Download Review Molecular pathogenesis of H5N1 influenza virus infections

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Antiviral Therapy 12:617–626
Review
Molecular pathogenesis of H5N1 influenza virus
infections
Gabriele Neumann1*, Kyoko Shinya2 and Yoshihiro Kawaoka1,3
1
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA
The Avian Zoonosis Research Centre, Tottori University, Tottori, Japan
3
Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, Institute
of Medical Science, University of Tokyo, Tokyo, Japan
2
*Corresponding author: Tel: +1 608 263 7114; Fax: +1 608 262 9641; E-mail: [email protected]
Highly pathogenic H5N1 influenza viruses have become
endemic in poultry populations throughout Southeast
Asia and continue to infect humans with a greater than
50% case fatality rate. So far, human-to-human
transmission of these viruses has been limited. Here, we
discuss the molecular features of H5N1 influenza viruses
that might affect their pathogenicity, and explain the
current lack of efficient human-to-human transmission.
Such knowledge is critical in evaluating the pandemic
risk these viruses pose.
Introduction
Influenza
viruses
belong
to
the
family
Orthomyxoviridae, which is comprised of five genera.
Only one genus – Influenzavirus A – causes pandemic
disease and will, therefore, be the focus of this article.
Influenza A viruses are further classified into subtypes
based on the antigenicity of their haemagglutinin (HA)
and neuraminidase (NA) molecules (reviewed in [1]).
To date, 16 HA subtypes (H1–H16) and nine NA
subtypes (N1–N9) have been described. Despite the
large number of potential combinations, viruses of
only a limited number of subtypes (H1N1, H1N2,
H2N2, H3N2) have circulated in humans.
The genome of influenza A viruses is composed of
eight segments of single-stranded, negative-sense RNA
that each encode one or two proteins (reviewed in [2]).
The enveloped viruses possess two surface glycoproteins
(HA and NA). These proteins are critical for virus
binding and internalization, as well as for the release
of newly assembled virions from infected cells. The
M1 matrix protein is thought to form a matrix
underneath the lipid bilayer. The virions contain eight
viral ribonucleoprotein (vRNP) complexes, each
composed of a viral RNA (vRNA) segment and its
attached polymerase proteins (PB2, PB1 and PA). The
vRNAs are wrapped around the nucleoprotein NP.
Other viral proteins include the M2 ion channel
protein, the interferon antagonist NS1, the nuclear
© 2007 International Medical Press 1359-6535
export protein NEP and the PB1-F2 protein, which is
encoded by a second reading frame in segment 2 and
might induce apoptosis [3].
Influenza pandemics
Influenza viruses cause a highly contagious respiratory
disease that manifests in local epidemics or global
pandemics. Last century, there were three pandemics
that killed an estimated 20 million–50 million people
worldwide in 1918/1919 (‘Spanish influenza’), about
70,000 people in the US in 1957 (‘Asian influenza’)
and about 33,800 people in the US in 1968 (‘Hong
Kong influenza’). How new pandemic viruses emerge is
poorly understood, although the introduction of a new
HA subtype into immunologically naive populations is
a key factor. Viruses containing a new HA subtype
can be introduced into human populations via the
transmission of a wholly avian influenza virus (which
probably occurred in the 1918 influenza pandemic) or
from human/avian reassortant viruses (which caused
the 1957 and 1968 pandemics).
Highly pathogenic H5N1 influenza viruses
Most avian influenza viruses do not replicate efficiently
in humans [4] and their direct transmission to humans
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was therefore considered a rare event that posed little
threat. This assumption proved wrong in Hong Kong
in 1997, when 18 individuals, six of whom died, were
infected with highly pathogenic avian influenza
viruses of the H5N1 subtype [5–7]. In parallel, H5N1
virus outbreaks occurred in live poultry markets in
Hong Kong. From 1997 to 2003, these virus
outbreaks were confined to Southeast Asia and
involved few human infections.
In 2003, however, an outbreak of H5N1 avian
infections started that has since spread throughout
Southeast and South Asia and has been accompanied
by an increasing number of human infections. The
viruses responsible for this outbreak have become
endemic in poultry populations in Southeast Asia.
The next remarkable event in H5N1 virus evolution
occurred in 2005 at Qinghai Lake, China, when
thousands of waterfowl succumbed to H5N1 virus
infection [8–10]. Waterfowl are the natural reservoir of
influenza viruses and, as such, are typically asymptomatic
to infection with highly pathogenic influenza viruses.
