Fusion glycoprotein (F) of rinderpest virus: Entire nucleotide sequence of the F mRNA, and several features of the F protein

VIROLOGY

164,

523-530

(1968)

Fusion Glycoprotein (F) of Rinderpest Virus: Entire Nucleotide Sequence of the F mRNA, and Several Features of the F Protein KYOKO TSUKIYAMA.’ Laboratory

YASUHIRO YOSHIKAWA,

AND

KAZUYA YAMANOUCHI

Animal Research Center, institute of Medical Science, University Received December

16, 1987; accepted

of Tokyo, Minato-ku,

February

Tokyo 108, /apar,

19. 1988

The full-length cDNA corresponding to the mRNA for the fusion protein of rinderpest virus (RV) was cloned and its complete nucleotide sequence was determined. The mRNA for the F protein was composed of 2359 nucleotides and contained a single large open reading frame which was capable of encoding 566 amino acids with a molecular weight (MW) of 58,929. The RV-F mRNA had a long noncoding region at the 5’ end (586 bases) which was C-rich like the measles virus (MV)-F mRNA but they did not appear to be homologous with each other. Their secondary structure with long G-C stems suggested that they are easily folded. The coding region of RV-F mRNA was significantly homologous with that of MV-F; 74% of the nucleotides and 78.9% of the amino acids were identical. The predicted RV-F protein had a basic amino acid region (104-l 08) which may be cleaved by protease to yield an activated form of F1,2. Three regions (l-1 9, 109-l 33, 418-513) were highly hydrophobic, and the N-terminal hydrophobic region of F, or the positions of cysteines were significantly conserved compared with those of the other paramyxovirus F proteins. Three potential sites for glycosylation existed only in the F2 protein. Several features of the predicted RV-F protein were confirmed in polyacrylamide gel electrophoresis. 0 1988 Academic Press. Inc.

INTRODUCTION

However, the features of rinderpest virus (RV)-F mRNA and its protein are not fully understood. The size of the mRNA of RV-F is approximately 2.4 kb (Tsukiyama er al., 1987) which is almost the same size as that of the MV-F mRNA (Barret and Underwood, 1985). The MV-F mRNA has the longest noncoding region at the 5’ end among the F genes of the other paramyxoviruses and its function is still not clear. In this paper, we cloned the full-length RV-F cDNA and characterized the RV-F mRNA by comparing it with that of the MV-F. Several features of the RV-F protein were also clarified.

The F protein of paramyxovirus causes membrane fusion that joins the virus with a target cell. This phenomenon reflects several biological activities of the virus: virus penetration, hemolysis, and virulence (Homma and Ouchi, 1973; Scheid and Choppin, 1974; Nagai er al., 1976). Immunization with purified paramyxovirus F protein exhibited a protective activity against the viral challenge (&vell and Norrby, 1977; Norrby eta/., 1986). Moreover, atypical measles which occurred naturally in those who had received the inactivated measles vaccine previously have been claimed to be relevant in the failrrre of antihndy t-esponse against r protein (Met-z et a/., 1980: Norrby et a/., 1975; Norrby and Penttinen, 1978). Thus, the F protein may have a significant role in viral pathogenesis. The F protein is synthesized as an inactive precursor, FO, that is cleaved by a host cell proteolytic enzyme at neutral pH to form the biologically active protein consisting of the disulfide-linked subunits F, and F, (Scheid and Choppin, 1977). After the cleavage, the hydrophobic N-terminus of the F, protein, which plays an important role during the virus-induced fusion, is exposed. The amino acid sequence of F, is conserved among paramyxoviruses including simian virus 5. Sendai virus, Newcastle disease virus, parainfluenza virus, and MV (Paterson et a/., 1984; Blumberg et al., 1985; Chambers et al., 1986; Spriggs er al., 1986; Suzu et a/., 1987; Richardson et al., 1986). ’ To whom

rcques?s

for reprints

should

MATERIALS

AND METHODS

Cells and virus Vero cells and Cos-7 cells were scribed previously (Tsukiyama et strain of RV which was adapted to was used at the 1 1th passage level

maintained as deal., 1987). The L grow in Vero cells (Ishii et a/., 1986).

