Fusion (F) protein gene of newcastle disease virus: Sequence and hydrophobicity comparative analysis between virulent and avirulent strains

VIRUS GENES 1:4. 333-350. 1988 0 Martinus Nijhoff Publishing, Manufactured in The Netherlands

Fusion (F) Protein Gene of Newcastle Disease Virus: Sequence and Hydrophobicity Comparative Analysis between Virulent and Avirulent Strains

LONG LE,’ ROBERT BRASSEUR.’ CLAIRE WEMERS,’ GUY MEULEMANS’ AND ARSENE BURNY’.4 ‘Laboruton, of Biological Chemi.wy, Department of Mole&w Biology, Free University of Brussels. ULB, Rhode-St-Genke, Belgium ‘LLaboratoy of Macromolecu1e.s at Ittte~ace, Free Univenity of Brussels. ULB, Brussels. Belgium ‘National Institute for Veterinan, Research, Bru.wls. Belgium 4Facult.v of Agronomy. Gemblow Belgium

Received February 10, 1988 Accepted April 5. 1988

Key words: paramyxovirua molecular cloning, membrane protein, hydrophobic&y. virulence.

Requests for reprints should be addressed to Arsene Burny. Laboratory of Biological Chemistry, Department of Molecular Biology, Free University of Brussels. ULB. Rhode-St-Genese, Belgium.

Abstract

The nucleotide and predicted amino acid sequences have been obtained for the fusion (F) protein gene of the avirulent strain La Sota of Newcastle disease virus (NDV). The F, N-terminus begins with the tripeptide Leu-Ileu-Gly instead of Phe- X-Gly as usually observed in fusion peptide. It was found that the cleavage- activation domain of the avirulent La Sota strain contained single (but no pairs of) basic residues in the sequence Gly-Arg-Gln-Gly-Arg. Hydrophobicity analysis suggested that the cleavage-activation domain became more hydrophobic and could be less accessible for host-specific protease( dibasic residues next to the F, N-terminus were shown to be important for keeping the cleavage-activation site in exposed positioning, suitable for F protein activation. Comparative sequence analysis of the NDV F proteins revealed a striking homology between lentogenic La Sota and mesogenic Beaudette C strains. Furthermore, 58 variable positions were recorded in the NDV F protein, excluding signal sequence; some of these mutations, in the cysteine-clustered region, were surmised to alter virulence.

334 LE ET AL.

Introduction

Newcastle disease virus (NDV) belongs to the paramyxovirus group of enveloped, negative-stranded RNA viruses (1,2). The genome of NDV, whose gene order is reported to be 3’-NP-P-M-F-HN-L-5’ (3), encodes three nucleocapsid-associated proteins: the structural protein NP, and two minor protein components, L and P; and three envelope proteins: the hemagglutinin-neuraminidase glycoprotein HN, the fusion glycoprotein F, and a nonglycosylated internal membrane protein M.

The fusion proteins of NDV and other paramyxoviruses such as Sendai virus, SV5, have been studied extensively and shown to mediate membrane fusion pro- cesses in viral penetration, hemolysis and cell fusion with the formation of syn- cytia (reviewed in 4). Proteolytic cleavage, by trypsin-like host proteases, of the F0 precursor glycoprotein to yield two disulfide-linked subunits F, and F, is neces- sary for the infectivity of paramyxoviruses (5-7). This cleavage occurs in trans- Golgi membranes (8,9) and generates a new N-terminus on the F, subunit that is hydrophobic and highly conserved among different paramyxoviruses: for exam- ple, NDV, SW, Sendai, parainfluenza 3, mumps, measles (10-16). The N-terminal hydrophobic region has been suggested to interact with the target cell membrane in the membrane fusion reaction (11,12). This was confirmed by the finding that the treatment of cells with oligopeptides that mimic the N-terminal hydrophobic region of F, prevents cell fusion and virus penetration (12,17). Recently, the use of hybrid proteins containing the F, N-terminus in a protein translocation study pro- vided direct evidence of interaction of this region with membranes (18).

