Download The Attachment Protein of Hendra Virus Has High

VIROLOGY
251, 227±233 (1998)
VY989302
ARTICLE NO.
The Attachment Protein of Hendra Virus Has High Structural Similarity but Limited Primary
Sequence Homology Compared with Viruses in the Genus Paramyxovirus
Meng Yu,* Eric Hansson,* Johannes P. M. Langedijk,² Bryan T. Eaton,* and Lin-Fa Wang*,1
*CSIRO Division of Animal Health, Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia; and ²Department of
Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, San Diego, California 92037
Received March 24, 1998; returned to author for revision May 15, 1998; accepted June 12, 1998
The complete nucleotide sequence of the attachment protein gene of Hendra virus, a new member of the subfamily
Paramyxovirinae, has been determined from cDNA clones derived from viral genomic RNA. The deduced mRNA is 2565
nucleotides long with one open reading frame encoding a protein of 604 amino acids, which is similar in size to the
attachment protein of the members of the subfamily. However, the mRNA transcript is .600 nucleotides longer than others
in the subfamily due to the presence of long untranslated regions at both the 59 and 39 ends. The protein is designated G
because it lacks both hemagglutination and neuraminidase activities. It contains a hydrophobic transmembrane domain close
to the N terminus, eight potential N-linked glycosylation sites, and 18 cysteine residues. Although the HeV G protein had low
sequence homology with Paramyxovirinae members, the predicted folding pattern of its extracellular globular head was very
similar to that of members of the genus Paramyxovirus, with the location of seven potential pairs of sulfide bonds absolutely
conserved. On the other hand, among the seven residues known to be critical for neuraminidase activity, only one was
conserved in the Hendra virus G protein compared with at least six in HN proteins of paramyxoviruses and rubulaviruses and
four in H proteins of morbilliviruses. The biological significance of this finding is discussed. © 1998 Academic Press
Paramyxovirus and Rubulavirus genera, the Morbillivirus
genus, and the Pneumovirus genus have been designated
HN, H, and G, respectively, depending on whether they
display haemagglutination (H) and neuraminidase (N) activities. Pneumoviruses lack both activities. Recently, the
neuraminidase-negative status of morbilliviruses was challenged by Langedijk et al. (1997), who demonstrated that
the attachment proteins of rinderpest virus (RPV) and peste
des petits ruminant virus (PPRV), members of the genus
Morbillivirus, manifest enzyme activity.
The function of the neuraminidase is not well understood in members of the family Paramyxoviridae. In influenza viruses, members of the family Orthomyxoviridae,
the H and N activities are encoded by two different
genes, and N has been shown to be necessary for the
release of progeny virus from infected cells by cleavage
of sialic acids (Palese et al., 1974). Another role for the
neuraminidase activity in orthomyxoviruses and paramyxoviruses may be for virus transport through the sialic
acid-rich mucus secretions that serve to protect the
respiratory tract, and therefore the substrate specificity
of the enzyme may be related to the host and the site of
infection (Langedijk et al., 1997).
In September 1994, a new member of the Paramyxoviridae family was identified as the causative agent of an
outbreak of respiratory disease that resulted in the death
of horses and one person (Murray et al., 1995; O'Sullivan
et al., 1996). The virus superficially resembled morbilliviruses and was provisionally named equine morbillivirus
(EMV). As information on the sequence of the genome
INTRODUCTION
The family Paramyxoviridae was reclassified into two
subfamilies in 1993: the Paramyxovirinae and the Pneumovirinae (Murphy et al., 1995; Lamb and Kolakofsky,
1996). Members of the Pneumovirinae can be distinguished from the Paramyxovirinae on the basis of their
nucleocapsid diameter (13±14 vs 18 nm, respectively),
length of glycoprotein spike protruding from the virus
surface (10±12 vs 15 nm, respectively), and genome organization. Pneumovirinae and Paramyxovirinae members contain more than seven and fewer than seven
transcriptional units, respectively. In the subfamily
Paramyxovirinae, there are three genera, Paramyxovirus,
Rubulavirus and Morbillivirus, which are differentiated on
the basis of (1) antigenic cross-reactivity, (2) the presence or absence of neuramidinase activity, and (3) the
coding strategies of their respective P genes (Murphy et
al., 1995).
