1 Department of Virology, Swedish Institute for Infectious Disease Control/Karolinska Institute, 171 82 Solna, Sweden
2 Division of Medical Microbiology, Department of Molecular and Clinical Medicine, University of Linköping, Sweden
Correspondence
Lennart Svensson
Lensve{at}mbox.ki.se
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rotavirus undergoes a unique maturation process in the ER. The assembly process, which includes translocation of double-layered 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
; Mirazimi & Svensson, 1998
; Mirazimi et al., 1998
; Poruchynsky et al., 1985
, 1991
). Two rotavirus proteins have been in focus with regard to assembly and pathogenesis, the VP7 outer capsid protein, which is luminal and ER-associated, 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 al., 1989
; Bergmann et al., 1989
), but also acts as a toxin (Ball et al., 1996
; Newton et al., 1997
; Tian et al., 1995
). NSP4 contains two N-linked high-mannose oligosaccharide residues that appear to be critical for the assembly function of NSP4 (Estes & Cohen, 1989
; Petrie, 1983
). The deduced amino acid sequence of NSP4 predicts the presence of three hydrophobic regions, H1, H2 and H3 (Estes & Cohen, 1989
), where the H1 region carries two glycosylation signals. Two models for the topology of NSP4 have suggested. Chan et al. (1988)
suggested a model where the H3 region constitutes the membrane-spanning domain, while H1 and H2 are partly embedded in lipid bilayers on the luminal side of the ER. The second model by Bergmann et al. (1989)
suggested that H2 serves both as a signal sequence and as a membrane-spanning domain, while H3 is embedded in lipid bilayers on the cytoplasmic side of the ER. While three amino acids in the N terminus have been proposed to function as a retention signal for VP7 (Mass & Atkinson, 1994
), no information is available to date on how NSP4 remains associated with the ER. Very recently, it was shown that NSP4 molecules are cleaved and that the cytoplasmic part of this cleavage product (aa 112175) is secreted from the infected cells (Zhang et al., 2000
). In another study, Xu et al. (2000)
showed that NSP4 binds to microtubules and blocks ER-to-Golgi trafficking. Furthermore, we have recently shown that NSP4 prevents development of a permeability barrier and lateral targeting of the tight-junction-associated Zonula Occludens-1 (Tafazoli et al., 2001
). Taken together, these new data show that the cytoplasmic tail of NSP4 possesses novel biological properties. In this study, we provide, for the first time, information about the ER retention of NSP4. Using a Semliki Forest virus (SFV) expression system and deletion technology, we found that a cytoplasmic region in the C terminus between aa 85 and 123 is involved in the ER retention of NSP4 in the ER. This region does not contain any previously recognized ER retention motifs.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Indirect immunofluorescence and confocal imaging.
Subconfluent BHK cells grown on coverslips were infected with recombinant SFV virus at an m.o.i. of 5. At 18 h post-infection (p.i.), cells were fixed overnight with 2 % paraformaldehyde in PBS. Cells were then permeabilized for 15 min at room temperature with 1 % Triton X-100 or kept intact for surface fluorescent staining. Cells were incubated with primary antibodies for 1·5 h in 0·02 % BSA in PBS at 37 °C. After rinsing three times with BSA/PBS buffer, cells were incubated with FITC-conjugated (diluted 1 : 50) or rhodamine-conjugated (diluted 1 : 60) secondary antibodies for 1 h at 37 °C followed by rinsing three times with PBS and inspection by fluorescence microscopy.
Confocal imaging was achieved using a Sarastro 2000 confocal laser scanning microscope (Molecular Dynamics) equipped with an air-cooled argon laser for fluorescence activation. For dual excitation of fluorescein- and rhodamine-tagged secondary antibodies, all the lines (including 488 and 514 nm) of the laser were used. The primary beam splitter was selective at 535 nm, the second at 565 nm. The interference filter in front of the green fluorescein detector was 545DF30 and the barrier filter in front of the red (rhodamine) detector was 570EFLP. Single labelling with either marker was used to optimize protein labelling and photomultiplier voltages, so that negligible bleeding between green and red channels occurred. The chosen settings were used throughout an experiment. A high numerical aperture (1·4) x60 Nikon objective was used.
