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RETENTION OF A RECOMBINANT GFP PROTEIN EXPRESSED BY THE YELLOW FEVER 17D VIRUS IN THE E/NS1 INTERGENIC REGION IN THE ENDOPLASMIC RETICULUM
GFP expression
ER retention
deletion and insertion mutants
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.
Abstract
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
recombinant yellow fever 17D virusGFP expression
ER retention
deletion and insertion mutants
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