Members of the family Paramyxoviridae contain two envelope glycoproteinsÐthe fusion protein (F) and the attachment protein, with the latter responsible for binding the
virus to specific receptor molecules on the host cell surface. In some cases, it has been demonstrated that the
attachment protein is also required for membrane fusion by
acting in concert with the F protein (Bousse et al., 1994;
Sergel et al., 1993). Attachment proteins of members of the
1
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YU ET AL.
and its organisation became available (Wang et al., 1998;
Yu et al., 1998), it was clear that although the virus is a
member of the subfamily Paramyxovirinae, it does not fit
comfortably into any of the existing genera. Indeed, it
seems likely that this new virus represents the prototype
of a new genus in the subfamily Paramyxovirinae (Wang
et al., 1998; Yu et al., 1998). As a result, the name of the
virus was changed to Hendra virus (HeV), after the location of the first outbreak in Brisbane, Australia (Murray et
al., 1998; Wang et al., 1998).
HeV naturally infects humans and horses and has
been isolated from fruit bats, which are believed to be
the natural hosts of the virus (Halpin et al., 1996). The
virus is also pathogenic in experimentally infected cats,
horses, and guinea pigs (Westbury et al., 1995; 1996). The
wide host range distinguishes HeV from other known
members of the Paramyxovirinae, which generally have
narrow host specificity. The sequence and putative structural features of the HeV attachment protein (G) were
determined and compared with those of other members
of the Paramyxovirinae to determine whether the HeV
attachment protein has any unique feature that may be
responsible for its wide host range.
RESULTS AND DISCUSSION
Sequence features of the HeV G gene and protein
The complete nucleotide sequence of the G gene was
first determined from overlapping clones isolated from a
cDNA library and then confirmed by direct sequencing of
polymerase chain reaction (PCR) fragments derived from
genomic RNA. The gene sequence with the deduced amino
acid sequence is presented in Figure 1. The mRNA is 2564
nucleotides (nt) in length and is flanked by the trinucleotide
intercistronic sequence CTT (not shown in the figure) as
described for the other HeV genes identified so far (Gould,
1996; Wang et al., 1998; Yu et al., 1998). Transcription initiation (59-AGGACCCAAG-39) and termination sequences (59ATTAAGAAAAA-39) were identified on the basis of their
conservation with other members of the subfamily and
other HeV genes characterized so far (Lamb and Kolakofsky, 1996; Wang et al., 1998). A search for open reading
frames (ORFs) indicated only one of significant size in the G
mRNA. The ORF encodes a protein that is 604 amino acid
residues in length and has a calculated molecular mass of
67,251 Da. There are three in-frame ATG codons (highlighted in Fig. 1) before the hydrophobic transmembrane domain. In theory, translation initiation from any of these three
sites could produce a functional attachment protein. According to Kozak (1991), the optimal context for translation
initiation has the consensus sequence of GCCA(G)CCATGG, in which the A (or G) residue at 23 and the G
residue at 14 play the most important role in modulating
initiation efficiency. For the G gene, the third ATG codon has
the most favorable Kozak sequence, followed by the second, located immediately after the first. The G gene may
therefore produce several attachment proteins that differ in
the length of their cytoplasmic domain. If the first (or second) ATG codon is used in translation, the HeV G protein
would contain a cytoplasmic domain of ;45 amino acids,
which is much longer than that of known Paramyxovirinae
members. Regardless of which ATG codon is used for
initiation, the cytoplasmic domain is rich in lysine and is
highly charged. The significance of this difference is not
clear, but a previous report by Parks and Lamb (1990)
demonstrated that the cytoplasmic domain of paramyxovirus HN protein does play a role in the assembly and
transport of this type II membrane protein. The overall
length of the potential translation products merits comment.
Initiation at the first ATG codon produces a protein of 604
amino acids, which resembles those of morbilliviruses
(604±617 amino acids), whereas initiation at the third ATG
codon produces a protein (573 amino acids) much closer in
size to that of paramyxovirus HN proteins (572±575 amino
acids). It is also interesting to note that there are two
in-frame ATG codons located within the hydrophobic transmembrane domain (see Fig. 1). Their location is reminiscent of the alternative translational initiation site used by
the respiratory syncytial virus G gene to produce a soluble
form of the attachment protein (Roberts et al., 1994).