DNA constructs.
cDNAs encoding the open reading frame of NSP4 (rotavirus strain SA11) were synthesized by RT-PCR. The primers used were 5'-AATGAATCCCCGGGATGGAAAAGCTTACC-3' and 5'-AATGAATTCCCCGGGACGGCAGCTCAACCT-3'. Underlined sequences represent restriction sites for EcoRI and SmaI. The PCR product was cloned into a PCR-script plasmid (Stratagene). A c-Myc tag was inserted after the ATG signal of NSP4 by PCR technology (Fig. 1), followed by cloning into a PCR-script plasmid (Stratagene). After transfection into E. coli (Stratagene), positive clones were identified and purified. To generate a defined set of C-terminal truncated protein fragments, PCR was applied to the full-length C-mycNSP4 gene (Fig. 1
). SpeI sites were designed in both 3' and 5' ends of the designed cDNAs with c-Myc tags (Fig. 1
). After transfection into E. coli, positive clones were purified and plasmids were digested with SpeI. The genes were then ligated into the SpeI site of the SFV expression vector plasmid. The recombinant plasmids were transfected into E. coli and sequenced as described (Nilsson et al., 1998
).
|
Treatment of cells with brefeldin A.
Brefeldin A (BFA) was purchased from Sigma and a stock solution was prepared in 95 % ethanol and stored at -20 °C. To obtain labelled protein in the presence of BFA, 2 µg BFA ml-1 were added 1 h p.i. and maintained throughout the experiments, as described (Mirazimi et al., 1996).
Metabolic labelling.
To produce metabolically labelled cell lysates, BHK cells were infected with various truncated C-mycNSP4 recombinant SFVs at an m.o.i. of 10, as described previously (Mirazimi & Svensson, 1998; Mirazimi et al., 1996
, 1998
). At 8 h p.i., infected cells were starved for 1 h in methionine/cysteine-free medium before being labelled for 1 h with 250 µCi [35S]methionine/cysteine (Tran35S-label; ICN). At the end of the radioactive pulse, cells were incubated with ice-cold PBS containing 40 mM N-ethylmaleimide (NEM) (Sigma) for 2 min to prevent disulfide-bond rearrangement (Mirazimi & Svensson, 1998
; Mirazimi et al., 1996
, 1998
). Cells were then lysed in ice-cold lysis buffer (10 mM Tris/HCl, pH 7·5, 150 mM NaCl, 1 % Triton X-100, 0·5 % SDS, 1 mM PMSF) and the lysate was clarified of cell debris by centrifugation at 13 000 g for 2 min in a microcentrifuge before use.
Radioimmunoprecipitation (RIPA).
Immunoprecipitation was performed essentially as described (Mirazimi & Svensson, 1998; Mirazimi et al., 1996
, 1998
). Briefly, radiolabelled lysates (100 µl) were incubated with 10 µl or 1 µl of the desired antibody and 400 µl RIPA buffer (10 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·6 M KCl, 4 mM EDTA, 1 % Triton X-100) overnight at 4 °C. Fifty µl Staphylococcus aureus protein A or G-Sepharose CL-4B (Pharmacia) was subsequently added to the mixture, which was then incubated for 2 h at 4 °C. The immune complexes were washed three times with RIPA buffer, suspended in reducing sample buffer (10 mM Tris/HCl, pH 6·8, 0·5 % SDS, 10 % glycerol, 2 %
-mercaptoethanol) and boiled for 5 min before separation by SDS-PAGE.
Endo--N-acetylglucosaminidase digestion.
Digestion with endo--N-acetylglucosaminidase (Endo H) (Boehringer Mannheim) was performed essentially as described (Mirazimi et al., 1996
). Briefly, immunoprecipitated proteins were directly treated with 50 µl sodium acetate buffer containing 0·01 % SDS and 5 mU Endo H for 4 h at 37 °C.
SDS-PAGE.