Construction of a cDNA library from RV mRNA and Northern blot analysis The cDNA library was constructed from RV-infected Vero cell mRNA according to Okayama-Berg’s method (Tsukiyama et a/., 1987). Northern blot hybridization with RV-infected Vero mRNA was performed with a cDNA probe of canine distemper virus (CDV) under low stringent conditions or with RV under high stringent conditions, as described previously (Tsukiyama et al., 1987).

bc addressed. 523

0042~6822/88 Cccyvyht

$3.00

0 1988 by Academic

Press. Inc.

TSUKIYAMA,

524

YOSHIKAWA,

AND YAMANOUCHI

IDEAS (Integrated Database and Extended Analysis System for Nucleic Acids and Proteins) were used on a Vax-1 l/750 (Biosequence Analysis System, University of Tokyo) or Floating Point System (FPS)-M64/30. Isotope labeling of virus-infected cells and radioimmunoprecipitation (RIP) assay Confluent monolayers of Vero cells were infected with RV at an m.o.i. of 0.5 PFU/cell. When 50% of the cells showed cytopathic effects, the medium was replaced with methionine-free MEM containing [“%Imethionine (50 &i/G-cm dish), and the culture was incubated for 6 hr. The monolayers were lysed with RIPA buffer and allowed to react with monoclonal CDV-F and RV-F antibodies (Hirayama et a/., manuscript in preparation). The RIP was performed as described previously (Sato et al., 1981; Hirayama et al., 1985; Yoshikawa et a/., 1983). To detect glycoproteins, RV-infected cells were labeled with [3H]glucosamine (100 &i/G-cm dish) in glucosefree MEM. FIG. 1. Northern blot analysis of poly(A)+ mRNA from RV-infected (lanes 2-3) or uninfected (lane 4) Vero cells with labeled cDNA clones: CDV-F (lane 2), RV-F (lane 3), and 3ZP-labeled RV mRNA (lane 1). The filters were exposed for 2 days (lane 3) or 2 weeks (lanes 1, 2).

Expression

of cloned genes in Cos-7 cells

The biological activity of the isolated cDNA clones was examined by transfection using a fluorescent antibody (FA) test with monoclonal anti-CDV-F antibody which cross-reacts with RV-F protein (Hirayama, manuscript in preparation), or RV-infected rabbit serum at 3-4 weeks postinoculation (Tsukiyama eta/., 1987). DNA sequence

analysis

The coding region of cDNA was subcloned into the Ml 3 phage, pUCl18 and 1 19, using appropriate restriction endonucleases or Exolll-Mung bean nuclease (Fig. 3). The nucleotide sequence was determined by the dideoxy chain-termination method of Sanger (1970). Since the 5’ noncoding region is easily folded, cDNA was first cleaved into small fragments (~200 bp) by using Aval, Avall, and Banll, and these small fragments were sequenced according to Mizusawa’s method (1986) using 7-deazaguanosine-triphosphate. Computer-assisted

analysis

The DNA and protein analysis system of UWGCG (University of Wisconsin Genetic Computer Group) and

RESULTS Identification of cDNA clones corresponding to RV-F mRNA Since RV-F mRNA (approximately 2.4 kb) was detected specifically by the CDV-F cDNA probe under low stringent conditions (Fig. 1, lane 2) we screened F cDNA using the same probe under these conditions from a library of cDNA clones which was constructed from RV-infected Vero cell mRNA (Tsukiyama et a/., 1987). Three clones exhibited strong hybridization signals and were designated as clones F-2, F-7, and F-l 20. The F-2 and F-7 had an insert of approximately 1.8 kb and the F-l 20 had approximately 2.4 kb. Clones F-2 and F-120 were nick-translated and hybridized with mRNA isolated from RV-infected or uninfected cells (Fig. 1, lanes 3, 4). Under stringent conditions, these clones hybridized strongly with 2.4-kb sized mRNA which was detected by a CDV-F cDNA probe. Dicistronic F-M and H-F mRNA with approximate sizes 4.0 and 4.4 kb, respectively, were also detected by these clones (Fig. 1). Transfection

of the cloned F genes

To examine whether the putative RV-F cDNA contained the coding region of the F gene, clones F-2, F-7, and F-l 20 were transfected into Cos-7 cells. The production of the F protein was shown by the FA test using anti-CDV-F antibody or anti-RV serum (Fig. 2). Therefore, these three clones contained the coding region of the F gene.

