JOURNAL OF VIROLOGY, Aug. 1996, p. 5005–5015 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 70, No. 8
Down-Regulation of Paramyxovirus Hemagglutinin-Neuraminidase Glycoprotein Surface Expression by a Mutant Fusion Protein Containing a Retention Signal for the Endoplasmic Reticulum YOSHIKAZU TANAKA, BEVERLY R. HEMINWAY,
AND
MARK S. GALINSKI*
Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 Received 11 October 1995/Accepted 23 April 1996
The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotypespecific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression. 15). In these studies, coexpression of both F and HN resulted in massive cell fusion of the monolayer, whereas expression of F or HN alone did not induce any detectable syncytia. In addition, the reported serotype-specific restrictions between HPIV3 and other paramyxovirus F and HN proteins (i.e., HPIV1 or HPIV2) has been interpreted to suggest that specific F-HN interactions may be involved in mediating cell-cell fusion (3, 5, 6). The absence of detectable hetero-oligomers suggests that putative protein-protein interactions between F and HN are transient or not very stable. An alternative explanation for the fusion-promoting activity of HN, not requiring direct protein-protein interactions, infers that HN may function as a molecular bridge to bring opposed membranes to a specific distance and thereby facilitate the intrinsic fusion properties of F. In this model, the function of HN is ancillary to the primary fusion properties of F. In this report, we described studies which indicate that physical interactions can be detected between F and HN in vivo, using a recombinant expression system expressing both glycoproteins. In these studies, expression of HN was found to be down-regulated by a mutant HPIV3 F protein which was retained in the endoplasmic reticulum (ER).
Studies characterizing the oligomeric forms of the human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins have shown that homo-oligomeric trimers of F and tetramers of HN can be readily identified (2, 11, 13). In contrast, the formation of heterooligomers between F and HN, or indeed other paramyxoviral F and HN proteins, has not been convincingly demonstrated. Russell et al. (11) examined the oligomeric forms of simian virus 5 (SV5) F proteins and showed that monomeric, homodimeric, and homotrimeric forms could be found and that hetero-oligomeric forms of F and HN could not be identified, even with the use of cross-linking agents. Similar results were also obtained in this study for Newcastle disease virus (NDV) and HPIV3. Malvoisin and Wild (8), using vaccinia virusdriven recombinant expression of the morbillivirus measles virus F and hemagglutinin (H) glycoproteins, reported that both glycoproteins could be cross-linked at the cell surface by using a reducible cross-linking agent, 3,39-dithiobis(sulfosuccinimidylproprionate) and coimmunoprecipitation with monoclonal antibodies generated to either F or H. However, if lysis of the cells preceded cross-linking, then the two proteins were not coprecipitable. The apparent absence of hetero-oligomers of these proteins is of considerable interest in understanding the reported fusion-promoting activity of HPIV3 HN in the formation of syncytia, which is seen in recombinant expression studies (5, 6,
MATERIALS AND METHODS Cell lines. The HeLa cell lines (HeLa b-gal and HeLa-tat) used in this study have been previously described (15). The HeLa cell lines and COS-7 cells were routinely passaged in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and maintained as previously described (5). Plasmid constructions and mutagenesis. (i) pcDL-SR beta 8.2. The vector pcDL-SR alpha-296 (14) was obtained from the American Type Culture Collection, Rockville, Md. (catalog no. 67663). To facilitate subcloning and extend the usefulness of this vector in expression studies, a polyvalent restriction site with flanking T7 and SP6 promoter sequences was introduced into the plasmid. Two
* Corresponding author. Present address: Merck & Co., Inc., P.O. Box 4, WP29M-4, Sumneytown Pike, West Point, PA 19486. Phone: (215) 652-1098. Fax: (215) 652-4472. Electronic mail address: Mark
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primers, 59-AGCTCTTCGTCGACCTTATGTAT-39 (SP6 promoter primer) and 59-AGCTCTTCGTCGTAATACGACTC-39 (T7 promoter primer), were used to amplify by PCR a portion of the plasmid pGEM-3 (nucleotides 2828 to 89; X65302). PCR amplification methods and agarose gel purification of the DNA product were performed as previously described (5). The purified PCR DNA was ligated into agarose gel-purified pcDL-SR alpha-296 following digestion of the plasmid with the restriction endonuclease BamHI, dephosphorylation of the restricted DNA, and repair of the cohesive ends by using Klenow fragment as previously described (3). Recombinant clones were identified and sequenced to confirm the orientation and sequence of the inserted DNA. A clone, pcDL-SR beta 8.2, which had the T7 promoter oriented in the same direction as the upstream SR alpha promoter was identified. (ii) HPIV3 F and HN genes. The HPIV3 HN and F glycoprotein genes used in this study have been previously described (5, 15). Both genes were transferred into pcDL-SR beta 8.2 by release of the gene inserts with BamHI and PstI and unidirectional cloning into