firus Research, 26 (1992) 167-175 0 1992 Elsevier Science Publishers

VIRUS

167 B.V. All rights reserved

0168-1702/92/$05.00

00836

Characterization of baculovirus-expressed hemagglutinin and fusion glycoproteins of the attenuated measles virus strain AK-C Kazuaki Takehara a, Hiroshi Hashimoto b, Tomio Ri b, Takayuki Mori b and Masao Yoshimura a a Laboratory

of Poultry Diseases, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori, Japan, and b Department of Virology, The Kitasato Institute, Shirokane 5-9-1, Minato-ky Tokyo, Japan (Received

6 July 1992; revision

received

and accepted

11 August

1992)

Summary

With measles virus cDNA of the avirulent vaccine strain AIK-C, two cDNAs of H or F genes were amplified by the polymerase chain reaction. The amplified cDNAs were inserted respectively to the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa califomica (AcNPV). After co-transfection of the transfer vectors with AcNPV DNA to Spodopteru fmgiperdu cells, recombinant baculoviruses were screened by plaque assay, and the viruses containing H-cDNA or F-cDNA were named H-AIK or F-AIK, respectively. By Western blot analyses, the band around 80 kDa and some smaller bands were appeared in the H-AIK infected S. fmgiperdu cells, and the band around 40 kDa was detected in the F-AIK infected cells. Immunofluorescence studies on unfixed S. frugiperdu cells infected with H- or F-AIK recombinants showed that both antigens were transported to the cell surface. When green monkey red blood cells were added to the recombinant infected cells, H-AIK infected cells showed haemadsorption, and cells infected with F-AIK lysed the red blood cells. The recombinant proteins elicited the neutralizing antibodies against measles virus. Baculovirus; Measles virus; Glycoprotein

Correspondence to: K. Takehara, Laboratory of Poultry Diseases, School Animal Sciences, Kitasato University, Towada, Aomori 034, Japan.

of Veterinary

Medicine

and

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Since measles virus was isolated in cultures of human and monkey renal cells by Enders and Peebles (19541, research on the development of measles vaccine has become extremely active. Their isolate strain Edmonston (Enders and Peebles, 19541, a virulent type measles virus was attenuated by special passages in primary sheep kidney cells at 33°C (Makino et al., 1970), then the attenuated measles virus was plaque-cloned once on chick embryo (CE) cells, subjected further two passages in CE cell culture and the strain “AIK-C”, a live attenuated candidate vaccine strain was obtained (Sasaki, 1974). Now the strain AIK-C is used as a live attenuated vaccine of measles world wide (Nakayama et al., 1990). Quite recently the complete cDNA sequence of the strain AIK-C genome was determined (Mori et al., 1992). The strain AIK-C is stable and exhibits no invasiveness to the central nervous system (Sasaki, 1974; Makino, 1983). To investigate the attenuation, the characters of all gene products and genetic elements must be studied. In measles viruses, the envelope glycoproteins of hemagglutinin (H) and fusion (F) play a key role in infection by mediating the attachment of virus to the host cell receptors, and the fusion of virus membrane to the host cell membrane, respectively. In the present study we cloned and expressed the cDNAs of H and F glycoproteins by baculovirus vectors to investigate the strain AIK-C further. A cDNA containing H and F genes of AIK-C was used as a template for amplification (Mori et al., 1992). To clone cDNAs of H and F gene (H cDNA and F cDNA1, a polymerase chain reaction (PCR) procedure was used with the cDNA and four synthetic oligonucleotides (H-ATG + and H-TAG- for H gene, F-ATG2 and F-TAG- for F gene). The H-ATG + sequence, 5’-ttgggatccgaccATGTCAC___ CACAACGAGACCGGATA-3’ was a 37-base-long oligonucleotide, corresponding to the 5’ end of the sense RNA strand of the H gene (upper case> which includes the initiation codon for the H protein with BamHI restriction enzyme site (underlined). The H-TAG- sequence, 3’-GGTTAGCGTCTATCctaggaga-5’ was a 22-base oligonucleotide in which 14 bases from 3’ end (upper case) are equivalent to viral RNA, complementary to the 3’ end of the sense RNA strand of the H gene. The F-ATG2 sequence, 5’-ttcagatctgacCATGGGTCTCAAGGTGA-3’, and the F-TGA-, 3’-TTAGGATACATICCAGCGAGACTctagagcc-5’ were prepared with initiation or stop codons and BglII restriction enzyme site (underlined). The polymerase chain reaction was carried out using Gene Amp RNA PCR Kit (Perkin Elmer Cetus, Norwalk, CT, USA). For the PCR the mixture was submitted to 30 cycles of amplification involving heating at 95°C for 1 min, 30°C for 1 min, 40°C for 0.5 min, 50°C for 0.5 min, and 72°C for 1 min. After phenol extraction and ethanol precipitation, the PCR products were purified by agarose gel electrophoresis. The amplified H cDNA was digested with BamHI and the F cDNA was digested with Bgf II, and the resulted fragments were inserted into the BamHI site of the transfer vector pAcYM1 (Matsuura et al., 1987) to place those cDNAs under the control of the polyhedrin promoter. The resulting recombinant vectors were named pAcYM1 MVH and pAcYM 1MVF respectively. To obtain recombinant viruses that would express foreign genes S. frugiperda cells were co-transfected with the recombinant transfer vectors and infectious AcNPV DNA using Lipofectin (Bethesda Research Laboratories, MD, USA) as

