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

Ali Mirazimi1, Karl-Eric Magnusson2 and Lennart Svensson1,2

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 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 -3 and either -4 or -5 (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 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 112–175) 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and antibodies.
BHK-21 (baby hamster kidney) cells were grown in Dubebecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10 % foetal calf serum. The antibodies used in this study included anti-c-Myc mouse monoclonal antibody (R950-25; Invitrogen) and a rabbit antiserum that recognizes aa 114–134 of NSP4 (Mirazimi et al., 1998).

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).



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Fig. 1. Schematic representation of the truncated NSP4 proteins used.

 
Generation of recombinant SFV.
Recombinant plasmids were purified using plasmid minipreps (Qiagen) and linearized by NruI digestion. Linearized plasmids were used as templates for in vitro RNA transcription by SP6 RNA polymerase, as described previously (Mirazimi & Svensson, 2000; Nilsson et al., 1998). In vitro transcripts made from recombinant pSFV-C-mycNSP4 plasmids were electroporated into BHK cells, together with equal amounts of mRNA transcripts from SpeI-linearized pSFV-Helper1 (Mirazimi & Svensson, 2000). Electroporated BHK cells were then diluted in MEM and seeded on to tissue culture dishes and incubated for 24 h at 37 °C. The medium including recombinant SFV was then collected and frozen at -70 °C until use. Virus titres were determined by peroxidase staining, as previously described (Ruggeri 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 % {beta}-mercaptoethanol) and boiled for 5 min before separation by SDS-PAGE.

Endo-{beta}-N-acetylglucosaminidase digestion.
Digestion with endo-{beta}-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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction and expression of the C-terminal truncated forms of NSP4
Based on previous information about ER retention signals and the localization of ER retention motifs of transmembrane proteins (Jackson et al., 1990, 1993; Nilsson et al., 1989; Pelham, 1991), the cytoplasmic region of NSP4 was used for the creation of truncated forms of NSP4. As no antibodies were available against the luminal part of NSP4, a 10-amino-acid c-Myc tag, previously used in transport studies (Andersson & Pettersson, 1998; Munro & Pelham, 1987), was inserted into the N terminus immediately following the ATG codon of NSP4 (C-mycNSP4; Fig. 1).

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.



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Fig. 2. (A) Antibodies to c-Myc and NSP4 recognize SFV-expressed NSP4. BHK cells were infected with SFV-C-mycNSP4.175 (m.o.i. 10), starved at 7 h p.i. for 1 h in methionine/cysteine-free medium and subsequently labelled with [35S]methionine/cysteine (250 µCi) for 1 h. At the end of the pulse, monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min followed by cell lysis. The cell lysates were immunoprecipitated with a monoclonal antibody against c-Myc and a rabbit anti-NSP4 antibody, followed by mock or Endo H digestion for 4 h at 37 °C. Proteins were then separated by SDS-PAGE. Molecular markers (kDa) are shown on the left. (B) Truncated NSP4 is efficiently expressed in BHK cells. BHK cells were mock-infected or infected with SFV-C-mycNSP4.175, SFV-C-mycNSP4.123 or SFV-C-mycNSP4.85 (m.o.i. 10). At 7 h p.i., monolayers were starved for 1 h in methionine/cysteine-free medium and subsequently labelled with [35S]methionine/cysteine (250 µCi) for 1 h. At the end of the pulse, monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min, followed by cell lysis and analysis by reducing SDS-PAGE. Molecular markers (kDa) are shown on the right. (C) C-terminal mutated NSP4 is glycosylated. Cell lysates from (B) were immunoprecipitated with anti-c-Myc antibody and mock- or Endo H (5 mU)-treated for 4 h and separated by SDS-PAGE. Molecular markers (kDa) are shown on the left.

 
In order to examine whether the cytoplasmic part of NSP4 was involved in ER retention, two C-terminal truncated forms of NSP4 were created. Deletion of the C terminus by 52 and 90 amino acids, respectively, resulted in the generation of mutants of 123 and 85 amino acids (C-mycNSP4.123 and C-mycNSP4.85, respectively; Fig. 1). As mentioned previously, two topologic models of NSP4 have been suggested (Bergmann et al., 1989; Chan et al., 1988). In the shortest mutant (C-mycNSP4.85), the entire or a large part of the cytoplasmic region, depending on the proposed topology, was removed.

