Fluorescent tagging the NS1 protein in yellow fever virus: Replication-capable viruses which produce the secretory GFP-NS1 fusion protein

• Published in: Virus research Vol. 294; p. 198291
• Main Authors: Syzdykova, Laura R., Binke, Stephan, Keyer, Viktoriya V., Shevtsov, Alexandr B., Zaripov, Mikhail M., Zhylkibayev, Assylbek A., Ramanculov, Erlan M., Shustov, Alexandr V.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-03-2021
• Subjects: Amino Acid Sequence; Flavivirus; Flavivirus - genetics; Fluorescent reporter GFP; Genetic tagging; Protein-protein interactions; Replicative complex; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism; Virus Replication; Yellow fever virus; Yellow fever virus - genetics; Yellow fever virus - metabolism; Genetic tagging; ORF; Fluorescent reporter GFP; NS; nt; FMDV 2A; MTT; Flavivirus; nPAGE; PDB; UTR; Replicative complex; FFU; a.a; RdRp; wt; dsRNA; eGFP; MOI; HGH; YFV; CM; p.i; CMV; ER; Protein-protein interactions; ECL; Mab; SEC; PA; RC; TSS; p.t; CPE; HDV-Rz; SEM; VP; Yellow fever virus
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2020.198291

•Green fluorescent protein was fused to the protein NS1 in yellow fever virus.•Viruses with GFP-tagged NS1 accumulate mutations restoring the replication.•Missense mutations in GFP, NS4A and NS5 rescue the replicative capacity.•GFP-NS1 colocalizes with double-stranded RNA.•GFP-NS1 is produced as a secretory protein resembling sNS1.
Yellow fever virus, the prototype in the genus Flavivirus, was used to develop viruses in which the nonstructural protein NS1 is genetically fused to GFP in the context of viruses capable of autonomous replication. The GFP-tagging of NS1 at the amino-terminus appeared possible despite the presence of a small and functionally important domain at the NS1′s amino-terminus which can be distorted by such fusing.
GFP-tagged NS1 viruses were rescued from DNA-launched molecular clones. The initially produced GFP-tagged NS1 virus was capable of only poor replication. Sequential passages of the virus in cell cultures resulted in the appearance of mutations in GFP, NS4A, NS4B and NS5. The mutations which change amino acid sequences of GFP, NS4A and NS5 have the adaptive effect on the replication of GFP-tagged NS1 viruses. The pattern of GFP-fluorescence indicates that the GFP-NS1 fusion protein is produced into the endoplasmic reticulum. The intracellular GFP-NS1 fusion protein colocalizes with dsRNA. The discovered forms of extracellular GFP-NS1 possibly include tetramers and hexamers.