RINDERPEST

FIG. 2. The expression of clone F-2 in Cos-7 cells (200X) detected antibody (B), and uninfected cells treated similarly (C)

Nucleotide sequence of RV-F gene and its homology with MV-F gene The nucleotide sequence of RV-F mRNA was determined on clones F-2, F-7, and F-l 20. Their restriction cleavage maps and sequence strategy are shown in Fig. 3. The entire nucleotide sequence of RV-F mRNA was composed of 2359 nucleotides (Fig. 4A). The nine F120 F2,7

0

2.0

525

VIRUS FUSION GLYCOPROTEIN

kbP

FIG. 3. The restriction cleavage map and sequence strategy of clones F-2, F-7, and F-l 20. F gene was sequenced by Ml 3 phage (black arrows), pUCll8 and 119 clones using Exolll-Mung bean nuclease (open arrows), and 7-deaza-GTP (hatched arrows).

by FA test using RV-infected

rabbit serum (A), monoclonal

anti-CDV-F

bases at the 5’ end were completely conserved between the F mRNA of RV and MV (Cattaneo et al., 1987). RV-F gene had three AUG at the 5’ end. The frames which started from the first and second AUG (nucleotides 151 and 558) finished within the short open reading frame (ORF). The QRF from the third AUG (nucleotides 587) was long enough to code for the RV-F protein, and existed in three clones although clones F-2 and F-7 lacked the 563 bases of the 5’end of the F mRNA. Therefore, the coding region of F mRNA was thought to start from the AUG at nucleotides 587-589. The flanking sequence around the AUG appeared to be favorable for the initiation of eukaryotic ribosomes (Kozak, 1983). The coding region of RV-F mRNA was relatively conserved when compared with that of the MV-F mRNA {approximately 74%). Although the nucleotide sequence of their 5’ noncoding region (l-586) had little or no homology (Fig. 4B), their nucleotide constitution was conserved and C-rich (Fig. 5). A possible poly(A) signal, AAAG, was located at the 3’ end of F mRNA followed by the poly(A) sequence (Fig. 4R). Predicted amino acid sequence d RV-F and its homology with F proteins of paramyxoviruses The large ORF (587-2227) encoded a protein of 546 amino acids with a MW of 58,929 a. In the predicted