The Qinghai Lake H5N1 virus sublineage is now
dominant in northern China and has spread to Europe
and Africa [9].
Since 2003, the number of human infections with
highly pathogenic H5N1 viruses has increased, with a
fatality rate in humans that exceeds 50%. Moreover,
several family clusters of H5N1 virus infections have
now been reported [11–13]. Equally alarming is the
increased pathogenicity of recent H5N1 viruses in mice
and ferrets [14,15], and the ability of some recent H5N1
viruses to cause systemic infections in humans [16]. Both
of these findings suggest that highly pathogenic H5N1
viruses are adapting to mammalian species. Here, we
review the molecular features that might determine the
pathogenicity and transmissibility of highly pathogenic
H5N1 influenza viruses. Pathogenicity is defined as
the ability to cause disease, while transmissibility
refers to the ability of the infectious agent to spread
among host organisms. Currently, the relationship
between these two properties is not understood. For
example, high pathogenicity might not be required for
transmissibility, but can result in high virus loads
and/or systemic infections which might facilitate
virus transmission.
Roles of the HA protein and virus receptor
distribution in virus pathogenicity
Efficient virus binding to host cells is critical to virus
dissemination during epidemics and pandemics. Both
the binding to host cell receptors and the subsequent
fusion of the viral and cellular membranes are mediated
by the HA protein, which is, for these reasons, an
important determinant of virulence and host restriction.
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Receptor specificity of the HA protein and receptor
distribution on host cells
Historically, host range restriction of influenza viruses
was explained as a mismatch between the HA receptor
binding specificity and the receptor distribution on host
cells: Epithelial cells in the human trachea contain on
their surface sialic acid (SA) that is predominantly
linked to galactose by an α2,6-linkage (SA-α2,6-Gal)
[17,18], which is preferentially recognized by human
influenza viruses [19–22]. By contrast, human
influenza viruses do not efficiently bind to SA-α2,3-Gal
sialyloligosaccharides, which are predominantly
expressed by epithelial cells in the intestinal tract of
waterfowl (the main replication site of avian influenza
viruses). SA-α2,3-Gal sialyloligosaccharides are efficiently recognized by avian influenza viruses [19–22],
resulting in the infection of avian, but not human, cells.
This explanation, however, proved too simplistic,
when a wholly avian H5N1 influenza virus isolated
from an infected individual in 1997 in Hong Kong was
shown to bind to SA-α2,3-Gal, but not to SA-α2,6-Gal,
sialyloligosaccharides [23], suggesting that viruses with
‘avian-type’ receptor binding specificity can infect
human cells. This prompted researchers to reexamine
the distribution of influenza virus receptors in human
respiratory organs, which led to the finding of a more
complex pattern than was previously thought. Studies
with in vitro-differentiated human epithelial cells from
tracheal and bronchial tissues suggested that non-ciliated
epithelial cells (that is, most epithelial cells) contain SAα2,6-Gal sialyloligosaccharides on their surfaces,
whereas ciliated cells (a minor epithelial cell population)
express SA-α2,3-Gal sialyloligosaccharides [24]. This
distribution of sialyloligosaccharides corresponds to
the preferential infection of non-ciliated cells by human
influenza viruses [24].
The finding that human cells contain ‘avian-type’
influenza virus receptors (that is, those expressing SAα2,3-Gal sialyloligosaccharides) raised two fundamental
questions: If avian influenza viruses can infect humans,
why are these infections rare, and why do these viruses
not spread among humans? Possible explanations
come from a study that examined receptor distribution
on human respiratory tissue and found appreciable
differences between tissues of the upper and lower
respiratory tract [25]. Epithelial cells in nasal mucosa,
paranasal sinuses, pharynx, trachea and bronchi
express primarily SA-α2,6-Gal sialyloligosaccharides
[25], whereas the cells that line the alveolar walls
express SA-α2,3-Gal sialyloligosaccharides [25]. This
finding, and a similar observation by others [26],
confirmed the presence of ‘avian-type’ influenza virus
receptors on human cells. In addition, it might explain
the low frequency of human infection by avian
influenza viruses and the current inability of these
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Factors for influenza H5N1 virus pathogenicity
viruses to efficiently transmit among humans: SA-α2,3Gal sialyloligosaccharides are confined to the lower
respiratory tract of humans, which greatly reduces the
odds of avian influenza virus infection. Moreover, the
lack of ‘avian-type’ receptors in the upper airways
probably prevents efficient virus replication in this
setting and thereby transmission, which occurs via
droplets generated by coughing and sneezing. Recently,
Nicholls et al. [27] challenged this concept by demonstrating infection of upper respiratory organs with an
H5N1 avian virus. Also, de Jong et al. [28] reported
that individuals infected with recent H5N1 viruses had
higher vRNA levels in the pharynx than individuals
infected with human H3N2 or H1N1 viruses. Overall,
further quantitative analyses should be carried out to
assess avian influenza virus binding to and infection of
cells in the upper respiratory tract of humans.