Although the length of the coding region of HeV G
protein is similar, the G gene transcript length is much
larger in HeV than that in other Paramyxovirinae members. This is due to the presence of longer untranslated
regions at both ends of the mRNA. The HeV G gene has
59 and 39 untranslated regions of 233 and 516 nt, respectively, compared with 20±73 and 85±111 nt, respectively,
for other members of the subfamily. Long untranslated
regions are also found in the N, P, M, and F genes of HeV
(Gould, 1995; Wang et al., 1998; Yu et al., 1998). Similar
regions characterize the genes of members of the family
Filoviridae, including Ebola and Marburg viruses (Feldmann and Klenk, 1996). The function of long untranslated
regions in negative-stranded RNA viruses is not known,
but a role in increasing mRNA stability is a possibility.
The deduced HeV G protein has eight potential N-linked
glycosylation sites. Other Paramyxovirinae attachment proteins have five to seven such sites. The distribution of these
residues seems to be evenly spread along the molecule,
rather than concentrated at a particular region, as in the
case of N-glycosylation sites for measles virus H protein
(Lamb and Kolakofsky, 1996). The viral G protein is glycosylated in vivo as shown by its mobility in SDS-PAGE and its
binding to lectins (Wang et al., 1998; W. Michalski and B. T.
Eaton, unpublished results). The HeV G protein has 18
cysteine residues compared with 12±17 for other subfamily
members. The importance of cysteine residue location is
discussed later.
Sequence and structural alignment of HeV G protein
with other Paramyxovirinae HN/H proteins
Comparison of primary sequences of HeV G and
Paramyxovirinae HN and H proteins indicated that the
THE HENDRA VIRUS G PROTEIN
229
FIG. 1. DNA and deduced amino acid sequence of the HeV G gene. Amino acid sequence is given in single letter code beneath the DNA sequence.
Conserved nucleotide sequences for transcription initiation and termination are underlined. The three in-frame ATG start codons together with their
encoded methionine (M) residues are indicated in bold. Important amino acid sequence features are highlighted as follows: cysteine residues,
shaded; N-linked glycosylation sites; underlined by a double straight line; hydrophobic transmembrane domain, underlined with a wavy line. The
nucleotide sequence reported here has been submitted to the GenBank/EMBL and assigned the accession number AF017149.
overall sequence conservation is low (data not
shown). The percentage sequence identities vary from
11% to 23%, with HeV G displaying a closer relationship to paramyxoviruses than to morbilliviruses and
rubulaviruses. The highest sequence identity (23%)
exists between HeV and human parainfluenza virus 1
(hPIV1). However, this is only half of that observed
among known paramyxoviruses (45% between human
PIV1 and bovine PIV3). This overall low level of sequence identity is consistent with previous analyses
based on the sequences of the N, P, M, and F proteins,
suggesting that HeV may represent the prototype virus
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FIG. 1ÐContinued
of a new genus in the subfamily Paramyxovirinae
(Wang et al., 1998).
The structure of paramyxovirus attachment proteins
can be divided into four regions: N-terminal cytoplasmic tail, hydrophobic transmembrane domain, stem,
and extracellular globular head. Recently, Langedijk et
al. (1997) conducted a detailed sequence and structure alignment of the globular head domains of
paramyxovirus and morbillivirus HN and H proteins
and defined a highly conserved structure containing a
canonical fold, which is conserved in neuraminidases
derived from viruses, bacteria, and eukaryotic organisms. Similar sequence and structural alignments
were conducted for HeV G protein using sequences
derived from the stem and globular head domains.
Several important observations were made from the
alignment presented in Figure 2: (1) HeV G protein
shares many structural features characteristic of viruses in the genus Paramyxovirus. The locations of all
seven proposed disulfide bonds are absolutely conserved between HeV and paramyxoviruses, whereas
five are conserved between HeV and rubulaviruses
THE HENDRA VIRUS G PROTEIN
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FIG. 2. Sequence and structure alignment of the globular head region of Paramyxovirinae attachment proteins. (See below for abbreviations and
sources of sequences used in this analysis.) The locations of the strands of b-sheets 1±6 are given above the sequence. Residues conserved in all
nine viruses are shaded, whereas the seven proposed active-site residues are indicated by numbers 1±7. Residues conserved between HeV and
neuraminidase-positive viruses (i.e., paramyxoviruses and rubulaviruses) are shown in bold capital letters, and those conserved between HeV and
morbilliviruses are shown in bold small letters. Proposed disulfide bonds conserved between HeV and other members of the subfamily are shown
by solid lines, with the long range bond indicated by an open box. Disulfide bonds unique to morbillivirus and rubulaviruses are shown in dashed
lines. Residue numbering is indicated after each line. Abbreviations (Databank accession number): CDV, canine distemper virus (P24306); PDV,
phocine distemper virus (Z36979); MeV, measles virus (P08362); RPV, rinderpest virus (M21512); DMV, dolphin morbillivirus (Z36978); SeV, Sendai virus
(P06863); PIV3, bovine parainfluenza virus 3 (P06167); MuV, mumps virus (M19933); and NDV, Newcastle disease virus (P32884).