169

described (Inumaru and Yamada, 1991). Then the cultures were harvested, plaque-assayed, and polyhedrin-negative plaques were selected as recombinant viruses. Recombinant virus H-AIK was obtained from co-transfection with the pAcYMlMVH plasmid DNA, likewise recombinant F-AIK from pAcYM1MVF. S. frugiperdu cells were infected with viruses at a multiplicity of 5 plaque-forming units (p.f.u.)/cell, in 35mm dishes and incubated at 28°C. At various time the cells were harvested, boiled for 5 min in dissociation buffer (2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM Tris-HCI, 0.01% bromophenol blue, pH 6.8) and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a discontinuous gel of 10%. Direct analysis of stained cell extracts recovered from S. frugiperda cells infected with H-AIK or F-AIK recombinants up to 96 h postinfection failed to reveal the presence of measles virus glycoproteins above background, except a faint band around 65 kDa in H-AIK infected cells (data not shown). So immunoblotting “Western” analysis was carried out as described (Takehara et al., 1990). Anti-AIK-C rabbit serum was prepared by immunizing rabbits with polyethylene glycol concentrated AIK-C virus. Bound rabbit antibodies were detected with goat anti-rabbit immunoglobulins conjugated to horseradish peroxidase (Pel-Freez, AR, USA), using 4-choloro-1-naphthol (Merck, Darmstadt, Germany). The immunoblotting showed several bands (80 kDa, 65 kDa, 46 kDa, 42 kDa, and 25 kDa) in cells infected with H-AIK recombinant virus, and one 40 kDa band in cells infected with F-AIK (Fig. 11. These several bands in H-AIK cell lysates should be H gene productus, because no band corresponding to those bands were seen in

MW

H-AIK 12341234

F-AIK

Ac Mock

117.2 75.5 48.0

28.2

Fig. 1. Time course analysis of proteins expressed by recombinant viruses. S. frugiperda cells were infected with recombinant viruses on wild type AcNPV. The cells were harvested at 1, 2, 3, and 4 days post-inoculation (at 4 days post-inoculation for AcNPV and mock infected cells), lyzed, resolved by gel electrophoresis, blotted to nitrocellulose membrane, detected immunologically with a rabbit serum against the strain AIK-C measles virus. Molecular weight markers (MW) were used to provide size estimation of the products. The positions of H and F proteins are indicated.

