Virus Research, 17 (1990) 119-130
Elsevier
119
VIRUS 00603
Hemagglutinin-neuraminidase ( HN) amino acid alterations in neutralization escape mutants
of Wham mumps virus
Jan Kiivamees, Robert Rydbeck, Claes hell and Erling Norrby
Department of Virology, School of Medicine, Karolinska Institute, Stockholm, Sweden
(Accepted 26 June 1990)
Summary
The hemagglutinin-neuraminidase genes of the Kilham strain of mumps virus and three neutralization escape mutants (Mll, Ml2 and M13) of this strain (Love et
al., 1985a) were sequenced using their genomes as template. The predicted amino acid sequences were compared. While one mutant had only one amino acid substitution the other two mutants had four and five respectively. A putative region for the epitope of the selected neutralizing monoclonal antibody was identified in a
hydrophilic region encompassing amino acids 352-360, since the single amino acid substitution of one mutant occurred in this region and the other two mutants
showed non-conserved amino acid changes in this part of the protein. The previ- ously sequenced prototype strain RW, which lacks capacity to react with the selected neutralizing monoclonal antibody also has one non-conserved amino acid change in the region of the proposed neutralizing epitope. The three mutants showed different biological characteristics. These particular characteristics were therefore interpreted to be primarily associated with strain-specific amino acid
changes outside the region of the presumed neutralizing epitope. The decrease in molecular weight in one mutant (Mll) was shown to be due to a substitution in position 329 of an asparagine for an aspartic acid, leading to abolishment of a potential N-linked glycosylation site. In the other mutants, one substitution in
position 239 of a lysine for a methionine was correlated with an increased neur- aminidase activity of strain M12, while a substitution in position 360 of an arginine
Correspondence to: J. KBvamees, Department of Virology, Karolinska Institute, School of Medicine, S-105 21 Stockbolin, Sweden.
016%1702/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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for a cysteine appeared to represent the most likely explanation for the reduced neurovirulence of strain M13.
Mumps virus; Hemagglutinin protein; Mon~lonal escapes
Mumps virus is a human pathogen, which infects a variety of different glands, the central nervous system (CNS), the respiratory tract, myocardia and possibly also muscle and joint tissue (Chou, 1986, Gordon and Lauter, 1985, Wolinsky and Server 1985). This spectrum of susceptible organs, as well as the many different species which can be experimentally infected (Wolinsky and Server, 1985), indicates the occurrence of a wide variation of host cells susceptible to infection by the virus. However, strain characteristics also affect the host range as shown by McCarthy et al. (1980a) where different strains of mumps virus varied in capacity to infect
neurons. The neuroadapted Kilham strain of mumps virus giving severe meningo- encephalitis in hamsters (Kilham and Overman, 1953) has been extensively studied and compared to non-neurovirulent strains such as O’Take, Lepow and RW in attempts to characterize the pathogenesis of the disease (Margolis et al., 1974;
McCarthy et al., 1980a; Wohnsky et al., 1985). The main difference appears to be an ability of the Kilham strain to exert a direct destructive effect on neurons and cause encephalitis while other strains only infect ependymal and choroid plexus cells (Johnson, 1968).
In mumps virus, as with other members of the Paramyxoviridae family, the hemagglutinin activity (HA) is responsible for viral attachment to the sialic acid in the cell membrane receptor (&vell, 1978) thereby determining the cell tropism. The
virion peplomers carrying HA also have a separately located neuraminidase activity (NA) (&vell, 1984). This multi-functional glycosylated surface protein therefore is referred to as the hemagglutinin-neuraminidase (HN) component (Jensik and Silver, 1976). Peptide mapping has shown that the amino acid sequence of the HN protein
of different mumps virus strains varies while the composition of the fusion (F) protein is relatively constant (Wolinsky and Server, 1985). Minor antigenic dif- ferences and slight variations of molecular weights have been reported only in the HN protein from studies with monoclonal antibodies (Nabs) (&vell, 1984; Wolin- ski and Server, 1985). Likewise, experiments in hamsters have shown that neutraliz- ing Mabs against the HN protein can protect against the otherwise lethal infection
with the neuroadapted Kilham mumps strain while Mabs against the F protein cannot (Wolinski et al., 1985). This suggests that the HN protein of the Kilham strain is of critical importance in the neutralization of the virus.
