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Journal of General Virology (1993), 74, 311 314. Printed in Great Britain
31 l
Importance of conserved amino acids at the cleavage site of the
haemagglutinin of a virulent avian influenza A virus
John A. W a l k e r and Yoshihiro K a w a o k a *
Department of Virology and Molecular Biology, St Jude Children's Research Hospital, 332 North Lauderdale,
PO Box 318, Memphis, Tennessee 38101-0318, U.S.A.
The virulence of avian influenza A viruses depends on
the cleavability of the haemagglutinin (HA) by an
intracellular protease at multiple basic amino acids.
Although previous studies have demonstrated the importance of these amino acids for processing by the
cellular protease, with emphasis on conserved residues
near the cleavage site, the minimal requirements for
cleavage remain unknown. By expressing site-specific
mutants of the HA of a virulent avian influenza virus,
A/turkey/Ireland/1378/85 (H5N8), in the simian virus
40 system and testing for their cleavability by an
endogenous protease in CV-1 cells, and their fusion
activity in a polykaryon formation assay, we were able to
show that glycine at the amino terminus of HA2 is not
essential for cleavage and that maximal cleavage requires
at least five basic residues at the cleavage site, when
carbohydrate is nearby. Moreover, we confirmed, that a
conserved proline upstream of the cleavage site is not
essential for HA cleavage or fusion activity, and that
lysine replacement of the carboxyl-terminal arginine of
HA1 abolishes cleavability. These findings should help
identify the proteases responsible for intracellular cleavage of the HA of virulent avian influenza viruses.
The haemagglutinin (HA) molecule of influenza viruses
is responsible for attachment of the virus to cell surface
receptors and subsequent fusion of viral and cell
membranes leading to internalization of the nucleocapsid
(Lamb, 1989; Wiley & Skehel, 1987). Before this process
can take place, the HA molecule must undergo a low pHinduced conformational change preceded by specific
cleavage of the molecule into two disulphide-linked
moieties, HA1 and HA2 (Lamb, 1989; Waterfield et al.,
1980). HA cleavage in tissue culture by an endogenous
cellular protease correlates with the presence of multiple
basic amino acids at the site of cleavage (Bosch et al.,
1981; Kawaoka & Webster, 1988). Moreover, this
property appears limited to virulent strains of influenza
virus (Bosch et al., 1981).
Intracellular HA cleavage requires basic residues at
the cleavage site (Kawaoka & Webster, 1988). Additional
basic amino acids are required upstream, in the so-called
connecting peptide, to prevent the interference by
glycosylation at the amino-terminal region of HA1
(Kawaoka & Webster, 1989). Although previous studies
established the positions of the basic residues needed for
complete HA cleavage (Kawaoka & Webster 1988,
1989), they did not address the minimal number or
nature of the additional residues required for maximal
HA cleavage.
In addition to the basic residues at the site of HA
cleavage, several other amino acid residues surrounding
the cleavage site are conserved in all 14 subtypes of
influenza A virus (Nobusawa et al., 1991; Kawaoka et
al., 1990), even in those HAs cleaved at a single arginine.
Although basic amino acids at this site are essential for
intracellular HA cleavage (Kawaoka & Webster, 1988),
the conserved residues may also be important for
recognition by an intracellular protease. In this study, we
investigated the importance of two structural features at
the cleavage site: conserved residues and the number of
basic amino acids.
To determine the structural requirements for HA
cleavage by an intracellular protease, we expressed
mutant HAs in the simian virus 40 (SV40) system in CV1 cells and examined their cleavability and fusion activity.
There were no appreciable differences in cell surface
expression as determined by haemadsorption between
the site-specific mutant and parent HAs. Furthermore,
when these mutant HAs had been trypsinized prior to the
subsequent assays, they were all cleaved into HA1 and
HA2 moieties and were indistinguishable by fusion
activity (data not shown).
Comparison of the amino acid sequences at the
cleavage site of the HA revealed conservation, among all
the HAs examined to date, of two amino acids: the
proline proximal to the carboxyl terminus of the HA1
and the amino-terminal glycine of the HA2. An arginine
residue, the carboxyl-terminal residue of the HA1, was
conserved in all but the H14 strains (Nobusawa et al.,
0001-1255 © 1993SGM
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312
Short communication
1
2
3
4
5
6
Table 1. Amino acid sequences at the cleavage site of
H A mutants and their biological properties*
HAO
HA1
:
....