Polypeptide separation was performed by SDS-PAGE with a 4·5 % stacking gel and 10 % separation gel, as previously described (Mirazimi et al., 1998). Electrophoresis was carried out at a constant voltage of 50 V at room temperature, followed by fixation with 10 % glacial acetic acid and 35 % methanol for 1 h at room temperature. Autoradiography was performed as previously described (Svensson et al., 1994
). Molecular mass standards (Amersham) included myosin (200 kDa), phosphorylase b (97 kDa), BSA (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa) and lysozyme (14 kDa).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To analyse the expression capacity for the full-length NSP4, BHK cells were infected with recombinant C-mycNSP4.175 SFV, followed by metabolic labelling for 1 h, cell lysis and immunoprecipitation with a mouse anti-c-Myc antibody and a rabbit anti-NSP4 antibody (Mirazimi et al., 1998).
Fig. 2 shows that both antibodies recognized a 30 kDa protein. To establish whether C-mycNSP4.175 was inserted into the ER and became properly glycosylated similarly to the corresponding native viral protein, immunoprecipitated C-mycNSP4.175 was digested with Endo H, which cleaves high-mannose oligosaccharide units (Mirazimi et al., 1996
). Fig. 2(A)
shows that C-mycNSP4.175 increased in mobility following Endo H treatment, indicating that the oligosaccharide units had been removed and that C-mycNSP4.175 had become glycosylated in the ER similarly to native viral NSP4.
|
To examine the expression efficiency of these mutants, BHK cells were infected (m.o.i of 10) with recombinant SFV. Fig. 2(B) shows that both truncated forms of NSP4 were efficiently expressed in BHK cells concomitant with a significant reduction in host-cell protein synthesis. The proteins also had the expected molecular masses of 21 and 17 kDa, respectively (Fig. 2B
).
To examine whether the deletion of the entire C terminus of NSP4 inhibited ER insertion, cell lysates from Fig. 2(B) were Endo H-treated. The results showed that both mutants were inserted in the ER and glycosylated (Fig. 2C
).
Amino acids 85123 at the C terminus of NSP4 are involved in ER retention
To examine the role of the cytoplasmic tail in ER retention, BHK cells were infected with SFV expressing native and mutated forms of NSP4. Following infection, cells were fixed at 18 h p.i and processed for immunofluorescence. Fig. 3 shows that all mutants of NSP4 were strongly expressed within the cell after permeabilization with Triton X-100. The most interesting observation was, however, that only the shortest mutant, C-mycNSP4.85, gave a positive staining reaction in non-permeabilized cells, suggesting cell-surface localization.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have shown that aa 85123 in the cytoplasmic tail of rotavirus NSP4 are involved in ER retention. These amino acids partly cover the domain (aa 114135) involved in membrane destabilization activity (Tian et al., 1995) and with a capacity to induce diarrhoea in young mice (Ball et al., 1996
). The region also covers part of a domain that has been shown to mediate oligomerization via formation of an
-helical coiled-coil structure (Taylor et al., 1996
) and also binds to the microtubule and blocks ER-to-Golgi trafficking (Xu et al., 2000
).
To study the role of the cytoplasmic tail in the ER retention of NSP4, SFV was used as an expression system (Berglund et al., 1993). As no antibodies against the N-terminal part of NSP4 were available, a c-Myc tag was used as a reporter (Andersson & Pettersson, 1998
). The results showed that all truncated forms of NSP4 were significantly expressed, concomitant with a reduction in host-cell protein synthesis. Proper glycosylation and membrane insertion of mutant NSP4 was monitored by Endo H cleavage. Immunofluorescent staining of non-permeabilized cells infected with full-length or truncated C-mycNSP4 mutants revealed that only C-mycNSP4.85 was expressed on the cell surface. Intracellular detection of all truncated and full-length forms of NSP4 excluded the possibility that all mutant proteins, except C-mycNSP4.85, were poorly expressed or degraded and therefore not detected on the cell surface. Double immunofluorescence studies were used to confirm that cell-surface-expressed NSP4, and not simply intracellular NSP4, was analysed in these studies. As seen in Figs 4 and 5
, we showed that C-mycNSP4.85 was ultimately located on the surface of an intact cell.
