Characterization of a Cleavage Mutant of the ... - Journal of Virology

Vol. 68, No. 10

JOURNAL OF VIROLOGY, OCt. 1994, p. 6770-6774

0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Characterization of a Cleavage Mutant of the Measles Virus Fusion Protein Defective in Syncytium Formation GHALIB ALKHATIB,l* JOHN RODER,1 CHRISTOPHER RICHARDSON,2 DALIUS BRIEDIS,3 RANDALL WEINBERG,4 DARLENE SMITH,4 JILL TAYLOR,4 ENZO PAOLETTI,4 AND SHI-HSILANG SHEN2 Division of Molecular Immunology and Neurobiology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital,

Toronto, Ontario, Canada M5G 1X5'; Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R22; Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada H3A 2B43; and lirogenetics Corporation, Troy, New York 121804 Received 7 February 1994/Accepted 6 July 1994

Membrane fusion caused by measles virus (MV) is a function of the fusion (F) protein. This process is essential for penetration into the host cell and subsequent initiation of the virus replicative cycle. The biological activity of the MV F protein is generated by endoproteolytic cleavage of a precursor protein (FO) into a large F1 subunit and a smaller F2 subunit held together by disulfide bonds. The cleavage site consists of a cluster of five basic amino acids (amino acids 108 to 112) within the predicted primary structure of the F protein. To investigate the role of the arginine residue at the carboxy terminus of the F2 subunit (arginine 112), site-directed mutagenesis was used to construct a cleavage mutant of the MV F protein in which this arginine residue was changed to a leucine residue. The mutated F gene, encoding four out of the five basic amino acids at the cleavage site, was inserted into the genome of vaccinia virus. The resulting recombinant virus was used to study expression of the mutant F protein in infected cells. Analysis of the Leu-112 mutant protein made in infected cells demonstrated that this single-amino-acid substitution resulted in a reduced rate of transport of the mutant protein to the cell surface, despite its efficient cleavage to yield F1 and F2 subunits. However, the electrophoretic mobilities of the Leu-112 polypeptides suggested that the protein was cleaved incorrectly. This aberrant cleavage appears to have abolished the ability of the F protein to cause syncytium formation. The data indicate that the arginine 112 residue is critical for the correct proteolytic cleavage that is required for the membrane fusion activity of the MV F protein. Measles virus (MV) is an enveloped RNA virus which belongs to the Paramyxoviridae family of viruses. The viral envelope contains a nonglycosylated matrix protein and two integral membrane glycoproteins, the hemagglutinin protein and the fusion (F) protein. Inside the viral envelope, there is a ribonucleoprotein complex consisting of a single-stranded RNA genome of negative polarity, a nucleocapsid protein, a polymerase-associated protein, and an RNA polymerase large protein (reviewed in reference 8). The MV F protein is synthesized as an inactive precursor, Fo, which is cleaved in vivo by a host protease to generate two polypeptide subunits, F, and F2, held together by disulfide bonds (13). Generation of F1 and F2 is required for syncytium formation, hemolysis, and virus penetration. Cleavage of Fo is an essential cellular event that results in the exposure of the hydrophobic amino terminus of the F1 subunit and the subsequent activation of cell fusion activity (13). This region of the F1 subunit is believed to mediate the membrane fusion activity of paramyxoviruses (12). The homologous hydrophobic region on the simian virus 5 (SV5) F1 subunit has been shown to be capable of interacting directly with cellular membranes (9). There is a high degree of amino acid homology among the F1 hydrophobic amino termini of paramyxoviruses and the hydrophobic amino terminus of the HA2 subunit of the influenza virus hemagglutinin (17). This region of the molecule has considerable homology with the corresponding amino-terminal

regions of simian immunodeficiency virus, human immunodeficiency virus type 1 (HIV-1), and HIV-2 gp4l (4, 5). The exact nature of the enzyme or enzymes involved in cleavage activation of the paramyxovirus F protein is not known. The site of proteolytic cleavage of the MV F protein has been previously identified by direct amino acid sequencing of cleaved F1 polypeptide (16). The cleavage site consists of five basic amino acids (Arg-Arg-His-Lys-Arg [amino acids 108 to 112]). This sequence is thought to have a marked sensitivity to a trypsin-like protease that cleaves the inactive precursor

A.

5'-GG AGA CAC AAG CTT

iTI

GCG GGA GTA G-3'

B.

