midbody during cytokinesis in cells with NS2B3 expression (Figure S4L). The results suggest cleavage of SEPT2 by the NS2B3 protease results in failed cytokinesis and delayed cel" @default.
• W2913398081 created "2019-02-21" @default.
• W2913398081 creator A5003411069 @default.
• W2913398081 creator A5006161477 @default.
• W2913398081 creator A5014458109 @default.
• W2913398081 creator A5016278872 @default.
• W2913398081 creator A5021223600 @default.
• W2913398081 creator A5023285318 @default.
• W2913398081 creator A5024015568 @default.
• W2913398081 creator A5026244741 @default.
• W2913398081 creator A5026476503 @default.
• W2913398081 creator A5040650986 @default.
• W2913398081 creator A5047788407 @default.
• W2913398081 creator A5048795213 @default.
• W2913398081 creator A5051387088 @default.
• W2913398081 creator A5058982955 @default.
• W2913398081 creator A5061750530 @default.
• W2913398081 creator A5069479626 @default.
• W2913398081 creator A5076232765 @default.
• W2913398081 creator A5084997936 @default.
• W2913398081 creator A5085936954 @default.
• W2913398081 creator A5088333603 @default.
• W2913398081 date "2019-03-01" @default.
• W2913398081 modified "2023-10-15" @default.
• W2913398081 title "Zika Virus Protease Cleavage of Host Protein Septin-2 Mediates Mitotic Defects in Neural Progenitors" @default.
• W2913398081 cites W1545235721 @default.
• W2913398081 cites W1613745801 @default.
• W2913398081 cites W1893142060 @default.
• W2913398081 cites W1966203663 @default.
• W2913398081 cites W1969651353 @default.
• W2913398081 cites W1971368161 @default.
• W2913398081 cites W1986656413 @default.
• W2913398081 cites W1988476536 @default.
• W2913398081 cites W1991660576 @default.
• W2913398081 cites W2003949450 @default.
• W2913398081 cites W2012156061 @default.
• W2913398081 cites W2014318827 @default.
• W2913398081 cites W2038554008 @default.
• W2913398081 cites W2042141743 @default.
• W2913398081 cites W2055376777 @default.
• W2913398081 cites W2060501484 @default.
• W2913398081 cites W2065758508 @default.
• W2913398081 cites W2070520948 @default.
• W2913398081 cites W2078116784 @default.
• W2913398081 cites W2087678443 @default.
• W2913398081 cites W2088594374 @default.
• W2913398081 cites W2093325895 @default.
• W2913398081 cites W2104218385 @default.
• W2913398081 cites W2105346492 @default.
• W2913398081 cites W2124492056 @default.
• W2913398081 cites W2130802358 @default.
• W2913398081 cites W2136627895 @default.
• W2913398081 cites W2147577072 @default.
• W2913398081 cites W2152542121 @default.
• W2913398081 cites W2163000142 @default.
• W2913398081 cites W2163320603 @default.
• W2913398081 cites W2163412375 @default.
• W2913398081 cites W2255153102 @default.
• W2913398081 cites W2260746516 @default.
• W2913398081 cites W2292422784 @default.
• W2913398081 cites W2311770845 @default.
• W2913398081 cites W2319900810 @default.
• W2913398081 cites W2339793246 @default.
• W2913398081 cites W2346484133 @default.
• W2913398081 cites W2356757232 @default.
• W2913398081 cites W2360131028 @default.
• W2913398081 cites W2372368841 @default.
• W2913398081 cites W2384699986 @default.
• W2913398081 cites W2474204065 @default.
• W2913398081 cites W2482354067 @default.
• W2913398081 cites W2505033294 @default.
• W2913398081 cites W2507412157 @default.
• W2913398081 cites W2509505498 @default.
• W2913398081 cites W2513559175 @default.
• W2913398081 cites W2530933205 @default.
• W2913398081 cites W2540460228 @default.
• W2913398081 cites W2558398235 @default.
• W2913398081 cites W2560106869 @default.
• W2913398081 cites W2566335769 @default.
• W2913398081 cites W2571151926 @default.
• W2913398081 cites W2572109366 @default.
• W2913398081 cites W2573203202 @default.
• W2913398081 cites W2588238986 @default.
• W2913398081 cites W2588268036 @default.
• W2913398081 cites W2602095193 @default.
• W2913398081 cites W2609127248 @default.
• W2913398081 cites W2621240879 @default.
• W2913398081 cites W2748279491 @default.
• W2913398081 cites W2754499459 @default.
• W2913398081 cites W2760364797 @default.
• W2913398081 cites W2809050289 @default.
• W2913398081 cites W4252976893 @default.
• W2913398081 doi "https://doi.org/10.1016/j.neuron.2019.01.010" @default.
• W2913398081 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6690588" @default.
• W2913398081 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30713029" @default.
• W2913398081 hasPublicationYear "2019" @default.
• W2913398081 type Work @default.
• W2913398081 sameAs 2913398081 @default.

• next