the same sites in the vector. Two clones, pcDL-HPIV3 F10-8.2 and pcDL-HPIV3 HN-8.2, were identified and used in these studies. (iii) HPIV3 F protein signal retention mutant. A mutant F gene, lacking the transmembrane anchor and cytoplasmic tail and containing an ER retention signal (KDEL) appended to the carboxyl terminus, was constructed by PCR amplification. Two synthetic oligonucleotides were used to PCR amplify the retention mutant F protein. The upstream oligonucleotide 59-AGCTCTTCGT CGTAATACGACTC-39 (T7 promoter primer) and downstream oligonucleotide 59-ccgtcgactacagctcgtcctttgtggtgctagattgat-39 (F-KDEL primer) were used to amplify the entire F gene DNA, using the plasmid pcDL-HPIV3 F10-8.2 as a source of template. The PCR DNA was repaired with T4 DNA polymerase and subsequently digested with BglII. The small (approximately 100 bp containing a BglII cohesive end and a blunt end) restriction fragment encoding the relevant mutations was gel purified and ligated into pcDL-HPIV3 F10-8.2 which had been digested with PstI, the ends were repaired with T4 DNA polymerase, and the plasmid was redigested with BglII and gel purified. The resulting F gene no longer contained the nucleotide sequence encoding the carboxyl-terminal amino acid residues 494 to 539 and in addition contained nucleotide sequences encoding the ER retention signal KDEL followed by a stop codon. The clone pcDL HPIV3 F-KDEL was identified, and the fidelity of the mutant F gene sequences was confirmed by DNA sequencing as instructed by the manufacturer (U.S. Biochemicals, Cleveland, Ohio). (iv) HPIV3 Flag-HN mutant protein. The HPIV3 HN protein was engineered to contain a linear epitope (Flag sequence) within the N terminus of the protein. The Flag epitope is an octapeptide (DYKDDDDK) for which a monoclonal antibody (M2) is commercially available (International Biotechnologies, Inc.), and since the amino acid sequence is highly unique, it is unlikely to be found in most proteins (10). We used paired PCR amplification primers to generate the mutant HN. The upstream primer, 59-ggatccatggactacaaggacgacgatgacaaggaat actggaagcac-39, contained a BamHI site on the 59 terminus followed by an initiating methionine codon (in boldface) and the sequences encoding the Flag epitope (underlined) followed by 15 nucleotides which were identical to the HN coding sequence, and the downstream primer was 59-AGCTCTTCGTCGAC CTTATGTAT-39 (SP6 promoter primer). The paired primers were used to amplify the entire HN gene DNA, using plasmid pGEM-3HN as a source of template as described above. The PCR DNA was repaired with T4 DNA polymerase and subsequently digested with BamHI and XcmI. The small (approximately 400 bp containing a BamHI-XcmI cohesive ends) restriction fragment encoding the relevant mutations was gel purified and ligated into pcDL-HPIV3 HN-8.2, which had been digested with BamHI and XcmI and gel purified. The clone pcDL-HPIV3 Flag-HN-8.2 was identified, and the fidelity of the mutant gene sequence was confirmed by DNA sequencing as instructed by the manufacturer (U.S. Biochemicals). Heterologous genes. A number of heterologous paramyxovirus and nonparamyxovirus genes were used in this study. All genes were subcloned into pcDL-SR beta 8.2 following the appropriate restriction endonuclease digestion, gel purification of the gene, and ligation into the expression vector which had been appropriately treated by restriction endonuclease digestion. The Sendai Fushimi HN gene (3) was subcloned into pcDL-SR beta 8.2 between the EcoRI and SmaI sites following release from the parental plasmid (pT7/T3 alpha-18) with EcoRI and PvuII. The respiratory syncytial virus (RSV) glycoprotein (G) gene (kindly supplied by Peter Collins, National Institute of Allergy and Infectious Diseases [NIAID], Bethesda, Md.), which has been previously described (4), was subcloned into pcDL-SR beta 8.2 between the EcoRI and XbaI sites. The SV5 HN gene (kindly provided by Robert Lamb, Northwestern University, Evanston, Ill.), which has been previously described (5), was subcloned into pcDL-SR alpha-296 following release from the parental plasmid (pGEM-3) inserts, using XhoI and subsequent religation into the SalI site of the plasmid. The measles virus (Edmonston strain) H gene in plasmid pcDL-SR beta 8.2 was constructed from a recombinant clone derived in our laboratory (data not shown). The vesicular stomatitis virus (VSV) G gene (kindly provided by John K. Rose, Yale University) was subcloned into pcDL-SR beta 8.2 into a BamHI site. Finally, as a control ER retention mutant protein, we used the chimeric CD8/E19 protein in coexpression studies (7) (plasmid pCMU-CD8/E19 was kindly provided by Rohan Teasdale, R. W. Johnson Pharmaceutical Research Institute at The Scripps Research Institute, La Jolla, Calif.). The CD8/E19 gene was released