Most secretory proteins of viral and cellular origin are transported through the ERGolgi pathway. In this respect, rotavirus is unique in its restricted maturation and retention in the ER. While it has been established that rotavirus matures in the ER, studies have shown that certain rotavirus proteins are transported beyond the ER but still remain Endo H-sensitive. We have previously shown that rotavirus VP7, but not NSP4, is transported from the cell body to axons in neurons by a transport pathway bypassing the Golgi complex (Weclewicz et al., 1993a). Furthermore, Jourdan et al. (1997)
have surprisingly found that rotavirus can be secreted into the extracellular environment by a transport pathway not including the Golgi complex. Recently, it has been shown that a cytoplasmic region of NSP4, representing aa 112175, is secreted from rotavirus-infected cells. The secretion is not affected by treatment of cells with Brefeldin A, but is abolished by nocadazole and cytochalasin D, suggesting that secretion occurs via a Golgi-independent mechanism (Zhang et al., 2000
). While the cytosolic and non-glycosylated region (aa 112175) of NSP4 appears to be secreted by a non-classical mechanism, we report here that the ER-associated and glycosylated mutated form of NSP4 is secreted through the established ERGolgi pathway. Xu et al. (2000)
have previously shown that NSP4 inhibits the vesicle transport from the ER to the Golgi structure, and we have more recently shown that NSP4 prevents development of the permeability barrier and lateral targeting of the tight-junction-associated Zonula Occludens-1 (Tafazoli et al., 2001
), observations that further support a role for NSP4 in the interference of protein transport through the ERGolgi pathway. A possible biological effect of NSP4 could be interference with MHC class I antigen transport to the plasma membrane and thus an immunological impairment.
Our observations led to the question of how the truncated NSP4 reached the plasma membrane. To address this question, we treated the cells with BFA, a specific inhibitor of protein transport from the ER through the Golgi to the plasma membrane (Chen et al., 1991; Kantanen et al., 1995
; Mirazimi et al., 1996
; Nuchtern et al., 1989
). We found that the transport of truncated NSP4 to the plasma membrane was significantly prevented by BFA, suggesting that C-mycNSP4.85 used the established ERGolgi pathway to reach the plasma membrane. We have previously shown that VP7, but not NSP4, becomes Endo H-resistant following treatment with BFA, suggesting that NSP4 does not constitute a substrate for
-mannosidase II (Mirazimi et al., 1996
), which also would confirm our present Endo H results.
An interesting question that arises from our observations concerns the mechanism by which NSP4 is retained in the ER. While there are well-known examples of retrieval signals (Nilsson & Warren, 1994; Nilsson et al., 1989
; Pelham, 1988
), only a few true (static) retention motifs have been identified (Cocquerel et al., 1998
, 1999
). The fact that NSP4 does not contain the established ER retrievalretention signal for type 1 transmembrane proteins two lysines at -3 and -4 or -5 position (Jackson et al., 1993
) or a tyrosine-based signal motif (Mallabiabarrena et al., 1995
) could indicate that NSP4 may contain a motif for a static retention signal or an as yet unknown retrieval signal.
Taylor et al. (1996) have shown that cytoplasmic tail of NSP4 not only binds to microtubules but also inhibits anterograde protein trafficking, as illustrated by the inhibition of transport of VSV-G protein to the plasma membrane (Xu et al., 2000
). We have recently found that NSP4 prevents the development of a permeability barrier, as well as lateral targeting of tight-junction-associated Zonula Occludens-1 (Tafazoli et al., 2001
). Furthermore, we have also shown that rotavirus alters the targeting of the microtubule-associated protein 2 in neurons (Weclewicz et al., 1993b
).
Taken together, these observations favour a hypothesis where structural motifs located in the cytoplasmic tail of NSP4 are involved in the inhibition of intracellular transport rather than preventing transport of a single protein. It should be mentioned, however, that current information does not ultimately prove the existence of a true retention signal, but rather suggests that NSP4 is retained in the ER by an as yet unknown mechanism that also includes a novel property to retain foreign proteins.
