School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK1
Author for correspondence: Mike Flint.Fax +44 118 931 6671. e-mail m.j.flint{at}reading.ac.uk
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Abstract |
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Truncation of E1 after residue 311 causes it to be secreted from expressing cells (Michalak et al., 1997 ), suggesting the ER-retention signal resides between E1 residues 311 and 383. To determine if this region of E1 could confer ER localization on heterologous proteins, chimeric glycoproteins consisting of the ectodomains of the cell surface-expressed glycoproteins influenza A virus haemagglutinin (HA) and CD4, fused to the HCV sequence, were expressed. To achieve this, cDNA cassettes were constructed by PCR mutagenesis, where unique restriction sites were introduced at the sequence encoding the N and C termini of CD4 and HA, and at the junction between the ectodomain and the transmembrane region (Fig. 1
a). Introduction of an ApaI site at the junction between the ecto-and transmembrane domains caused the substitution of an Ala residue for an Ile in the HA cassette (HA/CAS) and a Met for Gly in the CD4 cassette (CD4/CAS). PCR was used to generate inserts, which were ligated with the cassette DNA to form cDNA encoding a chimeric glycoprotein (Fig. 1a
). Sequencing confirmed the identity of the cDNA clones.
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To determine if the chimeric glycoproteins were expressed at the cell surface, 100 mm dishes of subconfluent HEK (293) cells were transfected with 10 µg plasmid DNA encoding the CD4 chimeras using the calcium phosphate method. Forty-eight hours post-transfection cells were harvested and resuspended in PBS containing 1% FCS and 0·05% sodium azide. 1x105 cells were incubated with the anti-CD4 MAb Q4120 (5 µg/ml). Cells were washed and incubated with anti-mouse FITC (1/250) and analysed by flow cytometry. Both the wild-type CD4 molecule and the CD4/CAS+HATMCT protein were detected at the surface of unfixed cells, whilst chimeric CD4/CAS+E1311383 and CD4/CAS+E2661746 glycoproteins were not (Fig. 1b). This demonstrated that introduction of the mutation during construction of the CD4 cassette did not prevent export to the cell surface. To confirm expression of the chimeric glycoproteins, cells were fixed and permeabilized (Leucoperm, Serotec) before incubation with antibody (Fig. 1b
). When visualized using fluorescence microscopy, the permeabilized cells expressing CD4/CAS+E1311383 and CD4/CAS+E2661746 exhibited a perinuclear staining characteristic of ER localization (data not shown).
To monitor the intracellular transport of the chimeric glycoproteins, the sensitivity of their glycans to endoglycosidase treatment after pulsechase labelling was assayed. Endo-ß-N-acetylglucosaminidase H (endo-H) removes high mannose, but not complex forms, of N-linked glycans (Tarentino & Maley, 1974 ), whereas peptide N-glycosidase F (PNGase F) removes both high mannose and complex N-linked sugars. Resistance to endo-H, and sensitivity to PNGase F, indicates transport of a protein from the ER to at least the medial Golgi compartment.
Subconfluent monolayers of HEK cells grown in six-well plates were transfected with 5 µg plasmid DNA using lipofectamine (Life Technologies, GibcoBRL). Twelve hours after transfection, the cells were incubated with Cys/Met-free DMEM for 20 min at 37 °C. Pulsechase labelling was carried out by incubating cells with Cys/Met-free DMEM supplemented with 50 µCi/ml [35S]-PROMIX (Amersham) for 30 min, followed by DMEM supplemented with 10x the usual amount of Cys and Met for 3 h. Cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0·5% sodium deoxycholate, 0·1% SDS, 50 mM Tris pH 8·0) and clarified by centrifugation in a microfuge for 15 min at 4 °C. The chimeric glycoproteins were immunoprecipitated from the supernatants using antibodies specific for the ectodomains of HA (Fig. 2a) or CD4 (Fig. 2b
) and protein Gagarose. Digestion with PNGase F and endo-H (New England Biolabs) was carried out according to the manufacturer's instructions. HA derived from both the wild-type and cassette vectors (HA/WT, HA/CAS; Fig. 2a
) became resistant to endo-H within a 3 h chase period, indicative of their transport through the cis-Golgi. Wild-type HA attained resistance to endo-H slightly faster than the HA/CAS protein. The mutation introduced during the construction of HA/CAS may have altered the kinetics of folding or transport. Since HA/CAS became resistant to endo-H during the 3 h chase period this phenomenon was not investigated further. The negative control molecule with the HA ectodomain fused to the transmembrane and cytoplasmic domains of CD4 (HA/CAS+CD4TMCT; Fig. 2a
) also became resistant to endo-H during the chase period. Thus, addition of a heterologous sequence to the HA ectodomain could be tolerated without causing misfolding and inducing ER retention by a quality control mechanism. In contrast to the wild-type and cassette molecules, the HA fusion protein containing E1 residues 311383 remained sensitive to endo-H after a 3 h chase period (HA/CAS+E1311383; Fig. 2a
). Sensitivity to endo-H was also observed for the HA fusion protein containing E2 residues 661746 (HA/CAS+E2661746). These results indicate that E1 residues 311383 induce retention in a pre-medial Golgi compartment, probably the ER.