J. VIROL. with HindIII and BamHI, repaired with T4 DNA polymerase, and subsequently subcloned into a SmaI site in pcDL-SR beta 8.2. Transfection and fusion assays. Transient expression of the various recombinant plasmids in tissue culture monolayers was performed as previously described (15). Various recombinant plasmid constructs, alone or in combination, were transfected into COS-7 cells as previously described (3, 15), using electroporation techniques as previously described (15). To maintain a constant amount of transfected DNA, the total amount of plasmid transfected into cells was adjusted with vector DNA (pcDL-SR beta 8.2 minus insert). Measurement of cell fusion events was semiquantified following fusion of HeLa-tat and HeLa b-gal cells as previously described (15). IF assays. Transiently expressing cell monolayers were stained by using an indirect immunofluorescence (IF) assay as described previously (4). For analysis of total protein expression, cells were fixed with acetone-methanol (1:1) for 10 min at 2208C. For detection of cell surface expression, cells were fixed with 3.7% formaldehyde at room temperature for 10 min. Primary antibodies used in this study were either monoclonal, monospecific, or polyclonal, as indicated in Results. Cells were examined on a Nikon microscope equipped for epifluorescence, and digital photographs were captured by using a computer imaging system (Oncor Image Systems, Inc.) at the magnifications indicated. Images shown in each figure, unless otherwise indicated, were captured for the same length of time so that the relative levels of IF staining between experimental samples could be compared. Protein analysis. Protein radiolabeling and immunoprecipitation were performed as previously described (5). Transfected cells were pulse-labeled for 30 min with 100 mCi of 35S-Express-methionine (NEN) per ml 40 h after transfection and chased for 0, 90, or 180 min in unlabeled methionine and cysteine. Labeled cells were washed twice with cold phosphate-buffered saline and lysed with radioimmunoprecipitation assay buffer containing 10 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.15 M NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 2 mg of aprotinin per ml. After centrifugation at 14,000 rpm for 10 min, appropriate antibody was added to the recovered lysates and reacted for 1 h at 48C. The immunocomplexes were precipitated with protein A-Sepharose beads (Pharmacia LKB Biotechnology, Piscataway, N.J.) and eluted with sample buffer after being washed five times with radioimmunoprecipitation buffer. The recovered protein samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 8% polyacrylamide resolving gels followed by fluorography as previously described (2). Endoglycosidase H (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) digestion was performed as described previously (15). Semiquantification of the immunoprecipitated proteins was performed by using Molecular Dynamics ImageQuant, version 3.3, on scanned images of the radioautographic films. Immunologic reagents. A number of different antibodies, either monoclonal, monospecific, or polyvalent polyclonal, were used in this work. They included monoclonal antibodies against HPIV3 HN (68/2) and F (c199/3, c110/11, and B102/4) (17) (kindly provided by Susan Hall, NIAID); rabbit monospecific antiSV5 HN antiserum (kindly provided by Robert Lamb, Northwestern University); mouse monoclonal anti-Sendai virus HN antibody (kindly supplied by Allen Portner, St. Jude Children’s Research Hospital, Memphis, Tenn.); rabbit polyclonal anti-VSV antiserum (Amiya Banerjee, Cleveland Clinic Foundation, Cleveland, Ohio); monoclonal antibody against measles virus H (B2; kindly provided by Paul Rota, Centers for Disease Control and Prevention, Atlanta, Ga.); and monoclonal antibody against RSV G (L9; kindly provided by Peter Collins, NIAID). Monoclonal antibody OKT8 against CD8 protein was obtained from Cappell Laboratories, Malvern, Pa. Monoclonal antibody RL-77 against rat liver protein disulfide isomerase was kindly supplied by Michael Lamm, Case Western Reserve School of Medicine, Cleveland, Ohio). Monoclonal antibody 10C3 against a synthetic peptide (KSDKDL) from rat grp78/BiP was obtained from StressGen Biotechnologies Corp. Rabbit polyclonal anti-erythrocyte beta-spectrin antiserum was kindly supplied by Kenneth Beck, Stanford University Medical Center, Stanford, Calif.). Finally, fluorescein isothiocyanate (FITC)-conjugated antimouse immunoglobulin G (IgG) was obtained from Vector Laboratories, Inc., and rhodamine-conjugated anti-mouse IgG/IgM was obtained from Kirkegaard & Perry Laboratories, Inc.
RESULTS HPIV3 F down-regulates expression of heterologous HN/H proteins. In coexpression studies using HPIV3 F or HN and other heterologous paramyxovirus glycoproteins derived from Sendai virus, SV5, or measles virus, an unexpected reduction in the relative amount of immunoprecipitable heterologous HN/H occurred when they were coexpressed with HPIV3 F (see below). Figure 1D to F show that the steady-state level of HN/H detectable at the surface (formaldehyde-fixed cells) of cells coexpressing HPIV3 F is reduced compared with the level of HN/H expression alone (Fig. 1A to C). In contrast, no effect
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FIG. 1. HPIV3 F protein down-regulates heterologous HN protein expression on the cell surface. Shown is indirect IF staining of COS-7 cells recombinantly expressing HPIV3 HN, Sendai virus HN, and measles virus H (A to C) or coexpressing HPIV3 HN plus HPIV3 F, Sendai virus HN plus HPIV3 F, or measles virus H plus HPIV3 F (D to F). Cells (magnification, 3154) were fixed with 3.7% formaldehyde and reacted with appropriate primary anti-HN or anti-H monoclonal antibodies and a secondary horse anti-murine IgG FITC-tagged antibody. All images were exposed for the same length of time.