Influenza virus receptor binding specificity is primarily
determined by the amino acids that line the receptor
binding pocket. Several studies have identified residues
that are crucial for binding to SA-α2,3-Gal or SA-α2,6Gal sialyloligosaccharides: For H2 and H3 influenza
viruses, Glu226 and Gly228 confer specificity for ‘aviantype’ receptors, whereas Leu226 and Ser228 mediate
efficient binding to SA-α2,6-Gal sialyloligosaccharides
[19,22,29,30]. Consequently, the HA proteins of avian
influenza viruses encode Glu226 and Gly228, whereas
human influenza virus HA encodes Leu226 and
Ser228. For H1 influenza viruses, the amino acid at
position 190 of HA is crucial: Glu190 in avian
influenza viruses mediates efficient binding of SA-α2,3Gal sialyloligosaccharides, whereas Asp190 in human
and swine influenza viruses confers specificity to
human-type receptors [29,31–33]. Recently, Tumpey et
al. [34] reported that conversion of Asp to Glu at
position 190 and Asp to Gly at position 225 converts
the receptor specificity of the pandemic 1918 virus
from the human to avian type. This conversion does
not significantly reduce virus titres in nasal washes and
is still associated with severe disease in inoculated
animals, but abolishes transmissibility of the virus
among ferrets, indicating the importance of receptor
specificity for efficient transmissibility.
Most avian H5N1 influenza viruses possess ‘aviantype’ amino acids in their HA and, therefore, bind to SAα2,3-Gal but not to SA-α2,6-Gal sialyloligosaccharides
[23,35]. All 1997 human H5N1 isolates tested showed
‘avian-type’ receptor binding specificities [23,35]. In
2003, however, two H5N1 viruses, isolated from
infected individuals in Hong Kong, showed decreased
levels of binding to SA-α2,3-Gal sialyloligosaccharides and low but detectable levels of binding to SAα2,6-Gal sialyloligosaccharides [35]. Both viruses
contained a unique Ser-to-Asn substitution at position
227 of the HA1 protein, confirming that a single
Antiviral Therapy 12:4 Pt B
amino acid change is sufficient to affect HA receptor
binding specificity. Stevens et al. [36] solved the threedimensional structure of the HA of an H5N1 virus
and showed that replacement of two amino acids at
positions 226 and 228 (H3 numbering) can convert
avian-like to human-like receptor binding specificity.
Another study demonstrated that three of 21 recent
human H5N1 isolates recognize both sialyloligosaccharides [37]; by contrast, none of the avian H5N1 viruses
tested recognized SA-α2,6-Gal sialyloligosaccharides.
Further analysis has identified two amino acid changes,
Asn182 to Lys and Gln192 to Arg (equivalent to
positions 186 or 196 in H3 numbering, respectively)
that independently converted ‘avian-type’ to ‘humantype’ receptor specificity [37]. The HA crystal structure of
the respective HA protein [37] provides an explanation
for how these amino acid substitutions alter receptor
specificity. These amino acid substitutions were also
found in recent human H5N1 viruses isolated from
two individuals in Azerbaijan and one individual in
Iraq. During replication in humans, highly pathogenic
H5N1 viruses can acquire amino acid changes that
support their replication in humans and potentially
increase their likelihood of becoming pandemic
(Figure 1). However, these viruses did not transmit
efficiently among humans, indicating that additional
factors are necessary.