Glycosylated F2 Cleavage site Unglycosylated Fl 108 109 110 111 112 1 113 114 115 116 117 118 119 120 Arg Arg His Lys ArgY Phe Ala Gly Val Val Leu Ala Gly Wild-type F AGG AGA CAC AAG AGA lTTr GCG GGA GTA GTC CTG GCA GGT Arg Arg His Lys Leu Phe Ala Gly Val Val Leu Ala Gly AGG AGA CAC AAG CTT iTr GCG GGA GTA GTC CTG GCA GGT

Leull2 mutant

FIG. 1. (A) Synthetic oligonucleotide Leu-112 used as a primer in in vitro mutagenesis reaction mixture. Mutant bases are underlined. (B) Nucleotide and amino acid sequences at the cleavage site of the F protein (11, 16). A portion of the molecule from residue 108 to residue 120 is shown. The F protein is cleaved at arginine residue 112, generating the F1 hydrophobic amino terminus that begins with a phenylalanine residue at position 113 (16). In the MV F protein cleavage mutant, arginine residue 112 was changed to a leucine. an

* Corresponding author. Present address: Department of Medical Genetics and Department of Microbiology and Immunology, University of British Columbia, 2125 East Mall, Vancouver, B.C., Canada V6T 1Z4. Phone: (604) 822-7499.

6770

VOL. 68, 1994

NOTES

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FIG. 2. Immunoprecipitation of MV F proteins expressed by recombinant vaccinia virus. Monolayers of CV1 or NCI-H460 cells were infected with either the wild-type F protein-expressing virus or the Leu-112 mutant protein-encoding recombinant virus. Infected cells were labelled with [35S]methionine and immunoprecipitated with MV F protein-specific antibodies. The precipitates were then analyzed in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel containing 10% (A) or 7.5% (B) polyacrylamide. In panel B, cells were labelled with [35S]methionine for 2 h, after which cell lysates were prepared for immunoprecipitation. A portion of each immunoprecipitate was incubated with endo-3-N-acetylglucosaminidase H (Endo H) at 37°C for 20 h as previously described (3) (CV1 and H460 lanes). MW, molecular weight (in thousands) markers; MV, MV-infected cell lysate; WUF, wild-type F protein-expressing virus-infected cell lysate; Leull2, mutant F protein-expressing virus-infected cell lysate; Fung, deglycosylated F protein.

protein into a biologically active molecule (12, 13). The site of endoproteolytic cleavage of the MV F protein has been found to occur between arginine residue 112 and phenylalanine residue 113 (16). For this communication, we examined the role of the carboxy-terminal arginine residue (arginine 112) of the F2 subunit in cleavage-dependent activation of membrane fusion. Changing this arginine to a leucine reduced the number of basic amino acid residues at the cleavage site and resulted in aberrant cleavage and loss of fusion activity. Construction of the Leu-112 mutant. To examine the importance of Arg-112 in cleavage-dependent activation of the MV F protein, site-directed mutagenesis was used to convert this arginine residue into a leucine (Fig. 1). The Leu-112 oligonucleotide was used as a primer in an in vitro mutagenesis reaction mixture containing the single-stranded DNA of plasmid SK/MV F. Plasmid SK/MV F was generated by subcloning of the MV F BamHI fragment (18) in the double-stranded replicative form of the pBluescript SK(+) phagemid. The Leu-112 mutation was confirmed by DNA sequence analysis, and the fragment containing mutant DNA was subcloned into pRW908 (3). Plasmid pRW908, which directs insertion to the ATI locus, contains the wild-type MV F gene linked to the vaccinia virus H6 promoter. The resulting plasmid containing the cleavage mutant was used for in vivo recombination (15) to generate a recombinant vaccinia virus that contains the altered MV F gene. The Leu-112 mutant protein-encoding recombinant virus was designated vP1072.

FIG. 3. Flow cytometric analysis of cell surface expression of Leu-112 mutant protein. Cells (1, CV1; 2, NCI-H460; 3, Vero) were infected with the indicated recombinant virus and then reacted with anti-Fpure antiserum (primary antibody) for 1 h at 4°C. Unbound antibodies were washed with phosphate-buffered saline, and cellsurface expression of MV F protein was detected with fluoresceinisothiocyanate-conjugated F(ab')2 goat anti-rabbit immunoglobulin G by flow cytometry. Results are expressed as percentages of stained cells positive for the presence of MV F protein at the cell surface.