in surface expression of RSV G or VSV G was observed in coexpression studies (data not shown). These results suggested that interactions between HPIV3 F and heterologous HN/H proteins were occurring, resulting in decreased surface expression of the affected proteins. An occasional brightly staining cell, as shown in Fig. 1E, was observed when several fields were examined, and such cells represent cells expressing low or undetectable amounts of F (see below). Figure 1 also shows that massive syncytium formation occurred when the homotypic HPIV3 F and HN proteins were coexpressed; however, no syncytia were detectable when any of the heterologous proteins were coexpressed with HPIV3 F. Figure 2 shows that the intracellular expression of measles virus H is also suppressed when coexpressed with HPIV3 F (Fig. 2A, B, D, and E). Double staining of these cells (measles virus H [FITC tagged] and HPIV3 F [rhodamine tagged]) indicated that the down-regulation of H required coexpression of F. Thus, the one bright cell seen in Fig. 2E is not expressing detectable levels of HPIV3 F (compare with the double stain for F [Fig. 2F]), and in this cell, H expression is comparable to levels found when H is expressed alone (Fig. 2D). Similar results were obtained with double staining of cells expressing
Sendai virus HN and HPIV3 F (data not shown). Finally, the relative degree of F expression shown in Fig. 2C and F was indistinguishable from that seen when cells were transfected and expressing HPIV3 F alone (data not shown). HPIV3 F-KDEL is transport defective and is retained in the ER. We commenced a series of experiments by engineering a mutant HPIV3 F protein (Fig. 3B) which lacks a transmembrane anchor and cytoplasmic tail and contains an ER retention signal (KDEL) appended to the C terminus. If F and HN physically interact, then the retention mutant protein might complex with HN and block its egress from the ER. Figure 3A shows the surface IF staining properties of the wild-type F, F-KDEL, and another ER retention mutant protein, CD8/E19, which we have used as a well-characterized retention mutant control in our studies (7). As can be seen in the upper panels of Fig. 3A, there is no surface expression of either ER retention mutant, compared with the abundant levels of wild-type F surface expression. The intracellular staining properties of the two retention mutant proteins were similar, and both proteins could be localized to the ER by comparison with IF staining using an anti-grp78/BiP monoclonal antibody or anti-protein disulfide isomerase monoclonal antibody, each of which is an
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FIG. 2. HPIV3 F protein down-regulates heterologous intracellular H protein expression. Shown is indirect IF staining of COS-7 cells recombinantly expressing measles virus H (D) or coexpressing measles virus (MV) H plus HPIV3 F (A to C, D, and E). Cells (magnification, 3200) were fixed with acetone-methanol and reacted with a primary anti-H (a-H) monoclonal antibody (B2) and polyclonal rabbit anti-HPIV3 F (a-F) antibody. A secondary horse anti-murine IgG FITC-tagged antibody was used to localize the HN immunocomplexes, while a goat anti-rabbit IgG rhodamine-tagged antibody was used to localize the F immunocomplexes. The images were exposed for the indicated times (seconds), and appropriate filters were used to excite the FITC-tagged H immunocomplexes or the rhodamine-tagged F immunocomplexes. Panels A to C are images of the same field but vary in exposure time and wavelengths of excitation and emission. Panels E and F are images of the same field but vary in exposure time and wavelengths of excitation and emission.
antibody to a well-characterized endogenous ER resident protein (data not shown). In addition, F-KDEL was not secreted into the medium, whereas a soluble F protein, identical to F-KDEL but lacking the ER retention signal, was secreted into the medium (data not shown). Figure 3C shows the biochemical analysis of F-KDEL synthesis in a pulse-chase experiment compared with the wild-type F protein. The retention mutant
protein was not proteolytically activated, and the high-mannose cores were not processed to complex carbohydrate, as judged by maintenance of endoglycosidase H sensitivity of the protein during the chase times. HPIV3 F-KDEL down-regulates HPIV3 HN expression. Figure 4A shows that the intracellular and surface expression of HN in cells expressing F-KDEL and HN was strongly sup-
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FIG. 3. Comparison of the HPIV3 F-KDEL with the wild-type F protein and CD8/E19. (A) Transiently expressing COS-7 cells were fixed with 3.7% formaldehyde (surface) or alternatively with methanol-acetone (total) and stained with appropriate primary monoclonal antibodies recognizing HPIV3 F or CD8/E19 and a secondary horse anti-murine IgG FITC-tagged antibody. Magnifications are 3144 and 3432 for surface and total staining cells, respectively. (B) Comparison the carboxyl-terminal regions of the wild-type F protein and the engineered F-KDEL retention mutant protein, which lacks the transmembrane anchor (overlined) and cytoplasmic tail and contains an ER retention signal, KDEL (underlined). (C) Comparison of HPIV3 F and F-KDEL biosynthesis in COS-7 cells. Forty hours posttransfection, cells were cultured in methionine-cysteine-free medium for 60 min, pulsed with [35S]methionine-cysteine for 30 min, and chased for either 0, 90, or 180 min. Cells were harvested, and recombinant proteins were recovered by immunoprecipitation with a monoclonal anti-F antibody (C-199/3) and protein A conjugated to Sepharose. The recovered immunoprecipitates were divided into two fractions; one was subjected to endoglycosidase H digestion (1), and the other was untreated (2). Proteins were analyzed by discontinuous SDS-PAGE on 8% polyacrylamide gels. The positions of glycosylated uncleaved precursors F0 and F1 and of endoglycosidase H-sensitive forms of these proteins, f0 and f1, respectively, are indicated. CC represents vector-transfected control cell lysates treated with C-199/3.