Many strains of NDV greatly differ from each other in their virulence (7,19). It had been shown that susceptibility of the F,, precursor protein to cleavage and the availability of the required cellular protease among different cell types are important faCtors in tissue tropism and pathogenesis in birds, and correlate strictly with viral virulence (5,7,10,19). The F, glycoprotein of virulent strains of NDV is cleaved in a wide range of host cells whereas that of avirulent strains is cleaved only in a restricted range of cell types such as chorioallentoic membrane cells. But, when treated with trypsin, avirulent strains become infectious with the F,, then cleaved, displaying full biological activity.

The susceptibility of F, of paramyxoviruses to host cell proteases is determined by a high structural specificity of the cleavage-activation domain: it is known as a connecting peptide, next to the F, N-terminal hydrophobic region, consisting of one to five basic amino acids depending on the paramyxovirus (20-23). This connecting peptide joins F, and Fz in the F,, precursor and is probably removed on cleavage by the actions of trypsin-like endopeptidases and an exopeptidase of the carboxy-peptidase B type, by analogy with the well established cleavage activation of influenza virus hemagglutinin HA (24,25). Observations of an acidic shift in the isoelectric point and of a conformational change of NDV F0 in proteolytic activation (26) favor the interpretation of cleavage and removal.

To develop a further understanding ofthe large differences in virulence between NDV strains, we have determined the sequence of the F gene of an avirulent NDV

FUSION PROTEIN GENE OF NEWCASTLE DISEASE VIRUS 335

strain La Sota from cDNA clones of poly A+-RNA isolated from NDV-infected BHK-21 cells. A comparison with those of virulent and moderately virulent strains revealed a significant difference in the connecting peptide at the site of cleavage and a strikingly great homology between La Sota and Beaudette C strains (23). Analysis of the deduced amino acid sequence of La Sota F protein using the Eisenberg method (27-28) showed the absence of a “cleavage-activation area” as compared to that of virulent strains. This method showed also the importance of the presence and the position of dibasic residues for F protein activation.

Materials and Methods

Virus, cells and preparation of poly A’-RNA

The NDV strain La Sota used in this study was a commercial vaccine (Smith, Kline). The virus was grown in the allantoic fluid of 9-11-day-old embryonated eggs from specific-pathogen-free-chicks. Harvest of the allantoic fluid was performed 96 hr postinoculation.

BHK-21 cells were used for preparation of RNA and poly A+-RNAs were isolated and tested as described previously (29).

Construction and identiJication of cDNA clones

The poly A+-RNAs were used as template and, after oligo (dT) priming, cDNA was synthesized by the RNase H method (30). The double-stranded cDNAs were EcoRI methylated, ligated with EcoRI linkers and inserted into the EcoRI site of the bacteriophage hgtl0. This DNA was packaged in vitro and the resulting phage particles were plated out on a high frequency of lysogeny strain of E. coli. a C600 HFL strain (31).

Plaques containing sequences derived from the fusion protein mRNA or polycistronic RNA were first identified with a well-characterized, 3’P-labelled F specific-cDNA of pFII-14/NDV Italian clone (29). &I-&I and HincII-PstI restriction fragments corresponding to the 5’- and 3’ ends of this FII-14 cDNA, respectively, were labelled and used to detect positive plaques containing full-length F gene cDNA.

All general manipulations were performed essentially as described by Maniatis et al. (32).

Sequencing

Known restriction fragments were recovered from acrylamide or agarose gels and subcloned into Bluescribe Ml3 plasmid vectors (from Stratagene). The trans-

336 LE ET AL.

formations were done with JM 109 strain. DNA sequence was performed by the Sanger dideoxy sequencing method (33).

Hydrophobic@ analysis

The mean a-helical hydrophobic moment. <nr,> and the mean hydrophobicity <Hi> were calculated according to the procedure of Eisenberg et al. (27,28,34). Then, a segment of 7 amino acids was moved through the protein sequence and the mean hydrophobic@ and mean hydrophobic moment per segment were calculated. The <un> was plotted versus [Hi] for all possible segments. These two parameters were also plotted as a function of the midpoint of the amino acid segment along the sequence. In most calculations we used the normalized consensus hydrophobic scale (34) as described by Eisenberg, because it is especially suitable for membrane-related proteins.