Analysis of Leu-112 mutant protein made in infected cells. Confluent monolayers of CV1 cells or NCI-H460 cells were infected with the recombinant vaccinia virus at a multiplicity of infection of 10 PFU per cell for 6 h (CV1) or for 24 h (NCI-H460) and then labelled for 2 h with [35S]methionine. Cells were pretreated with rifampin to inhibit the fusion activity observed with vaccinia virus infections (3). Cell lysates were prepared and immunoprecipitated as previously described (1). The wild-type F protein-expressing recombinant vaccinia virus expressed the expected three proteins in infected cells: Fo, F1, and F2 (Fig. 2). Similarly, the Leu-112-encoding recombinant virus synthesized three corresponding protein species, which, however, had migration rates different from those of the wild-type F polypeptides. The mutant Fo and F1 protein bands had faster migration rates than their wild-type counterparts, while the mutant F2 appeared to have a slower gel mobility than the wild type (Fig. 2A). These aberrant electrophoretic mobilities were even more evident on the lower-percentage gel shown in Fig. 2B. The multiple Fo protein bands seen in Fig. 2 have previously been observed (3) and may indicate a possible difference in the extent of glycosylation or differential usage of the glycosylation sites. The presence of multiple Fo bands in infected cells expressing the MV F protein has previously been reported (2, 3). Endo-,B-N-acetylglucosaminidase H analysis of the wild-type and mutant F polypeptides made in two different cell lines revealed that the multiple Fo bands seen in both wild-type and mutant proteins are most likely due to different extents of glycosylation and/or differential usage of the glycosylation sites. Upon endo-,B-Nacetylglucosaminidase H treatment, these bands were resolved into one major protein band (Fung band in Fig. 2B). The gel migration rate of the deglycosylated mutant F polypeptide (Fung) was faster than that of the wild type, indicating a structural difference in the amino acid compositions of the proteins. Unexpectedly, the Leu-11i2-encoded Fo protein was cleaved efficiently to yield F1 and F2 subunits (Fig. 2A). However, all mutant F protein cleavage products had electrophoretic mobilities different from those of the wild type. The altered gel mobilities may suggest that the mutant Fo precursor was cleaved at a new site. The altered cleavage pattern was

6772

J. VIROL.

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FIG. 4. Biological activity of the Leu-112-encoded mutant protein on NCI-H460 cells. Subconfluent cell monolayers were infected (F protein only) or coinfected (F protein plus hemagglutinin protein [HA]) at a multiplicity of infection of 20 PFU per cell for 18 to 20 h. Infected cell cultures were then examined for the presence of syncytia with an inverted phase-contrast microscope at a magnification of X285. UN, uninfected; WTF, wild-type F protein.

observed in two different cell lines infected with the Leu-112encoding recombinant vaccinia virus (Fig. 2B). The Leu-112 mutation may have structurally altered the F protein such that the specificity of the cleaving protease was affected. Previous studies by Guo et al. (6) identified an HIV-1 gpl60 cleavage mutant analogous to the mutant described in this study; however, the HIV-1 gpl60 mutant was defective in cleavage. The cleavage site of HIV-1 consists of only four basic amino acids. It is possible that the presence of all four of these basic amino acids is required for cleavage activation of the HIV-1 gpl60 protein. Similarly, other studies of the SV5 F protein demonstrated that the minimum number of basic amino acid

residues required for cleavage is four. SV5 F mutant proteins with two or three basic amino acids were not cleaved by host cell proteases (10). Cell-surface expression and biological activity of the Leu112 mutant F protein. Intracellular transport of the Leu-112 mutant F protein to the cell surface was analyzed by flow cytometry of three different cell lines infected with the Leu-112 mutant protein-encoding recombinant vaccinia virus. Infected cells were treated with anti-Fpure antiserum. The generation of these antibodies was previously described (3, 17). After the primary antibody treatment, cells were incubated with fluorescein isothiocyanate-conjugated F(ab')2 goat anti-rabbit immu-

VOL. 68, 1994

NOTES

TABLE 1. Hemolysis assay of cells infected with recombinant vaccinia virus encoding the wild-type MV F protein or the Leu-112 mutant protein

Hemolysis (0D540)b

Recombinant virus'

Control virus (vP993) .........

............................

0.045

Leu-112 mutant protein-expressing virus (vP1072) ................. 0.060 Wild-type F protein-expressing virus (vP1067) .0.400 MVC ........................................................... 0.520 a

NCI-H460 cells

were

infected with the indicated recombinant virus at

a

multiplicity of infection of 10 PFU per cell for 20 h. Infected cells were washed with phosphate-buffered saline and overlaid with 1.5 ml of 10% monkey erythrocyte suspension. Hemolysis was quantitated by measuring the optical density of supernatants at 540 nm (OD540). b Values represent the average of three different experiments. Deviations from the mean value were not significant. c NCI-H460 cells were infected with MV at a multiplicity of infection of 5 PFU per cell for 24 h.