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F-AIK, AcNPV, or mock-infected cell lysates. Although due to institutional regulations no radioactive experiments can be undertaken, the 80 kDa and 65 kDa bands appeared in the H-AIK infected cells probably corresponded to glycosylated and non-glycosylated H-protein, respectively. The bands around 46 kDa or smaller in H-AIK infected cells probably suggests a degraded form of H protein, because no other open reading frames that code more than 130 amino acids were seen in the H gene (Mori et al., 1992). In the time course experiments, 80 kDa band was faint by 96 h postinfection, and the 65 kDa and 46 kDa band accumulated until 96 h. The 40 kDa band in the F-AIK infected cells is probably the processed F, protein (Fig. 1). It accumulated up to 96 h postinfection. No bands corresponding to the expected F, or F, bands were detected. The measles precursor protein F, is normally cleaved to F, (C-terminal of F,; 40 kDa) and F, (N-terminal of F,; 20 kDa) (Norrby and Oxman, 1990). The presenting F, protein may mean that the precursor protein F, is processed to F, and F, proteins even though F, was not identified. According to the sequence data of the strain AIK-C, F, protein lacks any N-linked glycosylation sites, although F,, and F, have 4 potential glycosylation sites (Mori et al., 1992). Normally, glycosylated proteins were diffused because of partial glycosylation of these proteins, and it is difficult to detect such a protein by Coomassie blue staining or Western blotting. Vialard et al. (1990) suggested that F, protein was not detected by their polyclonal antibodies. By comparison to LCM N (Matsuura et al., 1987) or HBcAg (Takehara et al., 19881, the level of expression of H- or F-proteins was quite low. Some membrane proteins are expressed to high level by baculoviruses (Matsuura et al., 1987; Prehaud et al., 19891, some are not (Schmaljohn et al., 1989; Takehara et al., 1988, 1990). The reasons are not known. For the immunofluorescence analysis, S. frugiperda cells were infected with viruses at a multiplicity of l-3 p.f.u./cell. For non-permeabilized (unfixed) cell analysis, at 48 h postinfection the cells were harvested into microcentrifuge tubes (1.5 ml), rinsed with phosphate-buffered saline (PBS), then the cell number was adjusted to 1.0 X 105/tube, and incubated for 30 min at room temperature with anti-AIK-C rabbit serum. After rinsing with PBS, the cells were incubated for 30 min at room temperature with an appropriately diluted, fluorescent isothiocyanate (FITC)-labeled goat anti-rabbit immunoglobulin, then rinsed with PBS before analysis. The cells were spotted onto a heavy Teflon-coated slide (Bokusui Brown, NY, USA). Observation at 495 nm demonstrated measles virus protein in unfixed H-AIK (Fig. 2A) or F-AIK (Fig. 2B) infected cells, but not in AcNPV (Fig. 2C) or mock-infected cells (not shown). The immunofluorescence results indicated that the expressed proteins were processed and transported to the cell surface membrane. To investigate the biological characters of expressed proteins haemadsorption tests and haemolysis tests were performed. S. fmgiperda cells in 35-mm dishes infected with viruses at a multiplicity of 5 p.f.u./cell were washed with PBS at 48 post-inoculation and 0.5 ml of a 1% suspension of African green monkey erythrocytes were added to each dish. The cell monolayers were then examined by phase contrast microscopy for the presence of adsorbed erythrocytes. Within 5 mitt, haemadsorption was observed in H-AIK-infected cells (Fig. 3A), but not in F-AIK,

Fig. 2. Immunofluorescence of recombinant virus and AcNPV infected S. frugiperda cells. S. frugiperdu cells infected 48 h previously with H-AIK (A), F-AIK (B), or AcNPV (Cl were reacted with a rabbit serum against the strain AIK-C measles virus for indirect immunofluorescence analysis as described in Methods.

AcNPV (Fig. 3B), or mock-infected cells. One milliliter of a 10% suspension of African green monkey erythrocytes was mixed with 200 ~1 of virus-infected S. fmg@erda cells (1.5 x lo6 cells). After 12 h incubation at 37°C cells were sedimented by low-speed centrifugation, and the amount of haemoglobin released was quantitated by spectrophotometric measurement at 540 nm. A remarkable haemolysis was detected only in F-AIK infected cells as seen in Fig. 4. As shown above,

Fig. 3. Haemadsorption of S. frugiberda cells infected with H-AIK recombinant. S. frugiperda cells infected 48 h previously with recombinant H-AIK were mixed with African green monkey erythrocytes. (A): H-AIK infected S. frugiperda cells were surrounded by adsorbed monkey erythrocytes. (B): AcNPV infected S. frugiperda cells.