Following these concepts Love et al., in this laboratory, used an anti-HN Mab with effective hemagglutination-in~bition and neutrahzing capacity to select for monoclonal escape mutants (Love et al., 1985a). Four mutants were serologically and biologically characterized. Two mutants showed variations in hemagglutination
127
TABLE 1
Phenotypic characteristics of the K&am strain of mumps virus and the neutraii~tion escape mutants
derived from this strain (L&e et al., 1985a).
HA”
+22Oc +4Oc
MWh
@Da)
Encephalitogenic activities
Kilham + + 75 5-7 days p.i. grave neurological symptoms,
most animals dead within 48 h, showing
general infection in cerebrum, brain-
stem and ependymal cells
Ml1
Ml2
Ml3
++
0
+I-
+ 72-73 As Kilham strain
++ 75 As Kilham strain
++ 75 4-6 days p-i. slight neurologieai symptoms, most animais surviving 14 days then
developing hydr~ephaius. Only single
neurons infected. degeneration of
ependymal ceils
a Performed with guinea pig erythrocytes at the temperatures indicated. 0 i + -z 256, figures repre-
senting reciprocal values of dilution.
b Determined by SDS-PAGE analysis.
activity, one showed an altered molecular weight of the HN protein and still another mutant exhibited significantly lower ability to induce lethal encephalitis in hamsters probably as a result of decreased affinity for neuronal cells (Table 1).
The HN protein genes of two non-neurovirulent strains, RW and SBL-1, have recently been sequenced (Waxham et al., 1988; Kovamees et al., 1989). The
predicted proteins consist of 582 amino acids with a hydrophobic domain allowing its N-terminal end to anchor to the membrane. In the present study the genomic RNA coding for the HN protein of the neurotropic Kilham strain and three of the mutants derived from this strain were sequenced. Substitutions were identified in
the predicted amino acid sequences and these changes were correlated to the varied
phenotype of each mutant.
Methods
Virus and cell cultures
The Kilham strain was originally obtained from Dr Wolinsky tuniversity of Texas, Health Science Center, Houston, TX). The virus was passaged three times in hamster brains and plaque purified between each passage. Monoclonal escape mutants were selected by growing the virus in presence of the anti-HN monoclonal antibody 5500 (&veil, 1984). This Mab effectively inhibited hemagglutination and neutralized not only the SBL-1 strain but also the Kilham strain of virus (Love et al., 1985a). Virus was grown in VERO cells in large roller bottles using Eagle’s
122
minimal essential medium (Flow Laboratories) supplemented with 1% inactivated fetal calf serum, peniciilin and streptomycin (60 and 50 pg/l, respectively).
Enzyme linked immunosorbent assay (ELISA)
Tests were performed in microtiter plates coated with 20 pg purified vu-ions/ml, using previously described techniques and chemicals (Rydbeck et al., 1988).
Isolation of genomic RNA
Cell culture supematants were harvested 5-7 days postinfection when almost complete cytopathic effects were seen. The supematants were clarified three times at 3000 rpm for 10 min (Sorvall, GSA rotor), pelleted at 12,000 rpm for 1 h and then spun through 35% sucrose onto a 65% sucrose cushion at 35,000 rpm for 1 h
(Beckman, SW40 rotor). The interface was rebanded in a discontinuous (20-65%) sucrose gradient by centrifugation for 2.5 h at 35,000 r-pm. The virions were
collected and pelleted for 1 h at 35,000 rpm in TNE/STE buffer (1 X TNE: 10 mM Tris-HCI, pH 8.0, 100 mM NaCl, 1 mM EDTA, pH 8.0). The pellet was resus- pended in TNE buffer. Virion proteins were removed by SDS and proteinase K treatment (0.3% and 0.5 mg/ml) at 56°C for 20 mm and subsequently extracted
twice with phenol and once with phenol/chloroform. The genomic RNA was ethanol/NaCl precipitated three times, washed and dissolved in water to a final
concentration of 3 pg/ul.