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and between HeV and morbilliviruses. Rubulaviruses
and morbilliviruses also have a set of disulfide bonds
unique to their respective genera. (2) In addition to
cysteine residues, other structurally important residues (such as glycine, proline, and aromatic amino
acid residues) are also conserved to a large extent.
(3) Among the seven active site residues known to
be important for neuraminidase activity (Langedijk et
al., 1997), only one residue (residue 7) is conserved
in HeV G protein, whereas six are conserved in HN
proteins of paramyxoviruses and rubulaviruses and four
are conserved in H proteins of morbilliviruses. However,
conserved residues are found in the surrounding positions of most proposed active site residues.
It has been proposed that the conserved hexapeptide
NRKSCS located at 2L01 and 2S1 in paramyxovirus HN is
close to the sialic acid binding site and forms part of the
neuraminidase activity site (Jorgensen et al., 1987; Morrison and Portner, 1991; Mirza et al., 1994). Binding to
sialic acid-containing molecules on the surface of cells
initiates the process of infection by viruses in the
paramyxovirus and rubulavirus genera (Lamb and Kolakofsky, 1996). A number of gangliosides that contain one
or more sialic acids as N-acetylneuraminic acid attached
to an internal or a terminal galactose have been identified as isoreceptors for Sendai virus (Markwell et al.,
1986). HeV G protein contains only the last two residues
of the putative sialic acid binding site (TIHHCS instead of
NRKSCS), suggesting that HeV may not bind sialic acid.
This is consistent with our experimental findings. Indeed,
not only does the virus fail to demonstrate neuraminidase activity in vitro (Murray et al., 1995), but also the
removal of sialic acid from susceptible cells by treatment
with neuraminidase did not abrogate infection by HeV
(B. T. Eaton, unpublished results).
In summary, the results indicate that the HeV G mRNA
resembles other HeV mRNAs in having long untranslated
regions and that the HeV G protein has a longer cytoplasmic tail than other Paramyxovirinae members. Sequence and structural comparison revealed that although the overall primary sequence homology is low,
the HeV G protein shared high structural similarities with
the HN proteins of paramyxoviruses. Although the biological significance of this discovery is not clear at the
present time, a high structural conservation with disposal of neuraminidase activity site residues for the HeV
G protein is an interesting and unusual evolutionary step
that merits further investigation.
MATERIALS AND METHODS
Cloning and sequencing
The methods for purification of virus, cDNA library
construction, and screening have been described previously (Wang et al., 1998). Briefly, HeV was purified by
zonal centrifugation in sucrose gradients, and genomic
RNA was isolated using standard methods. The Time-
Saver cDNA synthesis kit (Pharmacia) was used to make
total cDNA using random priming, followed by the addition of an EcoRI adaptor. cDNA fragments were cloned
into pZEr0±1 vector (InVitrogen), resulting in a cDNA
library of ;100,000 independent primary clones. A total
of eight overlapping clones were isolated covering the
entire coding region of the G gene. DNA sequencing was
performed using the Sequenase Kit (U.S. Biochemicals),
and the nucleotide at each position was sequenced at
least twice, either by sequencing two overlapping clones
or by sequencing the opposite strand of the same clone.
The complete cDNA sequence was then confirmed by
direct sequencing of PCR fragments derived from
genomic RNA without cloning.
Sequence analysis and alignment
Primary sequence comparison and calculation of percentage sequence identities were done using the AlignPlus Program from Scientific & Educational Software
(Durham, North Carolina). Multiple sequence alignments
were performed using the Pileup program of the Genetic
Computer group (Devereux et al., 1984). Comparative
alignments were conducted between the HeV G protein
and several paramyxovirus HN proteins and morbillivirus
H proteins. A final alignment was conducted using the
same format and covering the same regions as in Langedijk et al. (1997). Phylogenetic analysis was carried out
using programs in the PHYLIP package (Felsenstein
1993). Distances were calculated with PROTDIST using
the alignment of Figure 2 as input.
ACKNOWLEDGMENTS
We thank Kaylene Selleck and Nadia Mayfield for technical assistance. The genome sequencing work was supported in part by a grant
from the National Health and Medical Research Council of Australia.
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