pressed. That this down-regulation was not due to a nonspecific block to protein egress from the ER is supported by coexpression of HN with the CD8/E19 retention mutant, which had no effect. The down-regulation of HN by F-KDEL was dose dependent (Fig. 4B), and with increasing amounts of F-KDEL, decreasing levels of HN (1:1 . 1:2 . 1:3) were detectable on the surface, although HN expression to the cell surface could not be totally eliminated. The kinetics and protein processing of HPIV3 HN when coexpressed with F, F-KDEL, or CD8/E19 are shown in Fig. 5. In this experiment, expressing cells were pulsed with [35S]me-
thionine-cysteine for 30 min and chased for 0, 90, or 180 min in the presence of excess unlabeled amino acids. The recovered proteins (obtained by using mixtures of monoclonal antibodies to F and HN) were also examined for endoglycosidase H sensitivity to determine whether they were processed through the Golgi complex. Figure 5A shows that expression of HN with the wild-type F had no significant effect on the synthesis, stability, or processing of HN through the Golgi complex. As shown in Fig. 5, HN processing to endoglycosidase H insensitivity is never complete, and partial products are generally seen at all times during the chase. By contrast, coexpression of HN
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FIG. 4. Down-regulation of HPIV3 HN expression by the ER retention mutant, F-KDEL, but not by CD8/E19. Shown is indirect IF staining of COS-7 cells recombinantly expressing HPIV3 HN, HPIV3 HN plus F-KDEL, or HPIV3 HN plus CD8/E19 proteins. (A) Cells (magnification, 3152) fixed with 3.7% formaldehyde (surface) or with methanol-acetone (total) and reacted with a primary anti-HN monoclonal antibody (68/2) and a secondary horse anti-murine IgG FITC-tagged antibody. (B) Cells expressing HPIV3 HN and HN plus F-KDEL coexpression at plasmid transfection ratios (HN/F-KDEL) of 1:1, 1:2, and 1:3. Cells (magnification, 3152) were fixed with 3.7% formaldehyde to detect surface expression of HN as described for panel A. All images were exposed for the same length of time.
with F-KDEL shows a marked reduction in the relative abundance of HN detectable during the 30-min pulse compared with that seen during the case (Fig. 5B). However, upon longer exposure of the gels, the HN which enters the ER is observed to be processed through the Golgi complex. The early block to the detection of HN suggested an alteration in the folding of HN into a conformation which was not detectable by the anti-HN monoclonal antibody or, alternatively, an early block in the introduction of the nascently produced HN into the ER. That this was not a nonspecific block to HN synthesis was supported by the biochemical results seen for coexpression of HN with the CD8/E19 retention mutant. To determine whether the reduction of detectable HN during the pulse was due to alteration in the folding of HN into a conformation containing the native epitope, we introduced a linear Flag epitope into the N terminus of HN (Flag-HN). Use of this epitope tag allowed us to monitor HN by using either an anti-Flag (M2) monoclonal antibody or a monoclonal antibody which recognizes a native epitope (68/2). Flag-HN was found to be comparable in all properties examined when compared in parallel with the native HN for IF staining, biochemical processing, and fusion-promoting activity (data not shown). Coexpression of Flag-HN with F-KDEL showed a pattern of protein expression, in pulse-chase experiments, which was identical to that of the native HN and was independent of the monoclonal antibody used to detect Flag-HN (M2 versus 68/2 [data not shown]). HPIV3 F-KDEL can suppress syncytium formation without affecting HPIV3 F expression. Since HPIV3 F has been shown to form homo-oligomeric trimers (10), we determined the ability of F-KDEL to interact and block wild-type F egress from the ER. Figure 6A shows biochemical analysis of coexpression
of F-KDEL with F and HN. Figure 6 shows an early block to HN synthesis, as was shown in Fig. 5B, but interestingly, the retention mutant protein has no detectable effect on wild-type F protein synthesis, stability, or subsequent processing through the Golgi complex (i.e., cleavage activation or carbohydrate processing). Surface IF staining of F was not altered, and the absence of effects on the wild-type F also occurred in the absence of HN (data not shown). These results suggest that F-KDEL does not oligomerize with the wild-type F, which can leave the ER and transit through the Golgi complex to the cell surface. The biochemical results shown in Fig. 6A suggested that F-KDEL ought to act as a negative regulator of syncytium formation when coexpressed with wild-type F and HN because of its ability to down-regulate HN surface expression but not F surface expression (15). Figure 6B shows that with increasing ratios of input plasmid F-KDEL DNA during transfection, the relative degree of syncytium formation is reduced. At an F/FKDEL ratio of 1:3, syncytium formation was inhibited by 90%, although it was not completely abolished. HPIV3 F-KDEL can suppress heterologous paramyxovirus HN/H expression. The ability of F-KDEL to down-regulate surface expression of heterologous receptor-binding proteins was examined to further characterize the specificity of F-KDEL effects on glycoprotein trafficking. Figure 7, like Fig. 4A, shows that accumulation of heterologous receptor-binding proteins on the surface of cells expressing both F-KDEL and HN/H was suppressed. In addition, the total amount of HN/H synthesized intracellularly was also reduced, although the suppression was less than that observed for HPIV3 HN. Coexpression of these HN/H proteins with CD8/E19 had no effect on protein egress from the ER or transport to the cell surface.