526

TSUKIYAMA,

YOSHIKAWA,

AND YAMANOUCHI

A 1

AGGGCCAAA~

@.TCACATCG

ACCCMGCCG

CCAGAMGGA

c.ucAccATc

GCCACCCMC

CAMCTGGM

ACCA-Gc

AGCCACCCGC

*GGGcccMA

101

GCCCGGGCCC

ACCCCACGAG

CAGGGCGGM

CCMGGGGCA

GGCCCGCCCC

ATGCA~GCA

ACAGMCACC

CCCAGATCCC

CCAC-G

CCCCCCCGAC

201

ACCGGGCAGA

ACCCGAGGGC

ACCCAGGAGA

ATCCCCCGAC

CCCAGACCCC

CMCAGCAGC

ATCCCCCACC

CACCCCCCCC

GTTCACTCCT

CCCGGMCTT

301

CCACTCCCAC

CCCCTCCCGA

GGAACAGAGG

GCCCCATCCC

CCCCKX+lT

CCCGCCCCGG

TCTCCULGCCC

GAGACAACCC

CCACACCCCA

ACACGGAAGG

401

TCTCGGTCGC

TCAGCGCCGC

CGMGATCCA

GCCAGAMGA

GCACCAAAGG

CAGACCACCG

GGCCCl’GTGG

TGGACCCCGC

CAiXCGGGAG

CCCCAGACCC

501

CCAIVLTCCAT

CCGGGCATCA

AATCACGGCA

GAGCAACCM

GGCACTGCTC

AACAAACATG

CCCAGACCAC

CTACAMGG+

CCTAG&@

GGATCTTATT

601

TGCTGCCCTG

CTAGCAATGA

CTAACCCACA

CTTAGCTACT

GGCCAGATCC

ACTGGGGCM

CCTTTCAAAA

ATTGGAGTCG

TAGGGACMG

TAGTGCCRGT

701

TATMGG'TGA

TGKCCAATC

GAGTCACCAG

TCILCTAGTTA

TRAAGCTGAT

GCCCAATATC

ACTGCTATAG

ACMTTGCAC

f.TAAPACAGAG

ATTATGGAGT

801

ACAAGAGGCT

GTTGGGGACR

GTGCTT&AGC

CTATCAGGGA

AGCTCTTAAC

GCTATTACTA

AGAACATAAA

ACCMTCCAG

AGTTCCACTA

CCAGTAGGAG

901

ACACAAGAGG

TTCGCGGGAG

TCGTTTTGGC

TGGAGCCGCT

CTCGGGGTTG

CAACCGCAGC

CCAGATTACA

GCAGGGATTG

CTCTTCATCA

GTClUTGATG

1001

AATXCCAAG

CTATCGAAAG

TCTTMGGCA

RGTCTGGAAA

CAACUVLTCR

AGCARTTGAG

GAAATACGAC

RAGCGGGTCA

AGAAATGCTC

CTAGCGGTTC

1101

AGGGTGTCCA

AGATTACATC

MCMTGAAC

TGGTGCCTGC

GATGGGCCAG

CTATCGTGTG

AAATTGTGGG

TCAAAAGCTR

GGGCTGAAAC

TGCTTAGATA

1201

TTACACTGAA

ATC-l-ETC?T

TATTTGGACC

CAGCCTCAGG

GACCCGGTCT

CGGCTGAGCT

TTCTAITCAG

GCCTTAAGTT

ACGCCl’TGGG

TGGGGACATT

1301

AATAMATAC

TAGAAMGCT

TGGGTATAGC

GGGAGTGATC

TCCTTGCTAT

ACTGGAAAGT

AAGGGTATCA

AGGCTAAGAT

AACCTATGTG

GATATCGAAA

1401

GTTACTTCAT

TGTGCTCAGC

ATAGCTTATC

CGTCACTCTC

CGWATTAAG

GGGGTGATAG

TGCACCGTCT

GGAGAGTGTC

TCTTACAATA

TAGGATCGCA

1501

GGAGTGGTAC

ACCACAGTAC

CGCGGTATGT

GGCRRCCCAG

GGTTACCTCA

TTTCAAATTT

CGATGACACA

CCCTOTGCAT

TCACTCCGGA

GGGCACTATC

1601

TGCAGTCkUL

ATGCAATATA

CCCGATGAGC

CCATPGCTCC

AAGAATGTTT

CCGTGGGTCA

ACCAGGTCAT

GTGCCCGTAC

ACTGGTTTTG

GGGTCCATAG

1701

GGAATAGGTI

TATACTATCT

AAGGGGAACC

TCATTGGCAA

CTGTGCCTCT

ATTTTGTGCA

AGTGCTACAC

CACTGGCTCA

ATRATAAGTC

AAGivXCl'TGA

1801

CAAGATCCTA

ACATRTATAG

CTGCAGACCA

ATGCCCCGTC

GTAGAAGTAG

GCGGTGTAAC

CATCCAGGTC

GGGAGCAGGG

AGTACTCCGA

TGCAGTGTAC

1901

CTGCATGAAA

TAGATCTTGG

CCCACCAATA

TCGCTAGAGF.

AGCTGGATGT

GGGGACTAAC

CTCTGGAATG

CAGTGACAM

ATTGGAGGAAA

GCCAAGGATC

2001

TACTAGATTC

ATCTGATCTG

ATCCTGGAAA

ACATCAAGGG

TGGTTTCAGTC

ACGAACACAG

G.TTZ,CATTCT

AGTCGGAGW

GGGTTGATTG

CAGTGGPGGG

2101

~MCcTcmv

ATTACCfGTT

GWGTAAGAA

GCGCAGGACT

GACAACAAAG

TCTCCACTAT

GGTCTTGAAC

CCGGGTCTTR

GACCAGATCT

~ACTGGTACA

2201

TCTMMCCT

ACGTACGGTC

G+?T@,@AG .a

GTGTGTmCC

CATGTGATCA

ATGTCCCCGA

ATACCTTATC

AAACCTTACA

ATGCTTTGTC

CTCCTCGCAG

2301

CCAGCTAGTC

GCTWl’CcTcA

GCAGGCACCA

TGCTCGATCG

CTCATTM.TT

GCTACt&,,,

M V

: 20 1 "I

’

_

:

; r I ...L

I

.. ' / .

j 4i

,, ,

,soe .

