Memorias do Instituto Oswaldo Cruz (Mar 2012)

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

  • Gisela Freitas Trindade,
  • Marlon Gilsepp Veloso de Santana,
  • Juliana Ribeiro dos Santos,
  • Ricardo Galler,
  • Myrna Cristina Bonaldo

DOI
https://doi.org/10.1590/S0074-02762012000200017
Journal volume & issue
Vol. 107, no. 2
pp. 262 – 272

Abstract

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The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

Keywords