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HPIV3 F or F-KDEL. These results confirm that the degree of heterologous HN/H down-regulation by F-KDEL was greater than that observed for HPIV3 F and that the block to expression of these proteins was not an early event in the synthesis of the proteins. Finally, suppression of HN/H expression by FKDEL (summarized in Table 1) had the apparent ordering HPIV3 . Sendai virus . measles virus and no effect on HPIV3 F, RSV G, or VSV G. DISCUSSION In this report, we have described a series of experiments which examine potential interactions between HPIV3 F and HN, using a non-virus-driven transient expression system. Recent studies by Russell et al. (11) were unable to detect any hetero-oligomers between HPIV3 F and HN, although homooligomers of F were readily identified in HPIV3-infected cells. One interpretation of these results is that HN does not directly participate in the formation of a fusion pore at the cell surface FIG. 5. Biochemical analysis of F-KDEL down-regulation of HPIV3 HN shows an early block to HN synthesis. COS-7 cells were transfected with plasmid constructs containing HPIV3 HN alone or HPIV3 HN plus F (A), and HPIV3 HN plus F-KDEL or HPIV3 HN plus CD8/E19 (B). Forty hours posttransfection, pulse-chase analysis and analysis of the endoglycosidase sensitivity of the expressed proteins were performed as described for Fig. 3C. Glycoproteins were recovered by using a monoclonal anti-HN antibody (68/2) and a monoclonal anti-F antibody (C-199/3). The positions of glycosylated HN, uncleaved precursors F0 and F1, and endoglycosidase H-sensitive forms of these proteins, hn, f0, and f1, respectively, are indicated. All panels were exposed to X-ray film for the same length of time. Times are indicated in minutes.
Similar results were also observed for SV5 HN; however, RSV G expression and VSV G expression were unaffected when the proteins were expressed with F-KDEL (data not shown). Figure 8 shows the results of biochemical analysis of the down-regulation by F-KDEL of measles virus H (Fig. 8A) and Sendai virus HN (Fig. 8B), as examples of heterologous HN/H proteins which were affected by coexpression of F-KDEL, and VSV G (Fig. 8C), as an example of a heterologous receptorbinding protein which was unaffected. There did not appear to be a suppression of HN/H protein synthesis during the pulse as was noted for HPIV3 HN; however, the HN/H was less stable in the chase, although some did traffic to the Golgi complex and cell surface, as judged by the partial resistance to endoglycosidase H sensitivity (1809** lanes). These results contrast with those seen for the homotypic HN (Fig. 4B), in which case a definite block appears to occur during the pulse-labeling. No effect on the synthesis or stability of VSV G or RSV G proteins was seen. The biochemical analysis of the down-regulation of heterologous HN/H proteins by HPIV3 wild-type F is shown in Fig. 8D to F. There is a notable suppression of measles H maturation (Fig. 8D) and Sendai virus HN (Fig. 8E) stability, relative to expression of individual HN/H proteins, but no effect on VSV G (Fig. 8F). While not as profound as the down-regulation effected by F-KDEL, decreased steady-state levels of protein synthesis, as judged by IF staining, were evident (Fig. 2). Semiquantification of the effects of HPIV3 F and F-KDEL on the synthesis of homotypic HN and heterologous HN/H/G glycoproteins is shown in Fig. 9. The graphs shown were derived from densitometric analysis of the amounts of HN/H/G protein shown in Fig. 5 and 8 for the endoglycosidase-untreated samples. The data shown illustrate the suppression of HPIV3 HN synthesis when coexpressed with F-KDEL and more clearly convey the effects on the stability and maturation of the heterologous HN/H proteins when coexpressed with the
FIG. 6. HPIV3 F-KDEL affects HPIV3 HN expression but not HPIV3 wildtype F expression, resulting in suppression of syncytium formation. COS-7 cells were transfected with HPIV3 F, F-KDEL, and HN. Forty hours postransfection, pulse-chase analysis and analysis of the endoglycosidase sensitivity of the expressed proteins were performed as described for Fig. 3C. Glycoproteins were recovered by using a monoclonal anti-HN antibody (68/2) and a monoclonal anti-F antibody (C-199/3). The positions of glycosylated HN, uncleaved precursor F0 and F1, and endoglycosidase H-sensitive forms of these proteins, hn, f0, and f1, respectively, are indicated. Times are indicated in minutes. (B) Series of fusion assays to determine whether the down-regulation of HN surface expression can influence the formation of syncytia in HeLa b-gal and HeLa-tat cells. Plasmids containing the HPIV3 HN, F, or F-KDEL gene were transfected alone or in combination into subconfluent monolayers. 1, Cells transfected at 1 mg per well with a selected plasmid; 2, nontransfected cells. The F-KDEL plasmid was tested at both 1 and 3 mg per well, as indicated. All points were done in duplicate and adjusted for the same amount of protein per reaction, and data are presented as percentages of control HPIV3 wild-type F plus HN fusion activity.