Results

Gene identljication and structural features of the fusion protein

A cDNA library was constructed from poly At-RNAs isolated from BHK-21 cells infected with NDV strain La Sota as described under Materials and Methods. Among positive plaques identified with a well-characterized 3’P-labeIled F specific cDNA of pFII-14 /NDV Italian clone (29) some clones could likely contain a complete copy of the F mRNA as they hybridized also to the PstI-PstI and HincII- PstI restriction fragments corresponding to the 5’- and 3’-ends, respectively, of the FII-14/NDV Italian clone. F VII-1A clone was chosen for analysis. Its insert was about 2 kb in agarose gel. After restriction analysis, along with Southern blot and hybridization to either 3’P-labelled F-specific probe or its two “P-1abelled restriction fragments (shown above), and based on restriction map of Italian F clone (29) it was found that clone F VII-1A contained a small fragment at the 5’-end in addition to the entire copy of F gene. This fragment could correspond to the 3’-end of the M gene according to the gene order of NDV genome (3). Thus, clone F VII- 1A was considered to derive from a polycistronic transcript 5’-M-F-3’and such transcripts have been shown to exist in NDV-infected cells (3). This assignment was confirmed by the first results of sequencing of 5’- and 3’-ends of F VII-1A insert.

The nucleotide sequence of clone F VII-IA, in the mRNA sense, and its deduced amino acid sequence are presented in Fig. 1. The clone contains 2,114 nucleotides spanning the 3’-end of the M gene and the entire F gene that is flanked by consensus mRNA start and polyadenylation sequences (35-37).

A sequence of 313 nucleotides of the NDV M gene most likely encodes for 66 amino acids (the other two reading frames are blocked frequently). This was con-

FUSION PROTEIN GENE OF NEWCASTLE DISEASE VIRUS

AL% cm TCA TOC MT OTT TTA TCC TTA 08, 000 AT& LF, , , A *co ETC I O , oco O A A TTC OAT CF. ACT TAT CA0 aho L”, Ol” 5.r C ” , A . ” V . , L.” 8.7 L.” f ! f 01” , I . Th, L.” ATI L.” S.7 01” 0,” Ph. A . P x;: Th7 TV 0,” L”. .n l .7 p” 170’

Fig 1. The nucleotide sequence of the partial M gene segment and the F gene of the Newcastle disease virus (NDV) avirulent strain La Sota and the deduced amino acid sequences. The consensus sequences of mRNA start and polyadenylation are underlined (broken line). Star indicates the M-F intergenic nucleotide. Presumptive signal sequence and membrane anchorage segment are underlined (continuous line). The Ft N-terminus is indicated by a bold continuous line and the cleavage-activation domain. by a double line. The arrow indicates the cleavage site between Fz and Ft. Potential glycosylation sites are boxed. The nucleotide changes in F Beaudette C are indicated above the La Sota gene sequence, with the corresponding amino acid substitutions below the F La Sota protein.

338 LE ET AL.

Fig, 1. (Continued)

firmed afterward by comparing with the M sequence available (37). After the M gene consensus polyadenylation signal ITAGAAAAAA and the M-

F intergenic nucleotide C, the F gene starts with the sequence ACGGGTAGAAG and terminates with the sequence TAAGAAAAAA. The are eight A residues in addition to this NDV F polyadenylation signal, assuming that it is a conserved consensus sequence with six A residues as it has been reported in cDNA cloning of NDV genomic RNA(23,35,37). Thus, the NDV F gene is 1,792 nucleotides long. In one long open reading frame, there are two other ATG in the frame at 34 nucleotides downstream of the first ATG triplet (nt. 361-363) as in the case of Beaudette C F gene (23), but this first one is found in the optimal structure for translation in the eukaryotic system (ANNATGG) (38). Assuming initiation at this point, La Sota F gene encodes an F0 protein of 533 amino acids with a molecular weight of 58,828 in nonprocessed form.

The N-terminus of the predicted protein sequence contains uncharged and hydrophobic residues (except for 2 arginine and 1 lysine residues) and could form a signal sequence for translocation of the F protein across the rough endoplasmic reticulum. It is subsequently removed by proteolytic cleavage that probably acts at the site next to residue 31 as predicted by the method of von Heijne (39).

There is a stretch of 32 hydrophobic, or unchanged, amino acids near the c- terminus (res. 495-526) that could represent the transmembrane anchorage domain. The last, more hydrophilic, 27 amino acids are likely to be the cytoplasmic tail of the F protein (22,40) and are thought to interact with the matrix protein M (41).