Primers
Synthetic oligonucl~ti~es (17-mers) were made on a Pharmacia Gene Assembler. The oligonucleotid~ were solub~lized and protective groups removed by NH,OH followed by detritylation by acetic acid. The oligonucleotides were then purified on
a polyacryiamide gel and eluted in buffer (0.5 M NH,Ac, 0.01 M MgAc,, 0.1% SDS, 0.1 M EDTA) for 24 h. Finally the product was desalted on a Pharmacia
NAP-5 column and diluted to 2 pg/ml in water.
RNA sequencing
50 ng primer was kinased using 50 PCi 32 P-ATP and T4 polynucleotide kinase.
One tenth of this product was heated at 95°C for 3 mm to 5-10 pg genomic RNA
in annealing buffer (250 mM KCl, 10 mM Tris-HCl, pH 8.3) and instantly cooled on ice. The annealed material was divided into four tubes each containing 1 ,ul of 0.25 mM A, C, G or T dideoxynucleotide, 3.3 ~1 of RT buffer (24 mM Tris-HCl, pH 8.3, 16 mM MgCl,, 8 mM DTT, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, 0.4 mM dTTP) and 3 units of AMV reverse transcriptase. After incubation at 52 o C for 45 n-tin the samples were stopped, denatured and run on a 6% polyac~la~de gel containing 8 M urea (Gelibter, 1987).
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Rt?!dtS
Sequencing strategy
Recent work has provided the complete nucleotide sequence of mumps virus (SBL-1 strain) genes except for the L gene (Elango 1988, 1989; Elango et al., 1988, 1989a, b, c; Kiivamees et al., 1989). For genomic sequencing of the Kilham strain and neutralization escape mutants derived from this strain {Love et al., 1985a), we selected a known sequence in the upstream region of the HN gene and sequenced all the way through the gene by primer extension. In order to cover the 1888 bases composing the HN gene in the Kilham strain we made ten 17 nucleotides long oligodeoxynucleotides which were used as primers.
To confirm that the mutants were non-reactive with Mab 5500, an ELISA was performed. This showed that Mab 5500 reacted with the Kilham strain but not with any of the mut~ts as expected (Love et al., 1985a) (results not shown).
0 100 200 300 400 500 600
I I I I I I IAt-4
AA Position 239 297 3;9
KILHAM L L N
Ml3 L L N
CORRELATI0.i TO PIIMOTYPK ALTERATIOS
G P
G Lsl
q p
G P D q D
INCREASED DECREASED REDUCED
NEURA~.~lNlDASE MOLECULAR NEURO-
ACTIVITY WEIGHT VIRULENCE
Fig. 1. Schematic presentation of amino acid substitutions in the HN protein of the mutants. Circles represent conserved substitutions while boxes indicate non-conservative substitutions. Boxed amino acid position 352-360 indicates the putative neutralizing epitope for Mab 5500. x) JBrgensen et al., 1987;
VanWyke Coelingh et al., 1987; Kijvamees et al., 1989.
124
Comparison of the amino acid sequences of the HN proteins of the Kilham, SBL-1 and R W virus strains
The predicted HN protein sequences of the Kilham strain were compared to the published sequences of the fusing active and non-neurovirulent SBL-1 strain (Kbvamees et al., 1989) and to the non-fusing, non-neurovirulent RW strain (Waxham et al., 1988). The putative membrane spanning region is identical in the
three strains and so are all of the major conservative areas described in paramy- xoviruses (Jorgensen et al., 1987; Kovamees et al., 1989; vanWyke Coelingh et al., 1987). Not only the sharing of fusion activity but also the HN protein sequence characteristics as well as the 5’ and 3’ non-coding regions suggested a relatively closer relationship between the SBL-1 and Kilham strains (results not shown).
Comparison of the amino acid sequences of the HN proteins of the mutants and the
parental Kilham strain
The predicted amino acid differences of the HN protein between the original Kilham strain and the three escape mutants (Mll, Ml2 and M13) are compared in
Fig. 1. A summary of the phenotype characteristics of the four viruses is given in Table 1.
Compared to the Kilham strain five amino acid substitutions were found in Ml1 (1 conservative and 4 non-conservative), four substitutions in Ml2 (2 conservative
and 2 non-conservative) whereas Ml3 only had one substitution (non-conservative). All amino acid substitutions were results of single base mutations. No differences were seen in the 5’ and 3’ noncoding regions. All of the Cys residues in the Kilham strain were conserved in the mutants. There were no deletions or insertions of bases.