In summary, we have shown that the cytoplasmic tail of NSP4 is involved in ER retention and that the mechanisms for this retention do not include previously established structural motifs. Our hypothesis, collating all current information, suggests that the retention of NSP4 in the ER is crucial for rotavirus pathogenesis and pathophysiology.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Au, K. S., Chan, W. K., Burns, J. W. & Estes, M. K. (1989). Receptor activity of rotavirus nonstructural glycoprotein NS28. J Virol 63, 45534562.[Medline]
Ball, J. M., Tian, P., Zeng, C. Q., Morris, A. P. & Estes, M. K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein [see comments]. Science 272, 101104.[Abstract]
Bellamy, A. R. & Both, G. W. (1990). Molecular biology of rotaviruses. Adv Virus Res 38, 143.[Medline]
Berglund, P., Sjoberg, M., Garoff, H., Atkins, G. J., Sheahan, B. J. & Liljestrom, P. (1993). Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Biotechnology 11, 916920.[Medline]
Bergmann, C. C., Maass, D., Poruchynsky, M. S., Atkinson, P. H. & Bellamy, A. R. (1989). Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum. EMBO J 8, 16951703.[Abstract]
Chan, W. K., Au, K. S. & Estes, M. K. (1988). Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. Virology 164, 435442.[Medline]
Chen, S. Y., Matsuoka, Y. & Compans, R. W. (1991). Assembly and polarized release of Punta Toro virus and effects of brefeldin A. J Virol 65, 14271439.[Medline]
Cocquerel, L., Meunier, J. C., Pillez, A., Wychowski, C. & Dubuisson, J. (1998). A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72, 21832191.
Cocquerel, L., Duvet, S., Meunier, J. C., Pillez, A., Cacan, R., Wychowski, C. & Dubuisson, J. (1999). The transmembrane domain of hepatitis C virus glycoprotein E1 is a signal for static retention in the endoplasmic reticulum. J Virol 73, 26412649.
Estes, M. K. & Cohen, J. (1989). Rotavirus gene structure and function. Microbiol Rev 53, 410449.[Medline]
Jackson, M. R., Nilsson, T. & Peterson, P. A. (1990). Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J 9, 31533162.[Abstract]
Jackson, M. R., Nilsson, T. & Peterson, P. A. (1993). Retrieval of transmembrane proteins to the endoplasmic reticulum. J Cell Biol 121, 317333.[Abstract]
Jourdan, N., Maurice, M., Delautier, D., Quero, A. M., Servin, A. L. & Trugnan, G. (1997). Rotavirus is released from the apical surface of cultured human intestinal cells through nonconventional vesicular transport that bypasses the Golgi apparatus. J Virol 71, 82688278.[Abstract]
Kantanen, M. L., Leinikki, P. & Kuismanen, E. (1995). Endoproteolytic cleavage of HIV-1 gp160 envelope precursor occurs after exit from the trans-Golgi network (TGN). Arch Virol 140, 14411449.[Medline]
Lewis, M. J. & Pelham, H. R. (1992). Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum. Cell 68, 353364.[Medline]
Mallabiabarrena, A., Jimenez, M. A., Rico, M. & Alarcon, B. (1995). A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure. EMBO J 14, 22572268.[Abstract]
Mass, D. R. & Atkinson, P. H. (1994). Retention by the endoplasmic reticulum of rotavirus VP7 is controlled by three adjacent amino-terminal residues. J Virol 68, 366378.[Abstract]
Mirazimi, A. & Svensson, L. (1998). Carbohydrates facilitate correct disulfide bond formation and folding of rotavirus VP7. J Virol 72, 38873892.
Mirazimi, A. & Svensson, L. (2000). ATP is required for correct folding and disulfide bond formation and folding of rotavirus VP7. J Virol 74, 80488052.
Mirazimi, A., von Bonsdorff, C. H. & Svensson, L. (1996). Effect of brefeldin A on rotavirus assembly and oligosaccharide processing. Virology 217, 554563.[CrossRef][Medline]
Mirazimi, A., Nilsson, M. & Svensson, L. (1998). The molecular chaperone calnexin interacts with the NSP4 enterotoxin of rotavirus in vivo and in vitro. J Virol 72, 87058709.