An internal, highly hydrophobic, region, located in positions 117-142, can be recognized as the known N-terminus of F,. Compared to the sequence of the F, N- terminus of the Hickman strain NDV determined by Edman degradation (12,16), of 20 first residues, there is only one difference: the N-terminal residue is leucine (res. 117) instead of phenylalanine. This hydrophobic N-terminus of F, is pre- ceded by the cleavage-activation site (res. 112-116) that, in the case of avirulent NDV strain La Sota, consists of the amino acid sequence Gly-Arg-Gln-Gly-Arg.

There are 6 potential N-linked glycosylation sites. One is on the F, subunit. Five


others are on F, but one of them is not likely to be glycosylated as it is located in the cytoplasmic tail of the protein.

Hydrophobic@ analysis

We investigated the NDV F protein sequence of strain La Sota and those of Beaudette C and Italian strains (data from 23, 29) using the Eisenberg method (27,28). A segment of seven amino acids was moved through the protein sequence and the mean hydrophobicity <H,> and mean hydrophobic moment <uu > per segment were calculated. When these two parameters were plotted versus the amino acid sequence (Fig. 2 and ref. 42,43) most of the F residues were characterized by values described by Eisenberg as characteristic of globular protein (27,28). The N-terminus segment of F, (117-135) of the three strains is mainly defined by a high hydrophobicity (Hi > 0.5) and a low hydrophobic moment (0 < uH < 0.4) associated with the transmembrane structure (28) (B on Fig. 2) as illus- trated for the seven transmembrane helices of bateriorhodopsin. Just to the left of this transmembrane peptide of Beaudette C strain (A on Fig. 2.2), and of Italian strain (42) a cluster of about 10 residues shows a very low hydrophobicity (Hi < -1) associated to a high hydrophobic moment (uu > 0.7). It represents the cleavage-activation domain recognized by the F activating host proteases that cleave the fusion protein, generating the disultide-linked F, and F, subunits (4,20- 23).

It is interesting to note that the hydrophobicity profile of the avirulent strain La Sota (Fig. 2.1) shows a striking difference in domain A: there is little if any “cleavage-activation domain.” It disappears while gaining about one unit of mean hydrophobicity <Hi>. Its mean hydrophobic moment <uu> has lost about 0.1 unit as compared to that of virulent strains.

When <uH> was plotted versus <Hi> for successive segments of seven amino acids on both sides of the cleavage site (116-I 17) the F1 N-terminus of all three strains is always present in and around an area of the plot, characteristic of peptides inserted into membranes (B on Fig. 3). The cleavage-activation domain is represented In area A of the plot foi velogenic and mesogenic strains but, in the case of the ic,itogenic strain La Zeta, the area A is empty (A on Fig. 3).

These calculations were applied to available NDV F protein sequences of Queensl: -4, Ulster, La Sota (after Toyoda et al.) (40) D26 (37) Australia-Victoria (22) Herts and Miyaderl (40) sxains. It should be noted that the partial sequence of F of La Sota strain of Toyod; It al. (40) presents some variations as compared to our strain La Sot :ee Discussion). The same results were obtained, namely the presence of the F, h .erminus representation in area B of the plot for all strains, and the presence or absence of the cleavage-activation domain in area A for virulent or avirulent strains, respectively (data not shown). Thus, variations in the sequence of the cleavage-activation domain can greatly affect the hydrophobic@ profile and the <uH> versus <Hi> plot of this domain.

340 LE ET AL.

1

.5

0

1. LA SOTA 2. BEAUDETTE C

Fig. 2. Hydrophobicity profiles. The <uB> and <Hi> of NDV F protein were plotted as a function of the midpoints of the 7-residue-long segments along the F protein sequence (x axis). Protein region in- cluding the cleavage-activation domain and the F,-terminus only is presented. (1) La Sota F protein. (2) Beaudette C F protein, The shaded area A between residues 107- I16 corresponds to the “cleavage- activation domain”; the shaded area B between residues 117-135 corresponds to the hydrophobic N- terminus of F, subunit. Data of Beaudette C F protein sequence was taken from ref. 23.