/,..'

, //I.

i ;_ F j

'.

I ,;

,' lOO0:".

,

.

.

*/I.. .. ,. '.a.,. :: .. .. . ,,i /'. .zc.. c. .'.L . .,';: .;. :; ::; . : 1. _1.<);' "T. * l.:,;:

‘:-;?;:;i;,-

,I.

'/

(,

:

.* 1

:' .,

,;.I

. +

:‘ .:

500.

,,( 1000

1500

dOd

t

( b)

RV FIG. 4. (A) The nucleotide sequence of RV-F gene. The initiation codon and termination codon are shaded and poly(A) signal (k) is indicated. (B) The nucleotide sequence comparison of RV-F and MV-F gene according to the UWGCG program (conditions: window 21, stringency 14).

RV-F protein (Fig. 6), there were three hydrophobic regions which consisted of amino acids l-l 9, 109-l 33, and 484-513. The RV-F had three potential sites for asparagine N-linked glycosylation at the amino acid positions 25-27, 57-59, and 63-65. The

basic amino acid sequences existed at amino acids 104-108and 514-517. Comparison of the sequences between MV-F and RV-F proteins indicates that 78.9% of amino acids were identical (Fig. 6). RV-F protein shared common

RINDERPEST VIRUS FUSION GLYCOPROTEIN 1) RV l-585 hlv I-584

2) RV m-2227 MV 585-2235

527

3) RV 2228-2359 r4v 2236-2377 C% I 50

C% I 60

FIG. 5. Nucleotide frequency in (1) 5’ noncoding region, (2) coding region, (3) 3’ noncoding region of RV-F (-) and MV-F (---) mRNA.

structural characteristics with other paramyxoviruses (Sendai virus, simian virus 5, Newcastle disease virus, human parainfluenza virus 3, bovine parainfluenza virus 3, and MV), especially in the second hydrophobic region (amino acids 109-l 33) and the cysteine positions. Fifteen out of twenty-five hydrophobic amino acids and the positions of 10 cysteines were completely identical to those of the F proteins of the other paramyxoviruses (Fig. 6). Characterization

of RV-F protein

Characterization of the RV-F protein was performed by RIP assay (Fig. 7). Nonreduced F protein had a MW of 67,000 Da and under reducing conditions, F, and F2 separated into a MW of 42,000 and 24,000, respec-

FIG. 7. Radioimmunoprecipitation of the RV-F protein. Lysates of [35S]methionine- (lanes A, B, D, E) or [3t-i]glucosamine- (lanes C, F) labeled RU-infected Vero cells were reacted with anti-RV-serum (A, D) or monoclonal anti-RV-F antibody(B, C, E, F) and electrophoresed in 1270 (A, B. C) or 15% (D, E, F) acrylamide gels. Samples in lanes D, E, F were treated with 2-mercaptoethanol.

tively. The F, protein was not labeled with C3H]glucosamine and the carbohydrate molecules were found in the F2 protein.

The entire sequence of the RV-F mRNA and some features of the RV-F protein were disclosed in this

220 230 240 190* 200 210 170 150 RQAGQEMVLAVQGVQDYINNELVPAMGQLSCElVGQKLGLKLLRYYT~lLSLFGPSl~l~DPVSA~LSiQALSYALGGDlN~ *******I****$*********I*S*N***+DLI******~****~~*~******~*;~*l~*~l******~~~~*~~*~ 210 220 230 240 200 170 180 190 290 310 320 270 280 * 300 260 260 i+RYVATC'GY ILEKLGYSGSDLLAILESKGIKAKITYVDIESYFlVLSlAYPSLSRIKGVlVHRL~SVSYNI~SQ~WYTTV V*~Z*~~***GH**G****R****R**H**H**T*~****~*****T****N********G*~**********~**K**~*~** 310 320 290 300 260 270 280 SQNA,YPMSPLLQE~FRGSTRSt;4RTLVLDSICNRFILSKGNLION~ASlLCKCYTTGSlI 350 360 370 380 =Jo* * 400 *c****ESS*T*M****V***r*Lc*********L****mK***~***S**F*~*****Q****A***********~*T~* 330 340 350 360 370 380

300

400

480 410 * 420 440 460 480 470 430 SQDPDKILTYlAADQCPVVEVGGVTlQVGSREYSDAVYLl~~lDL~PPlSLEKLDVGTNLWNAVTKLKKAKDLLDSSDLlL N**:~*O****~**+H****~*N*********R*P**~**~*R~**~***~*~R*~*****G**~A***D**E**E*~*E~~ .I 6 0 ‘IGO 480 420 430 470 440 490

.500 ,.

