Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Poruchynsky, Marianne S.; Tyndall, Chiara; Both, Gerald W.; Sato, Fumihiko; Bellamy, A R; Atkinson, Paul H.

doi:10.1083/jcb.101.6.2199

Cited by 104 publications

(93 citation statements)

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Mirazimi

Magnusson

Svensson

2003

Journal of General Virology

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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...

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Section: Introductionmentioning

confidence: 99%

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

Mirazimi

Magnusson

Svensson

2003

Journal of General Virology

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“…Although the nature of the structural changes caused by the deletion remains unclear, the removal of the hydrophobic N terminus of HA2 (fusion peptide) may have increased the solubility of HAl and thereby allowed the protein to be transported to the cell surface for eventual secretion into the medium. In the case of rotavirus VP7 protein, which remains localized in the RER, the deletion of a hydrophobic sequence makes the protein move from the RER to the cell surface and be secreted in the medium (36). Whether HA325 will exhibit a similar transport behavior (i.e., extracellular secretion) in mammalian cells remains to be determined.…”

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Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.

Jabbar¹,

Nayak²

1987

Mol. Cell. Biol.

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We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae. The cDNA sequences encoding variant forms of influenza HA were expressed in S. cerevisiae. The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated. In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S. cerevisiae. The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery. The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S. cerevisiae compared with the signal-minus, nonglycosylated HA molecules. Although both the anchor-minus HA (HA500) and HAl (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different. During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium. Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans. Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed.

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“…Earlier studies showed that removal of amino acids 47 to 61 from the polypeptide converted VP7 from a glycoprotein which located in the ER to one which was secreted from the cell and modified with complex (endo H-resistant) carbohydrate (18 (17). After transformation into E. coli TG1, colonies containing mutant phage were identified by hybridization to the radiolabeled oligonucleotide, and the presence of the mutation was confirmed by nucleotide sequencing (22).…”

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“…Electron microscopy has shown that rotavirus subviral particles assemble in cytoplasmic inclusions and mature by budding through the membrane of the rough ER, becoming transiently enveloped in the process. The temporary envelope surrounding the particles is later removed, leaving mature viruses within the lumen of the ER (5,8,18). VP7, the major serotype antigen of rotaviruses, is derived from a precursor which is cleaved as it is translocated across the ER membrane (7,12).…”

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confidence: 99%

“…2), giving mutant A42C. This plasmid was then used to prepare the 3.7-kb EcoRI-NcoI fragment so that the C mutation could be combined with other mutations in the VP7 gene by ligation with the 4.4-kb EcoRI-XhoI fragment and a -0.2-kb XhoI-NcoI fragment from the desired deletion mutant (18). In this way, variants A42C, A47SC, A42SC, and A23ZC were also constructed.…”

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Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

Whitfeld¹,

Tyndall²,

Stirzaker³

et al. 1987

Mol. Cell. Biol.

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The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (Hi and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell.For a variant retaining only the Hi domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking Hi and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.

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Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Cited by 104 publications

References 43 publications

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

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

Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae.

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

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