Biochemical and Biophysical Research Communications 315 (2004) 664–670

BBRCwww.elsevier.com/locate/ybbrc

Basis for fusion inhibition by peptides: analysis of the heptadrepeat regions of the fusion proteins from Nipah and Hendra

viruses, newly emergent zoonotic paramyxoviruses

Yanhui Xu,a Shan Gao,a,b,c David K. Cole,d Junjie Zhu,b Nan Su,a Hui Wang,c

George F. Gao,d,* and Zihe Raoa,*

a Laboratory of Structural Biology, Tsinghua University, Beijing 100084, Chinab Beijing Municipal Station of Animal Husbandry and Veterinary Medicine, Beijing, China

c Centre for Ecology and Hydrology, Oxford (CEH-Oxford), Mansfield Road, Oxford OX1 3SR, UKd Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DU, UK

Received 22 January 2004

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are novel zoonotic members of the Paramyxoviridae family and are the prototypes

for a newly designated genus, Genus Henipavirus. Recent studies have shown that paramyxovirus might adopt a similar mechanism

of virus fusion-entry. Under this mechanism, the two highly conserved heptad repeat (HR) regions, HR1 and HR2, in the fusion (F)

protein, seem to show characteristic structure in the fusion core: the formation of a 6-helix coiled-coil bundle. The three HR1s form

the a-helix coiled-coil surrounded by three HR2s. In this study, the two HR regions of NiV or HeV were expressed in an Escherichia

coli system as a single chain and the results do show that HR1 and HR2 interact with each other in both NiV and HeV and form

typical 6-helix coiled-coil bundles. This provides the molecular basis of HR2 inhibition to NiV and HeV fusion as observed in an

earlier report.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Nipah virus; Hendra virus; Heptad repeat; Fusion

Nipah virus (NiV) is an emerging pathogen identified

in 1999 responsible for the disease transmitted from pigs

to humans [1,2]. Hendra virus (HeV) emerged in 1994

and was transmitted to humans from close contact with

horses [3,4]. Therefore, they are potential zoonosis and

pathogens related to bio-terrorism [5–7]. Both NiV andHeV are unusual among the paramyxoviruses in their

abilities to infect and cause potentially fatal disease

(encephalitis) in a number of hosts, including human

beings [5,6]. These two viruses also have exceptionally

larger genomes than any other members of paramyx-

oviruses [5,8,9]. Phylogenetic analysis of their genomes

shows that they are distinct members of Family Para-

* Corresponding authors. Fax: +44-1865-222901 (G.F. Gao),

+86-10-62773145 (Z. Rao).

E-mail addresses: george.gao@ndm.ox.ac.uk (G.F. Gao), raozh@

xtal.tsinghua.edu.cn (Z. Rao).

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.01.115

myxoviridae, but are closely related to members in Ge-

nus Morbillivirus and Genus Respirovirus [8,9], in which

measles virus (MeV) and Sendai virus (SeV) are mem-

bers, respectively. They have been now classified as a

new genus, Genus Henipavirus, inside the Family Para-

myxoviridae [5,6].Paramyxoviruses are enveloped negative-stranded

RNA viruses, forming a large family (Family Para-

myxoviridae) sub-divided into two subfamilies with five

established and two newly defined genera (Genus Ru-

bulavirus, Respirovirus, Morbillivirus, Pneumovirus,

Metapnuemovirus, and the new Avulavirus, Henipavirus)

[6,10,11]. As with other paramyxoviruses, NiV and HeV

consist of two surface glycoproteins on the viral enve-lope, fusion (F) protein and glycoprotein (G protein,

also called attachment protein) [8,9,12,13]. These two

glycoproteins are responsible for the virus fusion and

entry into host cells [12,13]. G protein initiates the

Y. Xu et al. / Biochemical and Biophysical Research Communications 315 (2004) 664–670 665

infection by binding to the cellular receptor (attach-ment) but F protein mediates the actual virus–cell

membrane fusion process [12–14]. F protein is initially

synthesized as a precursor, F0, then being cleaved into

F1 and F2 by a furin-like enzyme derived from the host

(there is some evidence, at least in HeV, that the enzyme

involved in this cleavage is not furin) [15]. F1 and F2 are

linked by a disulfide bond forming a trimer on the virus

surface. In the attachment and subsequent fusion pro-cess of the paramyxoviruses, the F protein undergoes a

series of conformational changes under which virus and

cell membrane fusion occurs [14,16,17].