,N,K~~~~~~~~~~~~~~~~~~~~~~~~~KK,~~~NKVST,VL~"~,L,,DLT~:,",S,,,,, RSM*~~~~~~~~~~~~~~~~~~~~~RC*-SNK*C~QVGMSR***K***r*+********** 490 500 ." uid. 520 530

640

650

FIG.6. The predicted amino acid sequence of RV-F (top line) is compared with that of MV-F protein (bottom line) as reported by Richardson et al. (1986). Asterisks represent amino acid identity. Three hydrophobic regions are shaded. The basic amino acid sequences (V) which are the cleavage site of protease and the conserved positions of cysteine and proline among the paramyxoviruses (*) are indicated. The potential glycosylation sites are enclosed by rectangles.

528

TSUKIYAMA,

FIG. 8. Secondary

structure

YOSHIKAWA,

AND YAMANOLJCHI

of the noncoding

study. The F protein of RV appears to be cleaved by protease yielding two disulfide-linked polypeptides (F, and FJ. The best candidate for the cleavage site in the predicted RV-F protein is the sequence Arg-Arg-HisLys-Arg (amino acids 104-I 08). This sequence is thought to have a marked sensitivity to trypsin-like protease and may be cleaved to generate F, and F2 subunits. The MW of the unglycosylated F, and F2 protein was estimated from the nucleotide sequence to be 47,072 and 11,857 Da, respectively. Three potential sites for glycosylation existed only in the F2, and

region of RV-F and MV-F mRNA.

this was supported by the results of the RIP assay. The MW of the glycosylated FZ was found to be 24,000 by RIP, whereas the MW of the entire carbohydrate molecules was estimated to be approximately 12,000 by the sequence data. In general, the size of one carbohydrate chain is considered to be 3000-4000 Da. Therefore, these three glycosylation sites may be fully glycosylated (Keil et al., 1979; Horisberger et a/., 1980). The MW of the F, protein predicted by the sequence data is larger than the MW of 42,000 determined by SDS-PAGE. This discrepancy might be due

RINDERPEST

VIRUS FUSION GLYCOPROTEIN

to aberrant mobility in polyacrylamide gels which is probably due to the hydrophobic nature of the protein. Alternatively, the basic amino acids at 514-517 might be cleaved by protease yielding an F, protein with a MW of 43,000 Da. By comparing the F proteins of MV and the other paramyxoviruses, the hydrophobic region at F2 is considered to be a signal sequence and the one at the C-terminus of F, to be a transmembrane region. The hydrophobic region of F, at its amino terminus is highly conserved among paramyxoviruses and is believed to mediate the membrane fusion of paramyxovirus (Scheid and Choppin, 1974; Merz ef al., 1980). The RV-F, protein had this region in common at the N-terminus. The location of cysteine residues in the RV-F. protein was highly conserved (Fig. 6). The F2 protein contained a single cysteine residue (amino acid 64) forming the disulfide linkage between the F, and F2 subunits as indicated by the separation of F,,, proteins in SDS-PAGE under reducing conditions (Fig. 7). The RV-F protein may have the conserved features of the tertiary structure of F protein compared with those of the other paramyxovirus. In morbillivirus F, protein, 80.9, 69, and 70.5% of amino acids were identical between RV and MV, RV and CDV, and MV and CDV (Richardson eta/., 1986; Barret et al., 1987). The size of the F2 protein of CDV is about 120 amino acids longer than those of RV and MV. The secondary structure of RV-F had several hydrophobic regions and a-helix regions (data not shown). After cleavage by protease, the secondary structure of the N-terminus of F, may be changed: the hydrophobicity and a-helix structure increased at the N-terminus of F,. These conformational changes were also observed during the activation of the F. protein of the Sendai virus F,,, by proteolytic cleavage (Hsu et a/., 1981). RV-F and MV-F mRNA have the longest noncoding region at the 5’ end among paramyxovirus F genes. The function of this region is still unknown. There is one short ORF (nucleotides 151-585) but the flanking sequence around its AUG is not favorable for the initiation of eukaryotic ribosomes (Kozak, 1983). The noncoding region of CDV-F mRNA is not long (85 nucleotides) but the 5’ region of CDV-F (l-556) is also C- and A-rich (Barret et a/., 1987). In the 5’ noncoding region of RV-F and MV-F, the nucleotide contents are conserved and C-rich although their nucleotide sequences differ from each other. The most stable secondary structure of this region was estimated according to Zucker’s program (1981) and compared between RV and MV (Fig. 8). They have long G-C-rich stems, which are not shown in the noncoding region of the F genes of the other paramyxoviruses.