Fig. 3. Plots of <un> versus <Hi> for a 7-residue-long segment of a sequence spanning both sides of the cleavage site of the NDV F protein. (1) La Sota. (2) Beaudette C. (3) La Sota from Toyoda et al. (40) (4) Italian. Area A corresponds to the “cleavage-activation domain” and area B. the “membrane” region. M: the median of triangle B. The window in Fig. 3.1 shows area A. B and the median M. Calculations for the F sequence of the La Sota strain from Toyoda et al. (and those of Queensland, Ul- ster and Herts strains, data not shown), which is 26 amino acid long (40). were carried out by addition of approximately 10 corresponding amino acids of the La Sota F sequence at their NH; and COOH- ter- mini, assuming that there are no variations in these additional sequences (as observed with the six other strains, Fig. 4).

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342 LE ET AL.

Concerning the “membrane” region of these plots (area B), one can distinguish 2 groups of strains according to the shapes of their representation: the first group consists of La Sota (Fig. 3.1) Beaudette C (Fig. 3.2) and D26 (not shown); the sec- ond one includes Italian (Fig. 3.4), La Sota (after Toyoda et al. (40); Fig. 3.3) Queensland, Ulster, Herts, Miyadera and A-V strains (data of the last five are not shown). In group 1, the plot <uH> versus <Hi> of the peptide segment Ala (120) to Ala (130) lies below the median M. On the contrary, the plot of the corresponding sequence in the strains of group 2 falls on the median. It is possible that these two shapes have implications for the fusion capacity of the F, N-terminus. The only difference is the change of Ser into Gly.

The consensus sequence of NDV F connecting peptide could be established as X-R/K-Q-X-R. In order to estimate the importance of variations in this sequence as well as the importance of basic amino acid pairs, we set up combinations of hypothetical connecting peptides from those of La Sota and Italian strains, using variable positions. The <l.tH> was plotted versus the <Hi> for a segment of seven amino acids moved on both sides of the cleavage site with different hypothetical connecting peptides. Results are shown in Table 1. In this molecular context, Gly residue -5, counting from the cleavage site, does not induce change but Gly -2 can alter the hydrophobicity profile of the connecting peptide. Futhermore, it appears that Arg (-1)-Arg(-2) alone, can maintain the cleavage-activation domain in area A. This suggests that Arg in position -1, -2 are more important and that this pair of basic residues is required to keep hydrophobic features of the cleavage-activation domain unchanged and allow cleavage. Presence of leu instead of Phe, or vice versa, in the F, N-terminus had no effect on the graphic representation.

Table 1. Hydrophobicity analysisa of the cleavage-activation domain with different hypothetical connecting peptides

Combinations of connecting peptides Area A Area B

I. Based on La Sota sequence

1.-G RQ G R’L- (LaSota) -b +b 2.-RRQGR L- kc + 3.-GRQR R L- + + 4.-RRQRR L- + +

II. Based on Italien sequence 4 5. - R R Q R R F - (Italian) +b +b

6.-GRQRR F- + + I.-R RQGR F- + + 8.-G RQGR F- +

a<uH> was plotted versus <Hi> for a segment of 7 amino acids moved on both sides of the cleavage site (4) with different hypothetical connecting peptides (see text and Fig. 3). Results are represented as presence or absence of a portion of the graph in areas A and B of the plot. b+,-: indicate that the graph enters (+) or not (-) area A or area B of the plot. =+: graphic representation touches lightly the surrounding of area A.


Discussion

Homologies and variations in jiuion proteins of virulent and avirulent NDV strains

The F gene of NDV strain La Sota, derived from a polycistronic transcript 5’-M-F- 3’, was cloned and sequenced. It is 1,792 nucleotides long and encodes for a protein of 553 amino acids whose overall hydrophobicity profile is similar to those of other paramyxovirus F proteins.

To obtain information concerning the difference in virulence and the relation- ship between lentogenic, mesogenic, and velogenic strains, the amino acid sequence of the F protein of NDV La Sota, a lentogenic strain, was compared to those of lentogenic duck strain D26 (37) of mesogenic strain Beaudette C (23) and of velogenic strains Australia-Victoria (22), Italian (29), Miyadera (40) (Fig. 4).