It is known that some protein modules of the F

protein are crucial for the conformational changes

during the membrane fusion [16–21]. Among them, fu-

sion peptide (FP) is highly hydrophobic and is respon-

sible for direct insertion of the F protein into the cellularlipid membrane [16,17,22], whereas the highly conserved

heptad repeat (HR) regions in F1, HR1 and HR2, act

seemingly as scaffolding modules. During the confor-

mational changes in the fusion process, HR1 and HR2

interact with each other to form a so-called “trimer

of 2HR” or “6-helix bundle,” in which three HR1s

form a central trimeric coiled-coil surrounded by three

a-helix-structured HR2s in an anti-parallel manner[16,17,23,24]. The formation of this 6-helix bundle pulls

the viral and cellular membranes toward each other,

thereof facilitating the membrane fusion. Blocking of

the formation of this 6-helix bundle can inhibit the viral

fusion and infection. Introduction of exogenous soluble

HR1 or HR2 into the virus infection system in vitro

could inhibit viral infection by competing with the en-

dogenous HR1/HR2 interaction [17], holding the con-formational-changing F protein in the intermediate state

[17]. It has also been shown that the philosophy by in-

troducing soluble HR2 into HIV-infected patients does

work in vivo [25]. In a recent study involving HR2 of

NiV and HeV, strong inhibition was observed in the in

vitro cell-fusion system of the viruses [26]. However, the

interaction of HR1 and HR2 in NiV or HeV has not

been studied. Therefore in this study, we have used acomputer program called LearnCoil-VMF [27], de-

signed specifically for identifying viral membrane HR

coiled-coil regions, to predict the precise position of

both HR1 and HR2 in NiV and HeV F proteins. The

predicted HR1 regions are identical for NiV and HeV

but with a two-amino-acid difference in the HR2 re-

gions. Subsequently, a single chain of HR1 and HR2 for

both NiV and HeV was expressed in the Escherichia coli

system used previously for other paramyxoviruses [28–

34]. Biochemical and biophysical analyses of this poly-

peptide confirmed the formation of a 6-helix bundle,

indicating the typical characteristics of NiV and HeV F

proteins as members of the Paramyxoviridae family and

providing the molecular basis for HR2 inhibiting effect

on the viral fusion. The results also show that HR2 of

NiV and HeV are functionally and structurally inter-changeable.

Materials and methods

Prediction of the heptad repeat regions and construction of the ex-

pression vectors. The a-helix structure of the two heptad repeat regions

of the F proteins of NiV and HeV (GenBank Accession Nos.

AAK29087.1 for NiV and AAC83192.2 for HeV) was predicted by

LearnCoil-VMF program (http://nightingale.lcs.mit.edu/cgi-bin/vmf),

which was specifically developed for identification of potential coiled-

coil heptad repeat regions in viral fusion proteins by Kim and col-

leagues [27] at Massachusetts Institute of Technology.

The genes encoding the HR1 and HR2 regions of NiV were syn-

thesized by PCR using eight synthesized primers according to the

published sequences (GenBank Accession No. AAK29087.1). The

codon usage was optimized according to E. coli expression preferences.

HR1 and HR2 were connected by an eight-amino-acid linker

(GGSGGSGG, single-letter abbreviation of amino acids) and a 6-His

tag was introduced at the C-terminus to facilitate the protein purifi-

cation. A rhinovirus 3C protease cleavage site (EVLFQ+GP, single-

letter abbreviation of amino acids) [35] was introduced between the

end of the HR2 and the 6-His tag by PCR for the purpose of 6-His

removal to facilitate the crystallization. The gene of 2-Helix with NcoI–

BamHI sites (these sites were introduced by PCR primers) at the ends

was inserted into the NcoI–BamHI restriction sites of the pET ex-

pression vector pET-23d (Novagen). A stop codon was introduced

immediately before the BamHI restriction site. This construct was

named as 23NiV-2-Helix (Fig. 1). The construction was verified by

direct DNA sequencing.

