A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retention in the endoplasmic reticulum

Abstract

The rotavirus genome encodes two glycoproteins, one structural (VP7) and one non-structural (NSP4), both of which mature and remain in the endoplasmic reticulum (ER). While three amino acids in the N terminus have been proposed to function as a retention signal for VP7, no information is yet available on how NSP4 remains associated with the ER. In this study, we have investigated the ER retention motif of NSP4 by producing various C-terminal truncations. Deleting the C terminus by 52 amino acids did not change the intracellular distribution of NSP4, but an additional deletion of 38 amino acids diminished the ER retention and resulted in the expression of NSP4 on the cell surface. Brefeldin A treatment prevented NSP4 from reaching the cell surface, suggesting that C-terminal truncated plasma membrane NSP4 is transported through the normal secretory pathway. On the basis of these results, we propose that the region between amino acids 85 and 123 in the cytoplasmic region of NSP4 are involved in ER retention. INTRODUCTIONThe secretory pathway of eukaryotic cells provides a route for proteins to be secreted from the cells and to be targeted to various cellular organelles (Jackson et al., 1990;Nilsson & Warren, 1994;Paabo et al., 1987). Following synthesis on the endoplasmic reticulum (ER), certain proteins resist the bulk flow of the secretory pathway and become ER-associated and accumulate in the ER. Three sorting signals associated with ER retention and retrieval (Jackson et al., 1993;Lewis & Pelham, 1992;Pelham, 1994;Townsley & Pelham, 1994) have been proposed: (i) soluble proteins in the ER lumen carry a specific C terminus tetrapeptide retention signal, H/KDEL (Munro & Pelham, 1987); (ii) transmembrane proteins (type II) carry two arginines near the cytoplasmic N terminus (Schutze et al., 1994); and (iii) transmembrane proteins (type I) possess lysines at positions 23 and either 24 or 25 (Jackson et al., 1990). A tyrosine-based motif close to the C terminus has also been identified as an ER retention signal (Mallabiabarrena et al., 1995), but the retention mechanism for this motif is not yet known. In addition to such retrieval mechanisms, retention in the ER may also be static for glycoproteins that do not contain any of the above signal motifs (Cocquerel et al., 1999).Rotavirus undergoes a unique maturation process in the ER. The assembly process, which includes translocation of doublelayered particles across the ER and retention of mature virus in the ER, has provided a system in which post-translational events can be studied (Bellamy & Both, 1990;Estes & Cohen, 1989;Poruchynsky et al., 1985Poruchynsky et al., , 1991. Two rotavirus proteins have been in focus with regard to assembly and pathogenesis, the VP7 outer capsid protein, which is luminal and ERassociated, and NSP4, a non-structural glycoprotein that has been given significant attention in recent years. NSP4 is a novel type of trans-ER resident glycoprotein, which functions not only as a receptor for double-layered particles in the cytoplasm (Au et a...

Comparative efficacy of fluorescent antibody test, immunoperoxidase test and enzyme linked immuno sorbent assay in detection of rotavirus in cell culture

“…These findings are in agreement with observations made by Morera et al [7], Sestak and Musilov [10]. The intensity of the [2,6,10,11]. The usefulness of immunofluorescence is limited by the need for specialized equipment and experienced staff, and also by the difficulty in coping with large numbers of specimens [5].…”

Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca 2+ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

“…In spite of the fact that NSP4 is synthesized in the ER membrane, the activation of the putative channel would take place out of this compartment, since during infection there is an increase in total Ca 2ϩ in the ER. Supporting this view, a truncated form of recombinant NSP4 expressed in Vero cells corresponding to the transmembrane segment of the molecule (1 to 89 aa) was able to escape the ER via a brefeldin A-sensitive pathway and reach the plasma membrane (29). On the other hand, the fulllength, glycosylated NSP4 molecule has recently been de- FIG.…”

Molecular Biology of Rotavirus Entry and Replication

“…The viral protein would traffic from the ER to the plasma membrane, where this channel would become active by unknown mechanisms such as conformational changes, oligomerization, and/or proteolysis. Supporting this view, a truncated form of recombinant NSP4 expressed in Vero cells corresponding to the transmembrane segment of the molecule (aa 1-89) was able to escape the ER via a brefeldin A-sensitive pathway and reach the plasma membrane [169]. On the other hand, the full-length, glycosylated NSP4 molecule has been recently detected in plasma membrane rafts interacting with caveolin, in rotavirusinfected or NSP4-EGFP-expressing cells [170].…”