The comparison revealed a striking homology between La Sota and Beaudette C strains with 97.83% of amino acid identities and 97.26% of nucleic acid identities (Fig. 1). And, unexpectedly, lentogenic strain D26 is more closely related to velogenic strain A-V (23 different amino acids) than it is to La Sota (37 different amino acids), as if a lentogenic strain could derive from a mesogenic strain as well as a velogenic strain.

From data shown in Fig. 4,58 variable positions are recorded in NDV F protein, excluding the signal sequence. They could be divided into live categories: 1) Three subsitutions (112, 115, 117) are present only around the cleavage site of La Sota and D26, 2 of which may be directly responsible for the avirulence of lentogenic strains. 2) Four substitutions are found only in La Sota, D26 and Beaudette C strains. 3) La Sota and Beaudette C strains have exclusively 12 common substitutions. Some of the 16 variations in (2) and (3) may be of some significance to the moderation of virulence in Beaudette C strain (see below). 4) Thirty substitutions are distributed among these NDV strains (4,2,2,3,7 and 12 for La Sota, D26, Beaudette C, A-V, Italian and Miyadera strains, respectively). Strain Miya- dera is relatively more distant from the others as it has 12 new amino acid substitutions. These 30 variations presumably reflect strain variations or, both strain variations and differential pathogenic@. 5) Finally, nine other positions are found with substitutions that are not likely to be related to virulence features.

In all lentogenic strains (except La Sota strain from Toyoda et al. (40)) and in La Sota strain shown here, the F, N-terminus begins with Leu-Ileu-Gly whereas the common structure observed in paramyxovirus is the tripeptide Phe-X-Gly. That Phe-X-Gly, as well as the following amino acids in F, N-terminus, is directly involved in mediating cell fusion was supported by the finding that homologous synthetic peptides inhibit fusion and virus penetration (12). Recently, Gallaher has detected such a tripeptide, in tandem, within the N-terminal hydrophobic region of the transmembrane protein gp41 of human immunodeficiency virus (HIV) that is also able to cause cell fusion and cytolysis (44). However, lentogenic NDV strains can also exhibit fusion activity and replicate efficiently if they are treated with trypsin. Thus, Leu can replace Phe in the tripeptide Phe-X-Gly in fusion activity but this change might well contribute to low virulence character. In experiments using infrared spectroscopy and conformational analysis, F, N-

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Fig. 4. Comparison of the predicted amino acid sequences of six NDV F proteins. LS: La Sota. Data of Beaudette C (BEA). Australia-Victoria (A-V), Italian (ITA), Miyadera (MIY) and D26 strains were taken from ref. 23.22.29.40 and 37, respectively. Dashes indicate identity with La Sota strain; stars: common cysteine residues; circles: common potential glycosylation sites; arrow: the presumptive cleavage site of signal sequence; double arrow: the cleavage site between Fz and Ft.


terminus of NDV strain Italian and the N-terminus of gp41 of HIVs have been shown to adopt an oblique orientation with respect to the lipid acyl chains (42,43). This unusual orientation is expected to induce the disorganization of the surrounding lipids that has been suggested as one of the initial events in the virus-cell fusion. It would be of interest to know how the F, N-terminus of the lentogenic strain La Sota could lie in lipid membrane since it has some differences in the “membrane” region (Fig. 3.1, area B) due to the presence of Leu and Gly instead of Phe at the N-terminus and Ser (124) respectively (Fig. 4). Comparative inhibition studies of different strains with synthetic oligopeptides corresponding to the two distinct F, N-terminal sequences are underway (40) and will be helpful for understanding the role of the F, N-terminal region in the fusion mechanism.

As noted above, a partial sequence of F of La Sota strain published by Toyoda et al. (40) presents some variations as compared to the F sequence of the La Sota strain in this study (Figs. 1 and 4) and to the partial La Sota F sequence published by Glickman et al. (45). At positions 115, 117, and 124, Ser, Phe and Ser, respectively, are found instead of Gly, Leu and Gly in our strain and in that of Glickman et al. (45) These differences should be strain variations and it will be interesting to discover whether the full sequence of F protein of La Sota from Toyoda et al. (40) is available for comparison since it is the only lentogenic strain that has Phe at the F,N-terminus and its cleavage activation domain comes closer to the area A than in the case of our strain in a <uH> versus <Hi> plot (Fig. 3.1, 3).