The primers overlapped with each other for 8–16 nucleotide bases.

The NcoI and BamHI restriction sites were added in primers P1 and P8

(capitalized/italic denominators). The gene of NiV 2-Helix was ob-

tained by two-step PCR. First, the eight synthetic primers (0.2OD/ll)were mixed and the PCRs were run with the following conditions:

denatured at 94 �C for 5min and then 25 cycles of 94 �C 1.5min, 55 �C2min, and 72 �C 1min; For the second PCR, the first PCR product

was used as template and new PCR product was amplified by using

primers P1 and P8 at the same condition as the first PCR.

Primers used for making the NiV 2-Helix construct:

P1: 50-ggcatgCCATGGctatgaaaaacgctgacaacatcaacaaactgaaatcttcca-30

P2: 50-gaccgctctgcaggactatggtggctctggcggttccggtggcaaagttg-30

P3: 50-gctgcagagactggttcatagaagagatctgagaagagatgtcaactttg-30

P4: 50-ttaaactgcaggaaaccgctgaaaaaactgtttatgttctgaccgctctg-30

P5: 50-tccagcagacgctgagcttctttgatatagtctttagactgctgcagaga-30

P6: 50-aaactgaaatcttccatcgaatctaccaacgaagctgttgttaaactgca-30

P7: 50-atggtgatgcggaccctggaacagaacttcgttaacggtgtccagcagac-30

P8: 50-gggcccaagcttGGATCCctagtgatggtgatggtgatgcggaccctggaacag-30.

As the HR1 sequences of NiV and HeV are identical with only two-

amino-acid substitutions (RL)KI, single-letter amino acid abbrevi-

ation used) in HR2 between them (Fig. 1), the HeV 2-Helix construct

was made by using the QuickChange technique (Strategene) with the

following primers. The nucleotide sequences of the mutated amino

acids in the primers are indicated with bold italic capital letters in the

boxes. The construct was named as 23HeV-2-Helix.

Primer 1, 50-CAAAGAAGCTCAGAAGATC CTGGACACCGT

TAACG-30

Primer 2, 50-CGGTGTCCAGGATCTTCTGAGCTTCTTTGAT

ATAGTC-30.

Protein expression, purification, and gel-filtration analysis. The re-

combinant pET23d plasmids (23NiV-2-Helix or 23HeV-2-Helix) were

transformed into E. coli strain BL21 (DE3). A single colony of the

bacteria was grown at 37 �C in LB medium to an optical density of 0.6

Fig. 1. Prediction of the heptad repeat regions and the construction strategy of the 2-Helix protein construct for both NiV and HeV F proteins. (A)

Schematic diagram of NiV and HeV F proteins with the location of structurally significant domains indicated. The listed sequences of HR1 (137–178)

and HR2 (453–485) used in this study were derived from the LearnCoil-VMF prediction program [27]. There are only two amino acid differences

(479–480, RL)KI) in HR1 and HR2 regions (located in HR2) between NiV and HeV as indicated. (B) Two coiled-coil-like regions are predicted by

LearnCoil-VMF with high likelihood. (C) Schematic representation of the 2-Helix protein construct of HR1 and HR2 connected by an eight-amino-

acid linker is as indicated. (D) Helix wheel analysis of the predicted coiled-coil regions of NiV F HR1 and HR2. Please note the two mutations of

HeV relative to NiV were located at positions g and f of the helix wheel, not at a or d which are important for central HR1 trimer formation. The

mutations of RL)KI are also conserved changes.

