{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T04:36:54Z","timestamp":1781843814389,"version":"3.54.5"},"reference-count":47,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2021,8,30]],"date-time":"2021-08-30T00:00:00Z","timestamp":1630281600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002428","name":"Austrian Science Fund","doi-asserted-by":"publisher","award":["I1378-B13"],"award-info":[{"award-number":["I1378-B13"]}],"id":[{"id":"10.13039\/501100002428","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002428","name":"Austrian Science Fund","doi-asserted-by":"publisher","award":["P27501-B21"],"award-info":[{"award-number":["P27501-B21"]}],"id":[{"id":"10.13039\/501100002428","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called \u201cstem\u201d region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic \u03b1-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.<\/jats:p>","DOI":"10.3390\/v13091727","type":"journal-article","created":{"date-parts":[[2021,9,1]],"date-time":"2021-09-01T03:13:17Z","timestamp":1630465997000},"page":"1727","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles"],"prefix":"10.3390","volume":"13","author":[{"given":"Iris","family":"Medits","sequence":"first","affiliation":[{"name":"Center for Virology, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franz X.","family":"Heinz","sequence":"additional","affiliation":[{"name":"Center for Virology, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1902-8674","authenticated-orcid":false,"given":"Karin","family":"Stiasny","sequence":"additional","affiliation":[{"name":"Center for Virology, Medical University of Vienna, 1090 Vienna, Austria"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1038\/s41564-020-0714-0","article-title":"The continued threat of emerging flaviviruses","volume":"5","author":"Pierson","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"206","DOI":"10.15252\/embr.201745302","article-title":"The bright and the dark side of human antibody responses to flaviviruses: Lessons for vaccine design","volume":"19","author":"Rey","year":"2018","journal-title":"EMBO Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1038\/nsmb.2463","article-title":"Cryo-EM structure of the mature dengue virus at 3.5-A resolution","volume":"20","author":"Zhang","year":"2013","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"7700","DOI":"10.1128\/JVI.00197-13","article-title":"Immature and mature dengue serotype 1 virus structures provide insight into the maturation process","volume":"87","author":"Kostyuchenko","year":"2013","journal-title":"J. Virol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1038\/nature17994","article-title":"Structure of the thermally stable Zika virus","volume":"533","author":"Kostyuchenko","year":"2016","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1126\/science.aaf5316","article-title":"The 3.8 A resolution cryo-EM structure of Zika virus","volume":"352","author":"Sirohi","year":"2016","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1169","DOI":"10.1016\/j.str.2018.05.006","article-title":"Refinement and analysis of the mature Zika virus cryo-EM structure at 3.1 \u00c5 resolution","volume":"26","author":"Sevvana","year":"2018","journal-title":"Structure"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Wang, X., Li, S.H., Zhu, L., Nian, Q.G., Yuan, S., Gao, Q., Hu, Z., Ye, Q., Li, X.F., and Xie, D.Y. (2017). Near-atomic structure of Japanese encephalitis virus reveals critical determinants of virulence and stability. Nat. Commun., 8.","DOI":"10.1038\/s41467-017-00024-6"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Fuzik, T., Formanova, P., Ruzek, D., Yoshii, K., Niedrig, M., and Plevka, P. (2018). Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. Nat. Commun., 9.","DOI":"10.1038\/s41467-018-02882-0"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"DiNunno, N.M., Goetschius, D.J., Narayanan, A., Majowicz, S.A., Moustafa, I., Bator, C.M., Hafenstein, S.L., and Jose, J. (2020). Identification of a pocket factor that is critical to Zika virus assembly. Nat. Commun., 11.","DOI":"10.1038\/s41467-020-18747-4"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Renner, M., Dejnirattisai, W., Carrique, L., Martin, I.S., Karia, D., Ilca, S.L., Ho, S.F., Kotecha, A., Keown, J.R., and Mongkolsapaya, J. (2021). Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins. Nat. Commun., 12.","DOI":"10.1038\/s41467-021-21505-9"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Hardy, J.M., Newton, N.D., Modhiran, N., Scott, C.A.P., Venugopal, H., Vet, L.J., Young, P.R., Hall, R.A., Hobson-Peters, J., and Coulibaly, F. (2021). A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses. Nat. Commun., 12.","DOI":"10.1038\/s41467-021-22773-1"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e50069","DOI":"10.15252\/embr.202050069","article-title":"Extensive flavivirus E trimer breathing accompanies stem zippering of the post-fusion hairpin","volume":"21","author":"Medits","year":"2020","journal-title":"EMBO Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.virol.2015.03.043","article-title":"Viral membrane fusion","volume":"479\u2013480","author":"Harrison","year":"2015","journal-title":"Virology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.coviro.2017.06.009","article-title":"Flavivirus structural heterogeneity: Implications for cell entry","volume":"24","author":"Rey","year":"2017","journal-title":"Curr. Opin. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/bs.aivir.2020.08.003","article-title":"Structure-guided paradigm shifts in flavivirus assembly and maturation mechanisms","volume":"108","author":"Nicholls","year":"2020","journal-title":"Adv. Virus Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1834","DOI":"10.1126\/science.1153264","article-title":"Structure of the immature dengue virus at low pH primes proteolytic maturation","volume":"319","author":"Yu","year":"2008","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Blitvich, B.J., and Firth, A.E. (2017). A Review of Flaviviruses that Have No Known Arthropod Vector. Viruses, 9.","DOI":"10.3390\/v9060154"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1099\/0022-1317-78-5-1049","article-title":"Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr","volume":"78 Pt 5","author":"Mandl","year":"1997","journal-title":"J. Gen. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"W209","DOI":"10.1093\/nar\/gks396","article-title":"RF-Cloning.org: An online tool for the design of restriction-free cloning projects","volume":"40","author":"Bond","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/0022-2836(75)90213-2","article-title":"A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase","volume":"94","author":"Sanger","year":"1975","journal-title":"J. Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1007\/BF01703077","article-title":"Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR","volume":"8","author":"Allison","year":"1994","journal-title":"Virus Genes"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4549","DOI":"10.1128\/jvi.70.7.4549-4557.1996","article-title":"Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions","volume":"70","author":"Schalich","year":"1996","journal-title":"J. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"8495","DOI":"10.1128\/JVI.05129-11","article-title":"Mutational analysis of the zippering reaction during flavivirus membrane fusion","volume":"85","author":"Pangerl","year":"2011","journal-title":"J. Virol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"6365","DOI":"10.1128\/JVI.00447-16","article-title":"Membrane anchors of the structural flavivirus proteins and their role in virus assembly","volume":"90","author":"Blazevic","year":"2016","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4377","DOI":"10.1128\/JVI.02458-10","article-title":"The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion","volume":"85","author":"Fritz","year":"2011","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4099","DOI":"10.1128\/JVI.80.8.4099-4113.2006","article-title":"Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses","volume":"80","author":"Kofler","year":"2006","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/0168-1702(96)01325-1","article-title":"Characterization of Langat virus antigenic determinants defined by monoclonal antibodies to E, NS1 and preM and identification of a protective, non-neutralizing preM-specific monoclonal antibody","volume":"43","author":"Smith","year":"1996","journal-title":"Virus Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"12619","DOI":"10.1128\/JVI.01017-07","article-title":"Selection and analysis of mutations in an encephalomyocarditis virus internal ribosome entry site that improve the efficiency of a bicistronic flavivirus construct","volume":"81","author":"Orlinger","year":"2007","journal-title":"J. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/0092-1157(86)90032-6","article-title":"A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus","volume":"14","author":"Heinz","year":"1986","journal-title":"J. Biol. Stand."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1006\/viro.1994.1013","article-title":"Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM","volume":"198","author":"Heinz","year":"1994","journal-title":"Virology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1099\/0022-1317-57-2-263","article-title":"Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: Comparison with other flaviviruses","volume":"57 Pt 2","author":"Heinz","year":"1981","journal-title":"J. Gen. Virol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1128\/JVI.01063-09","article-title":"A trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination","volume":"84","author":"Taucher","year":"2010","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Haslwanter, D., Blaas, D., Heinz, F.X., and Stiasny, K. (2017). A novel mechanism of antibody-mediated enhancement of flavivirus infection. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006643"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/0042-6822(89)90044-5","article-title":"Epitope model of tick-borne encephalitis virus envelope glycoprotein E: Analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic pH","volume":"169","author":"Guirakhoo","year":"1989","journal-title":"Virology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1128\/jvi.63.2.564-571.1989","article-title":"Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model","volume":"63","author":"Mandl","year":"1989","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"9557","DOI":"10.1128\/JVI.00080-06","article-title":"Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites","volume":"80","author":"Stiasny","year":"2006","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"11526","DOI":"10.1128\/JVI.01041-07","article-title":"Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies","volume":"81","author":"Stiasny","year":"2007","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5605","DOI":"10.1128\/JVI.73.7.5605-5612.1999","article-title":"Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E","volume":"73","author":"Allison","year":"1999","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4268","DOI":"10.1128\/JVI.75.9.4268-4275.2001","article-title":"Mutational evidence for an internal fusion peptide in flavivirus envelope protein E","volume":"75","author":"Allison","year":"2001","journal-title":"J. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1083\/jcb.200806081","article-title":"Identification of specific histidines as pH sensors in flavivirus membrane fusion","volume":"183","author":"Fritz","year":"2008","journal-title":"J. Cell Biol."},{"key":"ref_43","unstructured":"Knipe, D.M., Howley, P.M., Cohen, J.I., Griffin, D.E., Lamb, R.A., Martin, M.A., Racaniello, V.R., and Roizman, B. (2013). Flaviviridae. Fields Virology, Lippincott Williams & Wilkins. [6th ed.]."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Newton, N.D., Hardy, J.M., Modhiran, N., Hugo, L.E., Amarilla, A.A., Bibby, S., Venugopal, H., Harrison, J.J., Traves, R.J., and Hall, R.A. (2021). The structure of an infectious immature flavivirus redefines viral architecture and maturation. Sci. Adv., 7.","DOI":"10.1126\/sciadv.abe4507"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Chen, L., Wang, M., Zhu, D., Sun, Z., Ma, J., Wang, J., Kong, L., Wang, S., Liu, Z., and Wei, L. (2018). Implication for alphavirus host-cell entry and assembly indicated by a 3.5\u00c5 resolution cryo-EM structure. Nat. Commun., 9.","DOI":"10.1038\/s41467-018-07704-x"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11362","DOI":"10.1128\/JVI.01679-06","article-title":"Functions of the stem region of the Semliki Forest virus fusion protein during virus fusion and assembly","volume":"80","author":"Liao","year":"2006","journal-title":"J. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"5159","DOI":"10.1128\/JVI.02099-10","article-title":"The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry","volume":"85","author":"Lin","year":"2011","journal-title":"J. Virol."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/9\/1727\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:52:53Z","timestamp":1760165573000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/9\/1727"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,30]]},"references-count":47,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["v13091727"],"URL":"https:\/\/doi.org\/10.3390\/v13091727","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,30]]}}}