title: Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus.
abstract: Flavivirus NS1 is a nonstructural glycoprotein that is expressed on the cell surface and secreted into the extracellular space. Despite its transit through the secretory pathway, NS1 is an essential gene linked to early viral RNA replication. How this occurs has remained a mystery given the disparate localization of NS1 and the viral RNA replication complex, as the latter is present on the cytosolic face of the endoplasmic reticulum (ER). We recently identified an N-terminal di-amino acid motif in NS1 that modulates protein targeting and affected viral replication. Exchange of two amino acids at positions 10 and 11 from dengue virus (DENV) into West Nile virus (WNV) NS1 (RQ10NK) changed its relative surface expression and secretion and attenuated infectivity. However, the phenotype of WNV containing NS1 RQ10NK was unstable, as within two passages heterogeneous plaque variants were observed. Here, using a mutant WNV encoding the NS1 RQ10NK mutation, we identified a suppressor mutation (F86C) in NS4B, a virally encoded transmembrane protein with loops on both the luminal and cytoplasmic sides of the ER membrane. Introduction of NS4B F86C specifically rescued RNA replication of mutant WNV but did not affect the wild-type virus. Mass spectrometry and coimmunoprecipitation studies established a novel physical interaction between NS1 and NS4B, suggesting a mechanism for how luminal NS1 conveys signals to the cytoplasm to regulate RNA replication.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Soonjeon
authors.initials: S
authors.lastname: Youn
authors.forename: Tuo
authors.initials: T
authors.lastname: Li
authors.forename: Broc T
authors.initials: BT
authors.lastname: McCune
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Edeling
authors.forename: Daved H
authors.initials: DH
authors.lastname: Fremont
authors.forename: Ileana M
authors.initials: IM
authors.lastname: Cristea
authors.forename: Michael S
authors.initials: MS
authors.lastname: Diamond
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D013489:Suppression, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication; D014902:West Nile virus / Q000502:physiology*
chemical_list: D050505:Mutant Proteins; C080107:NS4B protein, flavivirus; D017361:Viral Nonstructural Proteins; C531936:nonstructural protein 1, West Nile virus