666 Y. Xu et al. / Biochemical and Biophysical Research Communications 315 (2004) 664–670

(OD600nm) before induction with 1mM IPTG for 4 h. Bacterial cells

were harvested and lysed by sonication in phosphate-buffered saline

(PBS, 10mM sodium phosphate, pH 7.4; 150mM NaCl). The lysates

were clarified by centrifugation at 15,000 rpm for 15min at 4 �C. Theclarified supernatants were passed through Ni–NTA agarose column

(Qiagen) that was equilibrated with PBS. The histidine-tagged protein

bound column was washed with washing buffer (1� PBS, 100mM

imidazole) over 10 column volumes and eluted with elution buffer (1�PBS, 500mM imidazole) for five column volumes. The resultant pro-

tein 2-Helix was dialyzed against PBS and concentrated to a proper

concentration by ultrafiltration (5 kDa cut-off). Proteins were analyzed

by 15% SDS–PAGE. Protein concentration was determined using the

BCA protein determination assay (Pierce Biochemicals). The purified

2-Helix protein (0.5ml) was loaded onto a Superdex 75 HR10/30

(Pharmacia Biotech) column with an Akta Purifier System (Pharmacia

Biotech). The fractions of the peak were collected and analyzed by a

15% SDS–PAGE. The peak molecular weight was estimated by com-

parison with the protein standards run on the same column.

Chemical cross-linking of the complex. This was carried out essen-

tially as described earlier [28]. Briefly the gel-filtration purified 2-Helix

Y. Xu et al. / Biochemical and Biophysical Research Communications 315 (2004) 664–670 667

protein was dialyzed against cross-linking buffer (50mM Hepes, pH

8.3; 100mM NaCl) and concentrated to approximately 2mg/ml by

ultrafiltration (5 kDa cut-off). Proteins were cross-linked with ethyl-

eneglycol bis(-succinimidylsuccinate) (EGS) (Sigma). The reactions

were incubated for 1 h on ice at different concentrations of EGS, re-

spectively (0, 0.03, 0.1, 0.3, 0.7, and 1.5mM EGS), and quenched with

50mM glycine. Cross-linked samples were analyzed under reducing

conditions by 15% SDS–PAGE.

CD spectroscopic analysis. Circular dichroism (CD) spectra were

performed on a Jasco J-715 spectrophotometer. The working buffer

was PBS (10mM sodium phosphate, pH 7.3; 150mM NaCl). Wave-

length spectra were recorded at 25 �C using a 0.1-cm path-length cu-

vette. Thermodynamic stability was measured at 222 nm by recording

the CD signals in the temperature range of 20–90 �C with a scan rate of

1 �C/min. For the CD spectra of the re-natured protein, denatured

protein (at 90 �C) was cooled down on ice and then tested for CD

spectra at 25 �C.Protein crystallization. Proteins were concentrated to 20mg/ml in

25mM Tris–HCl and 25mM NaCl, pH 8.0. Initial crystallization

conditions were screened by using the hanging-drop vapor diffusion

method with sparse matrix crystallization kits (Crystal Screen I and II,

Hampton Research, Riverside, CA). One microliter of protein solution

was mixed with 1ll reservoir solution and equilibrated against 200llof the reservoir solution. The crystallization trays were kept at 18 �C.

Fig. 3. Gel-filtration analysis of the purified NiV 2-Helix protein. The

inset picture is of the proteins collected from the peak analyzed on 15%

SDS–PAGE. Clearly the symmetrical peak gives rise to a 2-Helix

protein (NP). From the estimated molecular weight in comparison

with the standards (14 and 52 kDa), the 2-Helix most likely forms

trimer (30 kDa). Results from HeV 2-Helix showed the same peak

(data not shown).

Results and discussion

HR1 and HR2 interact with each other to form a stable

complex

By using the LearnCoil-VMF program, two HR re-

gions were identified in the ectodomain of the fusion

protein for both NiV and HeV (Figs. 1A and B). HR1 is

located at the carboxyl terminus of the fusion peptide

which lies at the beginning of the F1 N-terminus. HR2 is

located adjacent to the amino terminus of the trans-

membrane domain. Consistent with that of other para-

myxovirus members, the two HR regions of these twoviruses span 275 amino acid residues, different from vi-

ruses of other families, such as Filoviridae and Retro-

viridae. Helical wheel drawing (Fig. 1D) showed that

positions “a” and “d” on both HR1 and HR2, which are

Fig. 2. SDS–PAGE analysis of the expression and purification of the 2-Helix

induced (final concentration is 1mM); lane 3, purified 2-Helix protein; and

important for coiled-coil structure [17,19], often consistof typical hydrophobic amino acids, e.g., leucine (L),

isoleucine (I) or valine (V).