Hydrophohicity analysis of the cleavage-activation domain

The susceptibility of F,, proteins of the NDV strains to proteolytic cleavage cor- relates well with their virulence and may be dependent on the presence of a highly basic peptide at the cleavage site (7,19). The cleavage-activation site of virulent strains consists of a double set of two dibasic residues with an intervening glutamine, Arg-Arg-Gin-Arg-/Lys-Arg (22,23,29,40) whereas sequencing data of our study and others (37,40) show that the connecting peptide of avirulent strains contains single basic residues, Gly-Arg/Lys-Gln-Gly/Ser-Arg. It has been shown that Sendai virus, often isolated with uncleaved F protein from certain cell types (6) has only a single Arg at the cleavage site (21). For avian influenza virus, avirulent strain HA protein contains a single Arg while, in virulent strains, a highly basic peptide precedes the cleavage site (25).

Hydrophobicity profiles, calculated according to the procedure of Eisenberg et al. (see Materials and Methods: 27,34). show a “cleavage-activation domain” in virulent strains (Fig. 2 domain A) and that the “cleavage-activation domain” tends to disappear in avirulent strains. When <uH> was plotted versus <Hi> for a sequence spanning both sides of the cleavage site, cleavage-activation domains of

346 LE ET AL.

virulent strains locate in area A and those of avirulent strains stand outside of this domain (Fig. 3). The same observations have been reported previously in the study of apolipoprotein E where graphic representation of “receptor binding domain” is found to be affected in apoE mutants (46). In addition, hydrophobicity calculations, when applied to hypothetical connecting peptides (Table I), indicate that the presence of a pair of basic residues in positions -1, -2 is more important and they are required for keeping the hydrophobic feature of the cleavage- activation domain unchanged and thus allow cleavage. This should correlate well with the fact that the cleavage-activation site of the human parainfluenza 3 virus F protein consists of Pro-Arg-Thr-Lys-Arg and the pair of Lys-Arg (-l,-2) could per- mit efticient cleavage (15).

The absence of the cleavage-activation domain of the F protein in area A (Fig. 3.1,3) corresponds to nonvirulence of NDV where F is found uncleaved in many cell types. As the cleavage-activation site of the lentogenic strain gets two glycines and becomes more hydrophobic, it could be folded into the protein and therefore could be less accessible to host-specific proteases. On the other hand, one can im- agine the inverse situation where the change of glycine to arginine (or lysine), in the cleavage-activation site of the virulent strain, increases the local hydrophilic@, thus bringing the cleavage-activation site into a more exposed position and facilitates the action of proteases. It has been found that mutations near or at the cleavage site of selected mutants of Sendai virus might alter local polypeptide conformation, allowing the proteases to have access to existing sites (47). However, the F, protein of avirulent strains can be activated in embryonated eggs and in chorioallantoic membrane cells or by trypsin. Perhaps a protease in these tissues has more similar activity to that of trypsin that cleaves after basic residues than the other protease( present in many cell types, that also have trypsin-like activity but could be more specific and show more restricted accessibility to substrates.

Alternatively it has been presumed, for fowl plaque virus, that the HA precursor is cleaved by an ubiquitous trypsin-like protease that requires a pair of basic amino acids, and that other influenza HA proteins that are activated only in a few host systems would be cleaved by a different enzyme that can act at a single arginine (48).

It should be noted that there are additional dibasic residues in all NDV strains shown in Fig. 4 (positions 100-101). They are probably not involved in proteolytic activation since avirulent strains have the same dibasic residues and their F0 Pro- teins are cleaved only in a limited host range. In addition, hydrophobic@ analysis showed that the graphic representation of the La Sota sequence containing these two Arg lightly touched the surrounding of area A in a <uH> versus <Hi> plot (data not shown). These additional dibasic residues could lie in a position not accessible for specific proteases. Moreover, it has been found that an avirulent strain of H5N2 avian influenza differs from a virulent strain of the same type by the presence of an Asn-linked carbohydrate in the vicinity of the connecting peptide. The


latter is the same in both viruses and is highly basic (Lys-Lys-Lys-Arg) (49). The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. Presence of carbohydrate may mask a recognition signal required by the cleavage enzyme or perturb a structural feature so that it no longer serves as a sub- strate for the processing enzyme (49).