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Since quality control mechanisms ensure that misfolded glycoproteins are not exported from the ER, it was important to establish if the observed retention was simply due to misfolding. To determine if the HA chimeras were misfolded, the proteins were expressed in HEK cells, labelled and immunoprecipitated under non-denaturing conditions (150 mM NaCl, 2 mM EDTA, 0·5% NP-40, 10 mM Tris pH 7·5) with a MAb against a conformational epitope (HC67; Daniels et al., 1983 ). All of the chimeras, including HA/CAS+E1311383 and HA/CAS+E2661746, could be immunoprecipitated by HC67 (Fig. 3
a), suggesting that the ectodomains of these glycoproteins had adopted a native conformation. In addition, we determined if the CD4/CAS+E1311383 chimeric glycoprotein could be recognized by MAbs directed against conformation-dependent epitopes within the CD4 ectodomain (Fig. 3b
). Both CD4/CAS and the chimeric protein could be immunoprecipitated by all five MAbs tested; 63G4 interacts with a linear epitope, and the four remaining MAbs recognize conformational epitopes. These data suggest that the CD4 ectodomain adopts a native conformation within the context of the CD4/CAS+E1311383 chimeric protein.
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Localization of HCV glycoproteins within the ER has been observed using a number of different expression methods, including recombinant vaccinia and Sindbis viruses (Dubuisson et al., 1994 ) and stable expressing cell lines (Duvet et al., 1998
). Duvet and colleagues (1998)
recently reported that the C-terminal region of E2 contains a signal mediating retention in the ER. We have demonstrated a similar property for the C-terminal region of E1. It is interesting that both the HCV glycoproteins contain sequences that mediate retention. Whilst the folding of E1 and E2 occurs slowly, these proteins associate whilst folding (Deleersnyder et al., 1997
). Since unfolded proteins are not permitted to leave the ER, the reason for two independent signals is not obvious.
Multiple mechanisms for retention of proteins within the ER have been described, including retention mediated through KDEL, di-lysine and di-arginine motifs. These motifs induce retention by signalling for proteins containing them to be retrieved from post-ER compartments. However, many ER resident proteins do not bear these retrieval sequences, suggesting that additional mechanisms for ER targeting also exist. The regions of HCV E1 and E2 which mediate ER retention do not possess similarity with other known ER localization signals, or with each other, except for the fact that many of these sequences contain stretches of hydrophobic residues, possibly functioning as transmembrane domains. The C-terminal regions of E1 and E2 shown to confer ER retention must contain the membrane-spanning domains for these type I transmembrane proteins. Whilst targeting of proteins to the Golgi apparatus may be mediated through the length of the transmembrane domain of a protein (Bretscher & Munro, 1993 ; Munro, 1995
), this property may also have a role in localizing proteins to the ER (Pedrazzini et al., 1996
; Yang et al., 1997
). This lipid-based sorting model suggests that, in the absence of dominant lumenal or cytosolic associations, proteins partition according to differences in membrane thickness and deformability between compartments of the secretory pathway. It may be that the HCV glycoproteins are ER-localized because of the properties of their transmembrane domains. This model predicts that localization is mediated in a sequence-independent manner, and consequently may be tested experimentally.
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Acknowledgments |
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References |
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Received 19 February 1999;
accepted 23 April 1999.