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.
Highlights
Yellow fever (YF) virus is the prototype member of the genus Flavivirus of the Flaviviridae family
Construction of recombinant viruses - The insertion of foreign proteins in the intergenic E/NS1 region must not interfere with the correct topology of the yellow fever (YF) viral proteins and precursor polyprotein processing in the endoplasmic reticulum (ER) membrane
The recombinant protein expressed by the YF17D/green fluorescent protein (GFP)/SA carrying the two TM segments at its carboxy terminus is associated with the luminal side of the ER membrane (Bonaldo et al 2007)
Summary
Yellow fever (YF) virus is the prototype member of the genus Flavivirus of the Flaviviridae family. The prM/E association appears essential for preventing protein E from suffering low-pH rearrangements during transport through the acidic compartments of the trans-Golgi network In this compartment, shortly before the virus is released from the cell, the pr portion is cleaved from prM by the cellular protease furin and the E protein dimer reaches its final native conformation, which leads to the formation of mature virions with E and M molecules on their envelope surface The same organelle-specific properties were determined in previous studies comparing the TM domains of early and late compartment proteins and indicated a difference in TM length and bilayer thickness (Bretscher & Munro 1993, Levine et al 2000, Sharpe et al 2010) These findings support a TM-dependent sorting mechanism for viral envelope proteins.
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