529

The secondary structure In the viral mRNA may have various effects on translation (Kozak, 1986). The c-erb-A gene has a similar long G-C-rich 5’ noncoding region which can form the stable secondary structure, and after the removal of this region, the translation efficiency of this gene was increased (Sap et al., 1986). Therefore, the 5’ noncoding region of RV-F and MV-F genes might have a regulatory role.

ACKNOWLEDGMENTS We thank Dr. S. 1. Martrn for hrs krnd grft of the cDNA clone to CDV-F gene and Dr. Miyajima for his kind help with the computerassocrated analysis. This work was supported by grants from the Ministry of Education, Science. and Culture and the Ministry of Health and Welfare of Japan.

REFERENCES BARRET.T., CLARKE, D. K., EVANS, S. A., and RI~,IA, B. K. (1987). The nucleotide sequence of the gene encoding the F protein of canine distemper virus: A comparison of the deduced amino acid sequence with other paramyxoviruses. Virus Res. 8, 373-386. BARRET. T., and UNDERWOOD, B. (1985). Comparison of messenger RNAs induced In cells infected with each member of the Morbilllvirus group. Virology 145, 195-l 99. BLUMBERG, B. B., GIORGI, C., ROSE, K., and KO~AKOFSKY.D. (1985). Sequence determination of Sendar vrrus fusion protein gene. J. Gen. Viral. 66, 317..331, CATTANEO, R., REBMANN, G., SCHMID. A., BACZKO, K.. TER MEULEN, V., and BILLETER, M. A. (1987). Altered transcnption of a defective measles virus genome derived from a diseased human brain. EM/30 /. 6,681-688. CHAMBERS, P.. MILLER, N. S., and EMMERSON, P. T. (1986). Nucleotide sequence of the gene encoding the fusion glycoprotern of Newcastle disease vrrus. J. Gen. Viol. 67, 2685-2694. CHOU, P. Y., and FASMAN. G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. In “Advances in Enzymology” (Merster, Ed.), pp. 45-148. Willey, New York. HI~YAMA, N., SENDA. M., YAMAMCTO. H., YOSHIKAWA, Y., and YA. MANOUCHI, K. (1985). Isolation and characterization of canine distemper virus-specific RNA. Microbial. Irnmunol. 29, 47-54. HONIMA, M., and OUCHI. M. (1973). Typsrn action on the growth of Sendar virus in tissue culture cells. I!(. Structural differences of Sendai viruses grown in eggs and tissue culture cells. /. Viol. 12, 1457-1463. HORISBERGER.M. A.. DESTRIZ. C., and CONTENT, 1. (1980). Intracellular glycosylation of influenza hemagglutinin: Tine effect of glucosamine. Arch. Viral. 64, 9-16. HSU, M., SCHEID, A., and CHOPPIN, P. W. (1981). Activation of the Sendar virus fusion protein (F) involves a conformational change with exposure of a new hydrophobrc region. 1. Viol. Chem. 256, 3557-3563. ISHII, H., YOSHIKAWA,Y., and YAMANOUCHI, K. (1986). Adaptation of the lapinized rinderpest virus to in vitro growth and attenuation of its virulence in rabbits. /. Gen. ‘Vird. 67, 275-280. KEIL. W.. KLENK. H. D., and SCHWARZ, R. T. (1979). Carbohydrates of influenza virus. Ill. Nature of oligosaccharide protein hnkage in viral glycoproteins. /. Viral. 31, 253-256. KOZAK, M. (1983). Comparison of initraticn protein synthesis in prokaryotes, eukaryotes, and organelles. Microbial Rev. 47, 1-45. KOZAK, M. (1986). Regulation of protein synthesis in virus-infected animal cells. Adv. Virus Res. 31, 229-292