noglobulin G and analyzed with an Epics flow cytometer (Coulter). The results of this analysis are illustrated in Fig. 3. Each histogram corresponds to the fluorescence distribution of 104 cells. Forward light scatter and right-angle light scatter were used to set a gate around the population of viable cells, excluding dead and clumped cells. The results shown in Fig. 3 are representative of at least three different experiments for each cell line used. The negative control (top histograms in Fig. 3) represents background staining of cells infected with the control vaccinia virus, which does not contain MV F proteinspecific DNA sequences. The other histograms represent specific staining of cells infected with either the wild-type F protein-expressing recombinant virus (middle) or the Leu-112 mutant protein-encoding recombinant virus (bottom). Positive staining is demonstrated by the shift of the curve to the right side relative to the background. The results indicate that cell-surface staining of the Leu-1 12-encoded F protein was always 10% less positive than that of the wild-type F protein, and there was a twofold reduction in intensity of fluorescence. This indicates that the Leu-112 mutation reduced the efficiency of cell-surface expression and may suggest a possible role of the arginine 112 residue in signalling intracellular transport. Alternatively, the observed differences in cell-surface expression could reflect a difference in the rate of transport of the due to structural changes. To determine whether the Leu-112 mutation affected the

mutant

biological activity of the MV F protein, NCI-H460 cells were infected with the recombinant vaccinia viruses and examined for the presence of syncytia. Cells infected with the wild-type F protein-expressing recombinant virus showed the typical syncytium formation that was enhanced by the presence of the MV hemagglutinin protein (Fig. 4). In contrast, cells infected with the Leu-1 12 mutant protein-encoding recombinant virus did not exhibit any significant cell fusion activity in the absence or presence of the MV hemagglutinin protein (Fig. 4). As expected, uninfected cells or cells infected with the control virus were negative for this biological activity. The syncytium formation results were confirmed by using a hemolysis assay as previously described (3). Cells expressing the wild-type F protein induced hemolysis of monkey erythrocytes, while cells expressing the Leu-112-encoded mutant F protein did not induce any significant hemolysis (Table 1). Recent studies of the hydrophobic amino terminus of HIV-2 gp4l demonstrated that mutations that decreased the hydrophobicity of this region severely reduced the capacity of the virus to induce syncytia (14). The different gel mobility of the Leu-112 F1 protein band (Fig. 2) indicates an aberrantly

6773

cleaved F protein compared with the wild-type F protein. The decreased size of the mutant F1 band may indicate that the aberrant cleavage resulted in a shorter hydrophobic F1 amino terminus. The decrease in the length of the hydrophobic F1 amino terminus could result in the loss of fusion activity. Previous studies with arginine deletion mutants at the cleavage site of the SV5 F protein demonstrated that cleavage of Fo did not necessarily result in biological activity of mutant F proteins (10). Furthermore, Paterson et al. (10) showed that the presence of only four basic amino acid residues at the cleavage site was sufficient for cleavage and fusion activity. Our data are in agreement with those reported by Paterson et al. (10) in that efficient cleavage of the Leu-112-encoded F protein did not result in cell fusion activity. However, the cleavage mutant reported in the present study had four basic amino acid residues remaining at the cleavage site and was still biologically inactive. It is possible that the carboxy-terminal arginine residue at the cleavage site of the MV F protein is critical for the local folding and final conformation of the F protein. Mutating this arginine may have induced a conformational change in the MV F protein that was sufficient to abolish its fusogenic activity. Recent work by Morrison et al. (7) indicated that changing the Newcastle disease virus F1 aminoterminal phenylalanine residue into a leucine abolished cleavage and fusion activity of the F protein. It seems that mutations at or near the cleavage site of paramyxovirus F protein result in modulation of its biological activity. This could indicate a unique amino acid structural requirement at the region of the molecule that is essential for inducing the right protein conformation recognizable by the host cleaving enzymes. In summary, a cleavage mutant of the MV F protein has been characterized. The data show that substituting a leucine for the arginine 112 residue resulted in an aberrantly cleaved molecule that was devoid of fusogenic activity. We speculated that substituting a leucine for arginine changed the F protein conformation, which subsequently altered the specificity of the host cell protease that cleaves Fo into a fusion active molecule. The Leu-112 mutation, in addition to other mutations of the cleavage site, will be useful in studying the specificity of the host cell proteases that cleave paramyxovirus F proteins. Furthermore, the findings reported here should be useful for the design of an effective recombinant MV vaccine. Antibodies generated against this region at the cleavage site that appears to be critical for fusogenic activity may prove very effective against MV infections. We thank Yaser Nimer for typing the manuscript and Jamal Nasir and Helen McDonald for useful comments. This work was supported by the Medical Research Council (MRC) of Canada. G.A. is an MRC postdoctoral fellow, and J.R. is an MRC Scientist. G.A. thanks the Samuel Lunenfeld Research Institute of the Mount Sinai Hospital for support.

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