The schematic representation of the construct

(23NiV-2-Helix or 23NiV-2-Helix) of HR1, linker, and

HR2 (2-Helix) is shown in Fig. 1C. The 2-Helix proteins

were expressed as soluble proteins in E. coli (Fig. 2) and

recovered from the supernatants of the cell lysates. The

purified proteins were concentrated to 20–30mg/ml inPBS and analyzed by gel-filtration and chemical cross-

linking for estimation of the molecular weight. The

2-Helix protein was eluted between the corresponding

position of 52 and 14.4 kDa (Fig. 3). In comparison, the

computed molecular weight of 2-Helix (with 6� histi-

dine) is about 10 kDa, which indicates that the 2-Helix

might form oligomers (30 kDa). Subsequently, chemical

cross-linking demonstrated that the 2-Helix protein ol-igomer was, in fact, a trimer (Fig. 4), although the

monomer/dimer bands could also be found even in high

concentrations of the cross-linker. In addition, the

content of trimer increased with the concentration

proteins of NiV (A) and HeV (B). Lane 1, Non-induced; lane 2, IPTG

lane M, molecular weight standards in kDa.

Fig. 4. Chemical cross-linking of the NiV 2-Helix protein with different

concentrations of chemical cross-linker, EGS (ethyleneglycol bis-

succinimidylsuccinate, from Sigma). Cross-linked products were sep-

arated on a 15% SDS–PAGE followed by Coomassie brilliant blue

staining. Protein markers (M) are shown in kDa. Numbers 1–6 indi-

cate the concentrations of the EGS used (1.5, 0.7, 0.3, 0.1, 0.03, and

0mM, respectively). Bands corresponding to monomer, dimer, and

trimer are indicated. Similar profiles were obtained with HeV 2-Helix

preparation (data not shown).

Fig. 6. Typical crystals of the NiV (A) and HeV (B) 2-Helix proteins

grown with hanging-drop method in 29% PEG 4000, 0.1M Tris, pH

8.5 (NiV) and 10% PEG 4000, 0.1M Hepes, pH 6.5 (HeV).

668 Y. Xu et al. / Biochemical and Biophysical Research Communications 315 (2004) 664–670

increase of the chemical cross-linker. All this indicates

that HR1 interacts with HR2, forming stable trimeric

complex.

2-Helix complex forms characteristic 6-helix bundle

The secondary structure of the 2-Helix protein was

examined by CD spectroscopy as described in Materialsand methods. The absorption curve showed that the 2-

Helix protein had double minima at 208 and 222 nm

(Fig. 5A), consistent with typical a-helix structure. The

thermal stability test indicated that trimer formation of

the 2-Helix was very stable in PBS (Figs. 5B and C),

suggesting that the 2-Helix trimer represents the core

structure of the post-fusion state of the coiled-coil

bundle, as thoroughly studied for other viruses [16–21].Denature–renature process could recover the stable he-

lix structure (Fig. 5C).

Crystals in different forms could be obtained in PEG

4000, pH 8.5, with the hanging-drop method as shown

Fig. 5. The CD spectra and thermal stability measurement of the NiV 2-H

secondary structure measured at 25 �C. (B) Thermal stability was recorded at

the re-natured 2-Helix protein from the denatured sample on 90 �C. Similar

in Fig. 6A for the NiV 2-Helix protein; in 10% PEG4000, 0.1M Hepes, pH 6.5, for HeV 2-Helix protein

(Fig. 6B). Efforts are being made currently to optimize

the crystallization conditions for better X-ray diffracting

crystals, ultimately to solve the atomic structure of this

6-helix coiled coil, for both NiV and HeV.