530

TSUKIYAMA, YOSHIKAWA, AND YAMANOUCHI

MERZ,D. C., SCHEID,A., and CHOPPIN,P. W. (I 980). The importance of antibodies to the fusion glycoprotein (F) of paramyxoviruses in the prevention of spread of infection. 1. Exp. Med. 151, 275-288. MIZUSAWA,S., NISHIMURA,S., and SEELA,F. (1986). Improvement of the dideoxy chain termination of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 14, 1319-l 324. NAGAI,Y., KLENK,H. D., and Roar, R. (1976). Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. virology 72, 494-508. NORRBY,E., ENDERS-RUCKLE, G., and TERMEULEN,V. (1975). Differences in the appearance of antibodies to structural components of measles virus after immunization with inactivated and live virus. J. Infect. Dis. 132, 262-269. NORRBY,E., and PENTTINEN,K. (1978). Differences in antibodies to the surface components of mumps virus after immunization with formalin-inactivated and live virus vaccines. J. infect. Dis. 138, 672-676. NORRBY,E., UTTER,G., ORVELL,C., and APPEL,M. 1. (1986). Protection against canine distemper virus in dog after immunization with isolated fusion protein. J. Viral. 58, 536-541. (~RVELL,C., and NORRBY,E. (1977). Immunologic properties of purified Sendai virus glycoproteins. 1. lmmunol. 119, 1882-I 887. PATERSON,R. G., HARRIS,T. J. R., and LAMS, R. A. (1984). Fusion protein of the paramyxovirus simian virus 5: Nucleotide sequence of mRNA predicts a highly hydrophobic glycoprotein. Proc. Nat/. Acad. Sci. USA 81, 6706-6710. RICHARDSON, C., HULL,D., GREER,P., HASEL,K., BERKOVICH, A., ENGLUND,G., BELLINI,W., RIMA, B., and LAZZARINI,R. (1986). The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): A comparison of fusion protein from several different paramyxoviruses. Virology 155, 508-523. SAP,J., MUNOZ,A., DAMM, K., GOLDBERG, Y., GHYSDAEL, J., LEUTZ,A., BENG, H., and VENNSTROM,B. (1986). The c-erb-A protein is a

high-affinity receptor for thyroid hormone. Nature (London) 324, 635-640. SATO, T. A., HAYAMI,M., and YAMANOUCHI,K. (1981). Analysis of structural proteins of measles, canine distemper, and rinderpest viruses. Japan J. Med. Sci. Biol. 34, 355-364. SCHEID,A., and CHOPPIN,P. W. (1974). Identification and biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57, 475-490. SCHEID,A., and CHOPPIN,P. W. (1977). Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses. Virology 80, 54-66. SPRIGGS,M. K., OLMSTED,R. A., VENKATESAN. S., COLIGAN,J. E., and COLLINS,P. L. (1986). Fusion glycoprotein of human parainfluenza virus type 3: Nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses. Virology 152, 241-25 I. Suzu, S., SAKAI,Y., SHIODA,T., and SHIBUTA,H. (1987). Nucleotide sequence of the bovine parainfluenza 3 virus genome: The genes of the F and HN glycoproteins. Nucleic Acids Res. 15, 2945-2958. TSUKIYAMA.K., SUGIYAMA,M., YOSHIKAWA, Y., and YAMANOUCHI,K. (1987). Molecular cloning and sequence analysis of the rinderpest virus mRNA encoding the hemagglutinin protein. Virology 180, 48-54. YOSHIKAWA, Y., YAMANOUCHI,K., MORIKAWA,Y., and SAKAGUCHI,M. (1983). Characterization of canine distemper viruses adapted to neural cells and their neurovirulence in mice. Microbial. Immunol. 27, 503-518. ZUCKER,M., and STIEGLER,P. (1981). Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 9, 133-148.