It would be of great interest to obtain 3-dimensional structural information from crystallography studies to understand how the hydrophobic F protein is folded and to get further insight into the cleavage-activation process as well as fusion mechanism.

Eventual alteration of virulence

The difference between lentogenic and velogenic characters resides in the crucial cleavage-activation domain on the F protein. But how can we explain the mesogenic character (moderate virulence) of Beaudette C strain? Beaudette C strain has a cleavage site susceptible to proteolytic cleavage (Arg-Arg-Gln-Lys- Arg) that has little if any difference compared to velogenic strain Italian. Com- parison of F amino acid sequences reveals a great homology between La Sota and Beaudette C strains. Inspection of F protein sequences, excluding signal sequence, shows that La Sota, D26, and Beaudette C strains have 4 common substitutions, and La Sota and Beaudette C have 12 common changes (Fig. 4). These variations may be of some significance to virulence and pathogenicity. Recently, Portner et al. (50) in localizing an epitope involved in fusion and virus neutralization, by selecting Sendai antigenic variants with anti-F monoclonal antibody and sequencing their Fgene, have found a single identical mutation in all these variants: a proline to glutamine substitution at residue 399 (407 in our sequence) that is far removed from the hydrophobic F, N-terminus and located in a structurally and functionally important region where most cysteine residues are clustered (21,51) (res. 338-424 in our sequence). These variants changed in their plaque morphol- ogy and made small plaques of uniform size. This mutation has altered the ef- ficiency of the fusion function and thus limits virus cell to cell spread. Looking back to NDV F sequences (Fig. 4) in the region corresponding to the location of the mutation of Sendai variants, there are two common changes in La Sota and Beaudette C strains: Ala to Val and Asp to Asn substitutions (402,403). They are next to cysteine and proline residues that are likely to be involved in protein conformation. Not far from these substitutions, another one is found at position 422 of lentogenic and mesogenic strains (La Sota, D26, and Beaudette C): His to Gin substitution, also next to a cysteine residue. As the attenuation of the cytolytic effect in Sendai antigenic variants is due to single mutations, we surmise that one of the three mutations, or all three located in the same cysteine-clustered region in the F protein of lentogenic and mesogenic strains La Sota, D26, and Beaudette C, might have a causal relation to the moderate virulence. Further studies are required to

348 LE ET AL.

ascertain this relation and to develop further an understanding of the virulence and the pathogenicity since they could be altered by variations in other regions of the F protein (see above, variations in F protein sequence) and/or in other viral constituents such as the matrix protein M in which mutation can result in de- creased infectivity (41). Sequencing of F protein genes of NDV strain Italian antigenic variants, selected with monoclonal antibodies (52) with different pathogenicity, is underway and would be helpful for further understanding of the structure and function of the F protein as well as pathogenicity.

In summary, we have presented the nucleotide sequence of the fusion protein gene of the NDV avirulent strain La Sota and the resulting predicted amino acid sequence. Comparison of the F protein sequence with those of other NDV strains reveals a great homology between lentogenic La Sota and mesogenic Beaudette C strains, By analogy with a small plaque induction of a mutation in Sendai antigenic variants, we surmise that mutation(s) in the same region in the NDV F protein might alter the virulence of NDV. Hydrophobicity analysis showed the absence of a “cleavage-activation domain” in avirulent strains probably reflecting the nonaccessibility of protease to cleavage site. They also showed that dibasic residues next to the cleavage site are required for F protein activation.

Acknowledgments

We thank D. Espion, C. Neyt for stimulating discussions and P. Duquenoy for help in the preparation of graphs. This work was supported by a contract from the Institutpour I’Encouragement de la Recherche Scientljique dans I’lndustrie et I’Argricul- ture (ZRSL4)- Smith Kline Animal Health. L.L. held a fellowship from Relations Cultwelles Internationales de la Communaute’ Franqaise de Belgique and received financial support from the Foundation David and Alice van Buren. C.W. is “bour- siPre IRSIA” and R.B. is “chercheur qualifie’ au Fonds National de la Recherche ScientiJique.”

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Documents

Fusion (F) protein gene of newcastle disease virus: Sequence and hydrophobicity comparative analysis between virulent and avirulent strains