::
Mutant
HA
Parent
MT2J;
MTI2$
MT22
MT23
MT25
MT34
MT41
MT42
Amino acid sequence
at the cleavage site
HA1 / HA2
P QRK RKKR
/G
P QOI"]R K K R / G
P Q['T'][E-']R K K R / G
P QRKRKKR
/[A--]
[Y-]QRKRKKR
/G
['G']QR K R K K R / G
P QRKRKK[K-]/G
P QI'T"]K R K K R / G
P Q[]KRKKR
/G
Cleavage
+
+
+
+
+
-+
_+
Polykaryon
formation % t
100
11
29
40
100
92
10
90
60
- - HA2
Fig. 1. Comparison of the cleavage of mutant HAs by an intracellular
protease. The plasmid pTH29, which contains the full-length HA gene
from A/turkey/Ireland/1378/85 (Kawaoka & Webster, 1988), was
used to generate site-directed mutants by the method of Kunkel (1985).
SV4(~HA recombinant viruses were prepared as described (Kawaoka
& Webster, 1988). Cleavage of the HA was examined in infected
monolayers of CV-1 cells labelled with [aH]mannose and [aH]glucosamine, as previously described (Kawaoka & Webster, 1988). Cell
lysates were immunoprecipitated with anti-H5 antiserum and analysed
on a polyacrylamide gel. Lane 1, wild-type. Lanes 2 to 7, MT22, -23,
-25, -34, -41 and -42, respectively.
1991; Kawaoka et al., 1990). The strictly conserved
proline upstream of the cleavage site may be important
in presentation of the cleavage site to an intracellular
protease owing to its strong turn-breaking potential. We
therefore constructed two mutant HAs, MT23 and
MT25, in which the conserved proline was changed to
tyrosine and glycine, respectively. In neither case was
there a significant change in either cleavability by
intracellular proteases or in fusion activity (Fig. 1, Table
1), indicating that the proline residue is not essential for
recognition by the protease.
The amino acid residue immediately downstream of
the cleavage site affects the cleavability of prorenin,
which is processed at multiple basic amino acids (Oda
et al., 1991). We therefore examined the importance of
the conserved first residue of HA2, a glycine, for HA
cleavage. The semiconservative substitution from glycine
to alanine (MT22) did not affect the cleavability of the
HA (Fig. 1), although fusion activity was reduced to
about 40 % of the level of the wild-type HA (Table 1).
This indicates that the glycine at the amino terminus of
the HA2 is not conserved for cleavability but because it
promotes the fusion activity.
Although the H14 HA contains lysine at the carboxyl
end of the HA1 (Kawaoka et al., 1990), all known viral
glycoproteins and prohormones containing multiple
basic amino acids at the cleavage site have arginine
immediately upstream of the cleavage site (Barr, 1991).
To determine the importance of this residue for cleavage
* Mutated or deleted amino acids are shown in boxes; cleavage sites
are indicated by slashes (/), basic amino acids by boldface type. +,
cleaved; + , partially cleaved; - , uncleaved.
t The extent of cell fusion was determined by calculating the average
percentage of fused cells in three microscopic fields.
:~ These previously reported mutants (Kawaoka & Webster, 1988)
are included for comparison.
by a cellular protease, we made a mutant HA, MT34,
containing lysine substituted for arginine. The MT34
was totally resistant to cleavage by an endogenous
protease in CV-1 cells (Fig. 1), with a concomitant
absence of cell fusion activity (Table 1), indicating the
absolute requirement of an arginine residue for cleavage
by this protease.
Deletion or change of basic amino acids to non-basic
residues at both the fifth (P5) and sixth (P6) positions
from the carboxyl terminus of the HA1 either abolishes
or severely inhibits cleavage, when carbohydrate is in the
proximity of the cleavage site (Kawaoka & Webster,
1988). To determine the minimal structural requirement
for cleavage of the HA in the presence of carbohydrate,
we mutated arginine at the P6 position, leaving the P5
lysine intact. The mutants MT42 and MT41 contained a
deletion or a change from arginine to threonine,
respectively. The substitution at the P6 residue (MT41)
affected cleavability and fusion activity only marginally
(Fig. 1, Table 1), whereas deletion of this residue (MT42)
resulted in partial cleavage (Fig. 1) with a concomitant
reduction in fusion activity (Table 1). The difference in
cleavability between these mutants was reproducible
(data not shown). Hence, at least five basic residues are
required for maximal cleavage by an intracellular
protease.
In this study, the strictly conserved proline proximal to
the cleavage site was not essential for HA cleavage by
intracellular proteases; however, the conservative change
of the arginine at the carboxyl end of the HA1 to another
basic residue, lysine, prevented HA cleavage by proteases. All viral glycoproteins and prohormones cleaved
at multiple basic residues contain an arginine immediately upstream of the cleavage site (Hosaka et al., 1991,
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Short communication
Dong et aL, 1992, Kawaoka & Webster, 1988). In a
spontaneous mutation of this residue to a lysine in the
envelope protein of a murine leukaemia provirus,
infectivity was severely impaired (Freed & Risser, 1987),
supporting the absolute requirement for an arginine at
this position. Recently, Vey et al. (1992) obtained results
identical to ours by replacing the proline and arginine
residues in a virulent avian influenza virus of the H7 HA
subtype. The important role, in the cellular proteaseinduced cleavage, of the first (Vey et al., 1992), second
(Kawaoka & Webster, 1988) and fourth (Kawaoka &
Webster, 1988) basic residues from the carboxyl end of
the HA1, and the irrelevance of the third (Kawaoka &
Webster, 1988) basic residue, led Vey et al. (1992) to
recognize a conserved sequence motif, Arg-xxx-Arg/LysArg, found at the cleavage site of virulent avian virus
HAs (Kawaoka et al., 1987). Results of the present study
support that observation.