172

F-AIK

H-AIK

AcNPV

Mock

Fig. 4. Hemolysis of erythrocytes by insect cells infected with baculoviruses. African green monkey erythrocytes were mixed with S. frugiperdu cells infected 48 h previously with baculoviruses (F-AIK, H-AIK, AcNPV. or Mock). After 12 h incubation at 37”C, the amount of hemoglobin released was quantitated by spectrophotometric measurement at 540 nm.

the expressed H and F proteins had respectively the haemadsorption and the haemolysis activites characteristic of the authentic H and F proteins of AIK-C. However, fusion mediated by F protein was not detected. Wild et al. (1991) demonstrated using recombinant vaccinia viruses that both H and F proteins of measles virus are required for fusion. Tanabayashi et al. also found that both F and HN proteins of mumps virus were required for cell fusion using COS7 cells (1992). Low pH is required for cell fusion for the expressed G protein of vesicular stomatitis virus (Bailey et al., 1989). At pH 5.4 or lower, AcNPV also produces giant cells (data not shown). Vialard et al. (1990) have demonstrated cell fusion by only the F protein expressed by a baculovirus vector at pH 5.8. In preliminary studies recombinant F-AIK and H-AIK were super-infected to S. frugiperda cells, although no giant cells were detected at pH 5.6. Neutralizing ability of the monospecific antisera against F or H proteins were tested using measles virus strain AIK-C. S. @g@erda cells (5.0 X 10’ cells) infected with recombinant viruses were resuspended in PBS containing 0.1% of Triton X 100 lysed by sonication, mixed with an equal volume of Freund’s complete adjuvant, and then inoculated to rabbits subcutaneously 5 times at intervals of two weeks. Two weeks after the fifth injection, sera were collected, and inactivated at 56°C for 30 min. The rabbit serum immunized in the same manner with a recombinant baculovirus that contains a gene of Rift valley fever virus glycoproteins (Takehara et al., 1991) was used as a control. Neutralization tests were performed by the plaque-reduction method as described (Takehara et al., 1991). Serially diluted antisera were mixed with equal volumes of measles virus AIK-C. After 1 h incubation at 37°C the mixture was inoculated onto Vero cell cultures and the remaining viruses were titrated. The neutralizing antibody titer was calculated at 50% plaque reduction point by Behrens-Karber’s method (Matumoto, 1949). A neutralizing titer of the serum against H protein expressed by H-AIK recombinant was 80, and that of the serum against F protein by F-AIK was

173

48.0

28.2

Fig. 5. Western blot analysis of partially purified H and F recombinant proteins. Partially purified recombinant proteins expressed by H-AIK, F-AIK, or both infected .S. frugiperdu cells as described in Methods were analyzed with immunoblot Western analysis.

10. The titer of control serum was less than 5. However, the neutralizing activity of recombinant F protein was quite low, the titer of F protein seemed to be specific. To analyze the extracellular recombinant proteins, the culture medium was harvested at 4 day post-inoculation, and clarified by low-speed centrifugation, then the recombinant proteins were partially purified by the method of extracellular baculovirus purification as described (Summers and Smith, 1987). The partially purified recombinant proteins were subjected to SDS-PAGE and analysed by an immunoblotting Western analysis. The bands around 80 kDa and 65 kDa were detected in H-AIK-infected culture, but no smaller bands were detected (Fig. 5). No specific band was recognized in F-AIK, AcNPV, or mock infected cultures (Fig. 5). In H-AIK and F-AIK superinfected culture, only H-proteins (80 kDa and 65 kDa) were found. Smaller bands that were evident in H-AIK-infected cell lysates were not detected at all. These data suggested that the expressed H proteins (80 kDa and 65 kDa) were assembled into a particle by budding from the cell membrane and released into the media, so that they could be purified as a band in sucrose gradient, and that the smaller degraded bands could not be assembled into a particle. Nagy et al. (1991) expressed HN protein of Newcastle Disease virus (NDV) using a recombinant baculovirus and synthesized NDV-like envelopes. They suggested that a protein such as the 64 kDa major enyelope

174

glycoprotein of a baculovirus may substitute for the matrix protein of NDV by participating in the generation of the NDV-like envelopes. In this study the export of H proteins into the medium was extremely low. If II or F proteins were expressed in high amount, they would be exported as assembled into particles and should be highly immunogenic.

Acknowledgements We would like to acknowledge Dr. D.H.L. Bishop for constructive review of this manuscript.

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