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 85–123 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.



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Fig. 3. Cellular localization of the various truncated forms of NSP4 in BHK cells. BHK cells were infected with mutated forms of SFV-C-mycNSP4. At 18 h p.i., infected cells were fixed and permeabilized with Triton X-100 or left intact, as described in Methods. NSP4 was detected with a c-Myc antibody and visualized with an FITC-conjugated rabbit anti-mouse antibody.

 
To study the topological distribution of NSP4.85 on the plasma membrane, confocal microscopy was used. Following infection, cells were fixed and a plasma membrane staining of NSP4 performed as described in the Methods. Fig. 4 shows a uniformly distributed punctated staining of NSP4 from the top of the cell (a) to the ventral surface at the substratum (d). The intensity of NSP4 fluorescence in permeabilized cells was significant and was associated with the cytoplasm (Fig. 5A). However, in non-permeabilized cells, NSP4 was associated with the apical and basolateral plasma membrane (Fig. 5B). These results confirm the observations presented in Fig. 3.



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Fig. 4. Cellular localization of truncated NSP4 in BHK cells. The stained cells were analysed by horizontal confocal sectioning, displaying punctate membrane fluorescence from the uppermost, dorsal surface (a) to the ventral surface at the substratum (d). The vertical step between sections was 2 µm. Bar size, 10 µm.

 


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Fig. 5. C-mycNSP4.85 distribution in non-permeabilized and permeabilized cells. NSP4 fluorescence was assessed from vertical confocal sections on (A) permeabilized cells and (B) non-permeabilized cells. The y-axis shows arbitrary fluorescence (0–255 levels) and the x-axis shows pixel values from the top to the bottom of the cells.

 
The plasma membrane-targeted NSP4 is transported through the secretory pathway
We next examined whether C-mycNSP4.85 used the established secretory pathway to reach the plasma membrane. The question was raised after reports that VP7 of rotavirus is targeted to axons in neurons by a mechanism that bypasses the Golgi complex (Weclewicz et al., 1993a) and that rotavirus is released from the apical surface through vesicular transport that also appears to bypass the Golgi complex (Jourdan et al., 1997). To examine the transport pathway for mutated NSP4 to the plasma membrane, infected cells were mock- or BFA-treated from 1 h p.i. BFA is an antiviral agent that inhibits protein transport through the exocytic pathway from the ER to the plasma membrane (Chen et al., 1991; Mirazimi et al., 1996). Mock- or BFA-treated infected cells were fixed at 18 h p.i and processed for surface immunofluorescence. As illustrated in Fig. 6, BFA prevented most of the mutated NSP4 from reaching the plasma membrane, suggesting that truncated NSP4 reaches the plasma membrane through the secretory pathway.



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Fig. 6. Brefeldin A prevents NSP4.85 from being transported to the cell surface. BHK cells were infected with SFV-C-mycNSP4.85 and treated with BFA (2 µg ml-1) at 1 h p.i. Cells were fixed at 18 h.p.i and NSP4 was detected by surface immunofluorescence using a mouse anti-c-Myc antibody.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enveloped viruses acquire their envelopes by budding through one of several host cellular membranes (Pettersson, 1991). While most enveloped viruses bud through the plasma membrane, a few viruses, such as rotavirus, exploit the ER membrane. A strategy that several viruses have developed is to endow the glycoproteins with signals for compartment-specific targeting and retention. Identification of these retention and retrieval signals is of importance not only for understanding the mechanism of virus budding and protein compartmentalization, but also for the revelation of novel mechanisms in cell physiology and viral pathogenesis.

In this study, we have shown that aa 85–123 in the cytoplasmic tail of rotavirus NSP4 are involved in ER retention. These amino acids partly cover the domain (aa 114–135) 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 {alpha}-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 ER–Golgi 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 112–175, 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 112–175) 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 ER–Golgi 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 ER–Golgi 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 ER–Golgi 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 {alpha}-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 retrieval–retention 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
 
This study was supported by grants from the Swedish Medical Research Council, project No. 10392 (L. S.) and 6251 (K.-E. M.), the Karolinska Institute Research Fund (L. S.), the Swedish Research Council for Engineering Science (K.-E. M.), the Swedish Research Council (L. S., K.-E. M.) and the Swedish Society for Medical Research (K.-E. M.).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 20 August 2002; accepted 19 November 2002.