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FIG. 7. HPIV3 F-KDEL can down-regulate expression of measles virus H and Sendai virus HN. Indirect IF staining was performed on COS-7 cells recombinantly expressing selected glycoproteins. (A) Cells expressing measles virus H, measles virus H plus F-KDEL, or measles virus H plus CD8/E19 (magnification, 3200) fixed with 3.7% formaldehyde (surface) or with methanol-acetone (total) and reacted with a primary anti-H monoclonal antibody (B2) and a secondary horse anti-murine IgG FITC-tagged antibody. (B) Cells expressing Sendai virus HN, Sendai virus HN plus F-KDEL, or Sendai virus HN plus CD8/E19 fixed with 3.7% formaldehyde (surface) or with methanol-acetone (total) and reacted with a primary anti-HN monoclonal antibody mixture and a secondary horse anti-murine IgG FITC-tagged antibody. All images were exposed for the same length of time.
but has an ancillary effect on F which does not require the formation of a long-term stable F-HN complex. A consequence of this interpretation would be that a transient F-HN interaction could induce a conformational change (activation) in F into a fusion-competent form. As an alternative approach to determine whether F-HN interactions could be defined in a more sensitive and biologically relevant system, we recombinantly engineered an HPIV3 F gene which encoded a protein that would be retained in the ER. The F-KDEL protein was shown to be trapped in the ER on the basis of its intracellular localization, inability to be proteolytically cleaved, and endoglycosidase H sensitivity. Although F-KDEL was found to be less stable than the wild-type F protein (Fig. 3C), the mutant protein could accumulate to significant levels in the cell (Fig. 3A) and appeared to diffuse laterally from the rough ER (perinuclear region) into the smooth ER, as judged from its ubiquitous distribution throughout the cell. The observed instability of F-KDEL (Fig. 3C) may not be due to changes in folding (rendering it malfolded), since a soluble form of F, identical to F-KDEL but lacking the ER retention signal, is stably synthesized and secreted into the media (unpublished observations). Ortmann et al. (9) have shown that HPIV3 F is proteolytically activated by furin, an endoprotease thought to be responsible for processing of a variety of viral glycoproteins in the constitutive secretory pathway (16). In the steady state,
furin is believed to accumulate within the trans-Golgi network, where it cycles to the cell surface, or, alternatively, to lysosomes directly or indirectly following internalization form the plasma membrane (1). Since F-KDEL is never proteolytically processed, our results support the observation that F cleavage activation is an event occurring outside of the ER and cis-Golgi network. Biochemical characterization of the down-regulation of HPIV3 HN by F-KDEL suggests that an early block to HPIV3 HN synthesis was occurring. Since the Flag-HN showed similar kinetics in pulse-chase experiments, it seems unlikely that the inability to detect the nascent HN was due to changes in folding of the complete protein within the ER lumen. Whether this block is a translational block, a block to translocation across the ER membrane, or association with chaperones or other proteins involved in facilitating the processing of HN folding or glycosylation is unknown. In addition, once HN was introduced into the ER, it did not undergo a rapid degradation with F-KDEL, but rather the protein appeared to leave the ER with kinetics similar to that of HN expressed alone. The apparent down-regulation of HN synthesis may reflect normal and transient F-HN interactions. Release from this interaction allows some HN to escape and to be processed through the Golgi complex (conversion to endoglycosidase H insensitivity) and would explain why HN synthesis is never completely sup-
FIG. 8. Biochemical analysis of F-KDEL down-regulation of measles virus (MV) H and Sendai virus HN but not of VSV G. COS-7 cells were transfected as described in Materials and Methods with plasmid constructs as indicated at the top of each panel. Forty hours posttransfection, pulse-chase analysis and analysis of the endoglycosidase sensitivity of the expressed proteins were performed as described for Fig. 3C. All glycoproteins were recovered with appropriate monoclonal antibodies. The positions of glycosylated HN/H/G, uncleaved precursors F0 and F1, and endoglycosidase H-sensitive forms of these proteins, hn/h/g, f0, and f1, respectively, are indicated. The 1809 and 1809** lanes are identical except that the 1809** lanes were subjected to a longer exposure. Times are indicated in minutes.