2-Helix proteins of NiV and HeV behave similarly

As described in Materials and methods, the 2-Helix

construct of HeV was obtained by site-directed muta-

genesis of the NiV 2-Helix. This protein was then ex-

pressed and purified essentially with the same method as

that used for NiV 2-Helix (Fig. 2B). All the biochemicaland biophysical analyses of the HeV 2-Helix protein, in-

cluding gel-filtration, chemical cross-linking, and circular

dichroism, were carried out and the results show clearly

that HeV 2-Helix also forms stable 6-helix bundle (data

not shown). This indicates that the two-amino-acid

polymorphism in the HR2 region of HeV does not affect

the binding ofHR1 andHR2. In an earlier report, Bossart

et al. [26] showed that HR2 derived from either HeV or

elix proteins in PBS. (A) The 2-Helix proteins show a typical a-helix222 nm. The Tm is approximately 85 �C. (C) The CD spectra at 25 �C of

results were recorded with HeV 2-Helix protein (data not shown).

Y. Xu et al. / Biochemical and Biophysical Research Communications 315 (2004) 664–670 669

NiV gave cross-inhibition to NiV or HeV fusion withsimilar effects in an in vitro cell system. These data indi-

cate that the HR2 of NiV and HeV is structurally and

functionally interchangeable. This is clearly consistent

with the conserved two-amino-acid polymorphism

(RL)KI) in the HR2 region between the two viruses.

Implications for virus–cell fusion and entry of NiV and

HeV

NiV and HeV have been convincingly identified as

members of Family Paramyxoviridae with some specific

characters, forming a new genus in the family termed

Genus Henipavirus [5,6,8,9]. Our results here showedthat their heptad repeat regions, HR1 and HR2, interact

with each other to form typical stable 6-helix coiled-coil

bundle, which represents the fusion core of the F protein

[16,17]. This provides further evidence that both viruses

are typical paramyxoviruses in terms of virus entry.

Bossart et al. [26] found that HR2s derived from either

NiV or HeV are potent fusion inhibitors for both viruses

and the inhibition effects are interchangeable. This isconsistent with the observations made in this study. The

basis of this interchangeable interaction awaits a solu-

tion by crystal structures of the 6-helix bundles.

The fusion and entry of the paramyxovirus need two

envelope glycoproteins, attachment and fusion proteins.

In the case of NiV and HeV, they are G and F glyco-

proteins. The fusion of most paramyxoviruses requires

both glycoproteins but with some exceptions, e.g., SV5.It is shown that both G and F proteins are required for

NiV and HeV [12,13,26]. HR1 inhibition to membrane

fusion or virus infection of NiV or HeV has not been

tested yet. The inhibition by HR1 might correlate with

the attachment protein requirement: HR1 derived from

those viruses whose fusion absolutely requires attach-

ment proteins does not give rise to fusion inhibition

[32,36–38], whereas HR1 derived from other memberswhose fusion does not absolutely require the attachment

protein does show strong inhibition of virus fusion and

entry [32,39]. It will be of interest to test the HR1 in-

hibition of NiV and HeV in future studies. Evidence

from previous observations of other paramyxoviruses

would suggest that this would not lead to inhibition.

Clear structural analysis of HR 6-helix bundle of NiV

and HeV would provide a detailed picture of the viralfusion core structure and the molecular mechanism

underlying the interchangeable interaction of HR1 with

both NiV and HeV HR2. Our strategy here will be

followed for a structural solution. This will inevitably

add to the repertoire of paramyxovirus 6-helix bundle

fusion core structures, of which only two are available at

present (that of RSV and SV5) [40,41]. This will also

open a new avenue toward the structure-based fusioninhibitor design of peptides, or peptide analogues, e.g.,

small molecules, for these emerging infectious diseases.

Acknowledgments

We thank Dr. Jieqing Zhu for his help during the experimental

procedure and Dr. Catherine Zhang for critical reading of the manu-

script. This work was supported by Project 973 of Ministry of Science

and Technology of China (Grant No. G1999011902). G.F.G.’s stay at

the Tsinghua University was supported by Chunhui Project Scheme of

Ministry of Education, China.

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