One region spanning amino acid position 352 to 360 of the HN protein exhibited a high frequency of substitutions. This is likely to be the neutralizing epitope
identified by the selective monoclonal antibody since the majority of substitutions were clustered there and it also harbours the only change in Ml3 (Fig. 1).
Compared to the Kilham strain, mutants Ml1 and Ml2 exhibited corresponding amino acid substitutions in two positions. These substitutions were: the conservative
substitution of a leucine for a phenylalanine in position 297, and the non-conserva- tive substitution of an aspartic acid for an asparagine in position 523 (Fig. 1). These two shared substitutions were neither present nor close to any changes in Ml3 and therefore should not be the cause of the neutralization escape. Furthermore, while the phenotypes of Ml1 and Ml2 are strain specific and these two shared substitu-
tions are not, they are probably not involved in the differences of the biological characteristics either.
Beside the two shared amino acid substitutions, virus mutant Ml1 exhibited three unique substitutions. These were: an asparagine for an aspartic acid in position 329 affecting a glycosylation site; a proline for a serine in position 353 and an aspartic acid for an asparagine in position 358, both non-conserved and located in the putative epitope region of the neutralizing antibody. The two amino acid substitu- tions specific for virus mutant Ml2 were: a leucine for a methionine in position 239,
125
which is a conservative change close to one of the areas shared between paramy- xoviruses, and a glycine for a glutamic acid in position 352 increasing negative charge in the putative epitope region. Finally, the amino acid substitution unique for virus mutant Ml3 was the non-conservative change of an arginine for a cysteine in position 360 (Fig. 1).
Discussion
In the two HN monoclonal escape mutants Ml1 and Ml2 two identical amino acid substitutions were found as compared with the parental Kilham strain. In
addition, Ml1 had three individual substitutions and Ml2 had two (Fig. 1). The mutant Ml3 only had a single amino acid change (Fig. 1). No differences were seen
in the 5’ and 3’ non-coding regions between the Kilham strain and the mutants. This supports the conclusion by Love et al. (1985a) that no altered transcription or translation capacity of the HN protein between the parental strain and the mutants
should be expected. The number of amino acid substitutions presented in Ml1 and Ml2 in this study exceed those normally encountered in studies of monoclonal
antibody selected escape mutants (Seif et al., 1985; vanWyke Coelingh et al., 1987). Before and after the original selection experiment the viruses were plaque-purified
which implies that the substitutions were either due to the selection procedure or to subsequent passaging in cells. All of the mutants were subjected to the correspond- ing number of passages in cells after the selection procedure but still there was a discrepancy in the number of substitutions in Ml3 on the one hand and in Ml1 and Ml2 on the other. A comparison of the Kilham strain sequenced here and the 59 amino acids identified by direct amino acid sequencing of the Kilham strain HN protein by Waxham et al. (1988) showed no amino acid differences even though the passage history of the viruses varied. Since the Kilham strain (and M13) seemed to be quite stable, one possible explanation for the larger number of changes of amino
acids in Ml1 and Ml2 might be that the original substitutions leading to neutraliza- tion escape of these mutants needed to be stabilized by additional mutations.
The variable region in the HN molecule spanning amino acids 352 to 360 probably contains the area responsible for the neutralization negative phenotype of the mutants and the epitope of Mab 5500 should therefore be located within this region. In the span of these nine amino acids 4/7 of all non-conservative substitu-
tions and the single one in Ml3 occurred. The hydrophilicity of the region suggests a potential exposure allowing interaction with antibodies. The non-neutralization phenotypes of the mutants are derived from different individual amino acid sub- stitutions, changing the secondary structure or charge of the Mab 5500 epitope. These changes are: amino acid 353 in virus mutant Ml1 where a proline is substituted for a serine leading to abolishment of a knick in the structure (it also contains a charge change in position 358); amino acid 352 in virus mutant Ml2 where a negative charge is introduced by the insertion of a glutamic acid in place of a glycine and finally amino acid 360 in virus mutant Ml3 where the substitution of an arginine for a cysteine creates a possibility for additional intramolecular di-
126
sulphide bondings and for a local change of charge. The Mab 5500 was raised against the SBL-1 strain of mumps virus and neutralized the Kilham strain beside the SBL-1 strain. Like the three virus mutants the laboratory RW strain escaped neutralization by Mab 5500. It is of interest that this strain differs from the Kilham and SBL-1 strains in positions 354 and 356 in the proposed neutralizing epitope.