Munro, S. & Pelham, H. R. (1987). A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899907.[Medline]
Newton, K., Meyer, J. C., Bellamy, A. R. & Taylor, J. A. (1997). Rotavirus nonstructural glycoprotein NSP4 alters plasma membrane permeability in mammalian cells. J Virol 71, 94589465.[Abstract]
Nilsson, T. & Warren, G. (1994). Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curr Opin Cell Biol 6, 517521.[Medline]
Nilsson, M., von Bonsdorff, C. H., Weclewicz, K., Cohen, J. & Svensson, L. (1998). Assembly of viroplasm and virus-like particles of rotavirus by a Semliki Forest virus replicon. Virology 242, 255265.[CrossRef][Medline]
Nilsson, T., Jackson, M. & Peterson, P. A. (1989). Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 58, 707718.[Medline]
Nuchtern, J. G., Bonifacino, J. S., Biddison, W. E. & Klausner, R. D. (1989). Brefeldin A implicates egress from endoplasmic reticulum in class I restricted antigen presentation. Nature 339, 223226.[CrossRef][Medline]
Paabo, S., Bhat, B. M., Wold, W. S. & Peterson, P. A. (1987). A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell 50, 311317.[Medline]
Pelham, H. R. (1988). Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. EMBO J 7, 913918.[Abstract]
Pelham, H. R. (1991). Recycling of proteins between the endoplasmic reticulum and Golgi complex. Curr Opin Cell Biol 3, 585591.[Medline]
Pelham, H. R. (1994). About turn for the COPs? Cell 79, 11251127.[Medline]
Petrie, B. L. (1983). Biological activity of rotavirus particles lacking glycosylated proteins. In Double-stranded RNA Viruses, pp. 145156. Edited by R. W. Compans & D. H. L. Bishop. New York: Elsevier.
Pettersson, R. F. (1991). Protein localization and virus assembly at intracellular membranes. Curr Top Microbiol Immunol 170, 67106.[Medline]
Poruchynsky, M. S., Tyndall, C., Both, G. W., Sato, F., Bellamy, A. R. & Atkinson, P. H. (1985). Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein. J Cell Biol 101, 21992209.[Abstract]
Poruchynsky, M. S., Maass, D. R. & Atkinson, P. H. (1991). Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER. J Cell Biol 114, 651656.[Abstract]
Ruggeri, F., Johansen, K., Basile, G., Kraehenbuhl, J.-P. & Svensson, L. (1998). Antirotavirus immunoglobulin A neutralizes virus in vitro after transcytosis through epithelial cells and protects infant mice from diarrhea. J Virol 72, 27082714.
Schutze, M. P., Peterson, P. A. & Jackson, M. R. (1994). An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J 13, 16961705.[Abstract]
Svensson, L., Dormitzer, P. R., von Bonsdorff, C. H., Maunula, L. & Greenberg, H. B. (1994). Intracellular manipulation of disulfide bond formation in rotavirus proteins during assembly. J Virol 68, 52045215.[Abstract]
Tafazoli, F., Zeng, C. Q., Estes, M. K., Magnusson, K. E. & Svensson, L. (2001). NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized epithelial cells. J Virol 75, 15401546.
Taylor, J. A., O'Brien, J. A. & Yeager, M. (1996). The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains. EMBO J 15, 44694476.[Abstract]
Tian, P., Estes, M. K., Hu, Y., Ball, J. M., Zeng, C. Q. & Schilling, W. P. (1995). The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum. J Virol 69, 57635772.[Abstract]
Townsley, F. M. & Pelham, H. R. (1994). The KKXX signal mediates retrieval of membrane proteins from the Golgi to the ER in yeast. Eur J Cell Biol 64, 211216.[Medline]
Weclewicz, K., Kristensson, K., Greenberg, H. B. & Svensson, L. (1993a). The endoplasmic reticulum-associated VP7 of rotavirus is targeted to axons and dendrites in polarized neurons. J Neurocytol 22, 616626.[Medline]
Weclewicz, K., Svensson, L., Billger, M., Holmberg, K., Wallin, M. & Kristensson, K. (1993b). Microtubule-associated protein 2 appears in axons of cultured dorsal root ganglia and spinal cord neurons after rotavirus infection. J Neurosci Res 36, 173182.[Medline]
Xu, A., Bellamy, A. R. & Taylor, J. A. (2000). Immobilization of the early secretory pathway by a virus glycoprotein that binds to microtubules. EMBO J 19, 64656474.
Zhang, M., Zeng, C. Q., Morris, A. P. & Estes, M. K. (2000). A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells. J Virol 74, 1166311670.
Received 20 August 2002;
accepted 19 November 2002.