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FIG. 9. Quantitative analysis of HN/H/G expression and coexpression with HPIV3 F or F-KDEL. Scans of the radioautographs in Fig. 5 and 8 were used to determine the amount of immunoprecipitable protein recovered when HPIV3 HN, Sendai virus HN, measles virus H, or VSV G was expressed alone or coexpressed with HPIV3 F or F-KDEL. The data were obtained from the endoglycosidase H-untreated samples. Recoverable protein (HN/H/G) is expressed as a percentage relative to the amount of protein recovered in the 30-min pulse (09).
pressed. The fate of HN which never leaves the ER is probably similar to that of F-KDEL; i.e., it is targeted into a degradative pathway. The balance of HPIV3 HN synthesis, when coexpressed with F-KDEL, is ultimately seen as a suppression of steady-state levels of HN. The down-regulation of heterologous HN/H proteins by FKDEL or HPIV3 F appears to differ from that seen with the homotypic HN in that there does not appear to be an early block to the synthesis of these proteins. However, a more definite instability of the heterologous HN proteins within the ER results in the loss of both proteins (HN/H and F-KDEL) during the chase times (Fig. 8 and 9). Ultimately, the loss of protein HN/H affects the steady-state levels of expression sufficiently that a notable reduction in the intensity of IF staining is apparent (Fig. 1, 2, and 7). The reduced degree of downregulation of heterologous HN/H proteins may correlate with
J. VIROL.
differences in protein homology. Together, these results suggest that interactions between F and HN can occur in the ER and, in the case of the homotypic HN, most probably reflect authentic F-HN interactions. These interactions may normally function in the accumulation of both viral glycoproteins into discrete locations on the cell surface which would be required for virion morphogenesis. The ability of HPIV3 F to down-regulate heterologous HN/H proteins was notable in consideration of the previously reported serotype-specific restrictions (3–6). The results presented in this paper suggest that the inability of heterologous HN and F proteins to functionally replace a homotypic protein may be due, in part, to down-regulation of surface HN/H expression. As we have shown in this report (Fig. 6B) and previously (15), alterations in HN surface expression levels have measurable effects on syncytium formation. We would propose that down-regulation of HN surface expression may be one of the determinants for the reported serotype-specific restrictions. Although F-KDEL can interact with heterologous HN/H proteins, syncytium formation was not detected in coexpression studies. Nevertheless, the question remains whether the reduced amounts of F and HN/H expressed on the cell surface are sufficient to induce membrane fusion in assay systems more sensitive than those relying on syncytium formation. Down-regulation of heterologous HN protein expression may be of some biological significance in authentic virus infections, in which interference in HN expression might function as a mechanism to inhibit replication of heterotypic viruses following superinfection. Recently, Sergel and Morrison (12) studied a series of cytoplasmic deletion mutants of NDV F protein. All of the proteins studied were proteolytically activated (albeit to different levels) and expressed on the cell surface. Nevertheless, some of the mutant F proteins were dramatically reduced in syncytium formation in cotransfection experiments with the homotypic HN, despite their transport and cleavage competency. Whether these mutant NDV F proteins, which were defective for syncytium formation, were able to down-regulate NDV HN expression and thus down-regulate fusion was never ascertained. As shown in this report, down-regulation of HN in coexpression experiments is clearly a mechanism of regulating syncytium formation, and it would seem reasonable to suggest that a consequence of an altered NDV F conformation might affect transport of NDV HN to the cell surface and thus could contribute to the apparent loss of fusion activity. TABLE 1. Inhibition of protein surface expression following coexpression of selected viral envelope glycoproteins with HPIV3 F-KDEL, HPIV3 F, or CD8/E19a Inhibition when coexpressed with: Protein
HPIV3 F HPIV3 HN Sendai virus HN SV5 HN Measles virus H RSV G VSV G
HPIV3 F-KDEL
HPIV3 F
CD8/E19
2 1 1 1 1 2 2
ND 2 1 1 1 2 2
ND 2 2 2 2 2 2
a Coexpression of HPIV3 F-KDEL, HPIV3 F, or CD8/E19 with selected viral envelope glycoproteins was performed as described in Materials and Methods. Comparison between individually expressed envelope glycoproteins and coexpressed proteins was assayed by IF staining following methanol-acetone (total) or 3.7% formaldehyde (surface) fixation and by biochemical analysis using pulsechase experiments. 1, down-regulated; 2, not affected; ND, not done.
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The results reported herein suggest that there is a common feature present in paramyxovirus and morbillivirus HN/H proteins which can interact with a specific feature in HPIV3 F or F-KDEL proteins. The F-KDEL protein differs from the wildtype F in that the protein is no longer associated with a membrane and is soluble. This finding suggests that the interactive domain, if identical in F and F-KDEL, is not in the cytoplasmic tail or transmembrane anchor and resides somewhere in the ectoplasmic domain of F. The recombinant construction of F-KDEL protein and gene resulted in repositioning a helical domain (leucine zipper motif which is adjacent to the transmembrane anchor in all F proteins examined to date) to the carboxyl terminus of the retention mutant protein. Whether this domain is involved in the F-HN interaction is unknown, but its conservation in all F proteins makes it an ideal candidate for further studies.
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