The significance of the glycine residue at the HA2
amino terminus for recognition of the HA cleavage
enzyme was not clear at the outset of this study.
Alteration of the amino acid residue immediately
downstream of the prorenin processing site affected the
cleavability of the proprotein (Oda et al., 1991),
suggesting a possible role for this residue in cleavage by
a cellular protease at a site of multiple basic amino acids.
This prediction was not substantiated by our results; that
is, substitution of alanine for the highly conserved
glycine at the amino terminus of the HA2 (MT22) did not
alter cleavability. Thus the glycine is conserved not for
HA cleavage but for fusion activity (Garten et al., 1981 ;
Gething et al., 1986), although alanine residues are found
at the analogous position of human immunodeficiency
virus type 1 gp41, the fusion peptide of the molecule
(McCune et al., 1988).
Ohuchi et al. (1989) reported that the HAs of variant
H5 viruses containing five basic residues at the cleavage
site are cleaved by intracellular proteases, but the extent
was limited and the importance of the P6 arginine
residue was not examined. In the present study, we
systematically examined the relation of the number of
basic amino acid residues at the HA cleavage site to the
cleavability of this molecule. We showed that the
deletion, but not the alteration, of the P6 arginine
reduces cleavability when the HA contains the carbohydrate near the cleavage site. Hence the P6 residue may
be important for the abolition of possible steric hindrance
of HA cleavage by the carbohydrate, but it does not have
to be basic in nature, at least for cleavage by an
endogenous protease in CV-1 cells. The basic nature of
the P5 position, by contrast, is critical for HA cleavage,
because alteration of the P6 and P5 residues to non-basic
residues appreciably reduces cleavability (Kawaoka &
Webster, 1988). Thus, for the H5 HA-containing
313
carbohydrate, at least five basic amino acids are required
for maximal HA cleavage.
Because of the structural similarities noted for cleavage
of viral glycoproteins and prohormones, understanding
the processing of prohormones by eukaryotic cellular
proteases would probably contribute to our understanding of viral glycoprotein activation. The search for
mammalian enzymes that cleave prohormones recently
led to identification of calcium-dependent subtiiisin-like
proteases (Barr, 1991). After identification and structural
determination of a yeast protease, Kex2, that cleaves the
killer toxin at dibasic sites (Fuller et al., 1989), three
eukaryotic homologues, furin, PC2 and PC1 or PC3,
were identified (for a review, see Barr, 1991). These
enzymes differ in cleavage specificities and tissue tropism.
Whereas both PC2 and PC3 cleave at the dibasic residues
R-R, K-R or K-K (Korner et al., 1991 ; Benjannet et al.,
1991; Thomas et al., 1991), furin cleaves at tetrabasic
(R-X-K/R-R) (Nagahama et al., 1991) and/or dibasic
(K-R or R-R) sequences (Bresnahan et al., 1990).
Moreover, furin is ubiquitously and constitutively expressed (Hatsuzawa et al., 1990) and located in the Golgi
complex (Misumi et al., 1991), whereas both PC2 and
PC3 are inducible and tissue-specific (Birch et al., 1991),
and both appear to act beyond the Golgi complex
(Nagahama et al., 1991). Thus, the structural requirements (Kawaoka & Webster, 1988; Dong et al., 1992;
Hosaka et al., 1991) and intracellular location (Walker et
al., 1992; Nagahama et al., 1991) of viral glycoprotein
cleavage suggest that furin, or a closely related enzyme,
is the cleavage protease. Indeed, Stieneke-Gr6ber et al.
(1992) recently reported that human furin and a protease
antigenically cross-reacting with furin in Madin-Darby
bovine kidney cells cleave the HA of another virulent
virus, A/FPV/Rostock/34 (H7N1). The availability of
genes encoding prohormone processing enzymes, together with mutant influenza virus HA genes, should
allow a detailed study of HA-protease interactions.
We thank Dr Robert G. Webster for TN/USSR virus and an antiH5 serum, and John Gilbert for editorial assistance. This work was
supported by Public Health Service grant AI-29599 from the National
Institute of Allergy and Infectious Diseases, Cancer Center Support
(CORE) grant CA-21765 and American Lebanese Syrian Associated
Charities (ALSAC).
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