The amino acid change in position 354 is non-conserved (proline to glutamine). The low molecular weight of Ml1 can be explained by the loss of a potential
glycosylation site as a consequence of the substitution of an asparagine in position 329 for an aspartic acid, thereby abolishing the Asn-X-Ser/Thr triad required for N-linked glycosylation sites (Neuberger et al., 1972). The substitution is located a short distance from the putative epitope but since this site seems to be glycosylated in the parental strain it is not likely to be part of an epitope.
A significantly increased NA activity in Ml2 is the most attractive explanation for the low HA and hemadsorption capacity exhibited at room temperature but not at + 4°C as described earlier (Love et al., 1985a; Merz and Wolinsky, 1981). Interestingly, this mutant has a leucine in position 239 conservatively replaced by a
methionine. The area consisting of amino acids 240-245, in the mumps virus HN protein, has been proposed to be an active NA site by many authors (Jorgensen et al., 1987; Thompson and Portner, 1987; vanWyke Coelingh et al., 1987) (Fig. 1). Recently though, Waxham and Aronowski (1988) have proposed that the region encompassing amino acids 177-184 might be of crucial importance for the NA activity, possibly with amino acid 181 as key residue. In relation to the increased NA activity our results can be interpreted to indicate that the biological effect most likely was modified by the substitution next to the 240-245 region.
Ml3 was the only virus mutant which exhibited a reduced neurovirulence (Love
et al., 1985a, b), probably as a result of decreased neurotropism since Ml3 showed no difference in capacity to infect ependymal cells compared to the parental Kilham strain (Table 1). The only amino acid substitution in Ml3 was of an arginine for a cysteine in position 360. Both the phenotypic alterations in this mutant; neutraliza-
tion escape and reduced neurotropism therefore should result from this amino acid
substitution. The substitution results, as already mentioned, in a change of charge and might also create a new disulphide bond. In work with rabies virus Dietzschold
et al. (1983) found that substitution of an arginine for a glutamic acid or a glycine in position 333 of the G-protein reduced the neurovirulence. Both changes affected the charge of the molecule. Prehaud et al. (1988) also in work with rabies virus found five G-protein monoclonal escape mutants which exhibited reduced pathogenicity. Four of these displayed single amino acid substitutions altering charge. The reduced neurovirulence of Ml3 exhibits similarities to attenuating mutations in reovirus (Bassel-Duby et al., 1986). In this work a monoclonal antibody against the hemag- glutinin with efficient neutralization was used to select escape mutants. Four mutants were markedly less virulent when injected intracerebrally on neonatal mice as a result of altered tropism. When these mutants were analyzed all had a single
amino acid substitution that caused a change of charge. Pathogenicity is a complex multifactorial characteristic but one potential cause of
variation of this property is the capacity of the virus to bind to receptors on
127
different cells (Fields and Green 1982). The attachment protein, in mumps virus the HN protein, is of decisive importance for the interaction with receptors and further knowledge of structural-functional characteristics of this protein may therefore highlight hgand features of pathogenic significance.
In this study we found an unexpected large number of amino acid substitutions after mon~lonal antibody selection. However, most substitutions could be interpre- ted to be of lesser importance leaving certain amino acids for correlation between their occurrence and the presence of a particular activity. The possibility remains
that some substitutions reflect a heterogeneity in the parental population, occurring in spite of the plaque purifications applied. If this indeed would be the case it would
not detract from the projected localization of the epitope but due to possible alterations in other viral proteins the correlation between protein changes and the biological activities should be deduced with certain caution.
Acknowledgements
We thank Mrs Agnes Foo for the excellent technical assistance and Dr. Per Stalhandske for constructive criticism. This work was supported by the Swedish
Medical Research Council (Project No. 0011 6).
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(Received 22 February 1990; revision received 26 June 1990)