HA cleavage
The HA protein is synthesized as a precursor protein
(HA0) that is post-translationally cleaved into two
disulphide-linked subunits, HA1 and HA2. The
hydrophobic N-terminus of HA2 (the so-called ‘fusion
peptide’) inserts into the endosomal membrane, initiating
fusion between the viral and endosomal membranes.
Therefore, without HA cleavage, influenza virus is
not infectious.
HA cleavability is a critical determinant of virulence
and pathogenicity [38–40]. The cleavability of the HA
protein is determined by the amino acid sequence at the
cleavage site. Single Arg residues at the HA cleavage
site of avirulent avian and non-avian influenza A
viruses [41,42] (with the notable exception of H7N7
equine influenza viruses), are cleaved by a limited
number of proteases in the respiratory tract and/or
intestinal organs, resulting in localized infections with
typically mild or no disease symptoms. By contrast,
highly pathogenic H5 and H7 viruses cause systemic
infections because the multiple basic amino acids at the
HA cleavage site of these viruses are recognized by
ubiquitous proteases, such as furin and PC6. The
proposed consensus motif for furin recognition is QR/K-X-R/K-R (where X is a non-basic amino acid) in
the absence of a nearby carbohydrate chain. The
presence of such a carbohydrate chain requires
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Figure 1. Hypothetical scenario for the emergence of pandemic influenza viruses
HA, PB2, etc
Cells in the lower respiratory tract of humans contain ‘avian-type‘ influenza virus receptors, in contrast to cells in the upper respiratory tract. This may explain the
ability of avian viruses (pale grey) to infect humans and cause disease. The inability of these viruses to replicate in the upper respiratory tract might prevent efficient
human-to-human transmission. If the avian viruses acquire mutations in HA, PB2 and other proteins that allow efficient replication in the upper respiratory tract
(dark grey), efficient human-to-human transmission may occur, resulting in a pandemic.
extended motifs of the following kind: B(X)-X(B)-R/KX-R/K-R or Q-X-X-R-X-R/K-R (where X represents
non-basic amino acids and B, basic amino acids).
Avian influenza viruses of low pathogenicity can
become highly pathogenic through the acquisition of
multibasic HA cleavage sites, a finding that further
demonstrates the importance of HA cleavability for the
pathogenicity of avian influenza viruses. Examples
include outbreaks of highly pathogenic H5N2 viruses
in Pennsylvania in 1983 [43] or in Mexico in 1994
[44], of highly pathogenic H7N1 viruses in Italy in
1999 [45], or of highly pathogenic H7N3 viruses in
Chile in 2002 [46] or in Canada in 2004 [47].
The role of terrestrial poultry in the acquisition
of HA mutations
Influenza viruses from terrestrial poultry display
reduced affinity for SA-α2,3-Gal sialyloligosaccharides
relative to viruses isolated from waterfowl [23]. Like
human virus isolates, land-based poultry viruses of
different subtypes (including H7N1 and H9N2) have
an extra glycosylation site in their HA and a deletion in
their NA stalk. These features were also found in the
HA and NA proteins of highly pathogenic H5N1
poultry viruses [23,48–50]. Terrestrial poultry might,
therefore, serve as an intermediary host for avian
influenza viruses to acquire mutations that support
their transmission to humans [23,51]. Avian H5N1
influenza viruses can also be transmitted directly from
aquatic birds to humans, as was demonstrated by the
isolation from infected individuals of two H5N1
influenza viruses isolated in 2003 that lacked the
typical features of poultry adaptation [52].
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Role of the replication complex in virus
pathogenicity
Given that the ability of an influenza virus to cause
disease probably depends on its ability to replicate
efficiently to produce high virus loads and outpace
cellular antiviral responses, it is not surprising that the
components of the viral replication complex are
involved in viral pathogenicity.
Influenza virus replication is mediated by the three
polymerase proteins (PB2, PB1 and PA) and the nucleoprotein NP. PB2, which binds to type 1 cap structures
of cellular mRNAs, and NP, which encapsidates
influenza vRNAs, have been implicated in host range
restriction [53–55]. A human virus containing an
avian virus PB2 gene required a ‘human-like’ amino
acid at position 627 of the PB2 protein to form
plaques in Madin–Darby canine kidney cells [55].
This finding was not fully appreciated until 2001,
when the pathogenicity of H5N1 influenza viruses in
mice was linked to the nature of the amino acid at
position 627 of PB2 [56]. Most human influenza
viruses contain Lys at this position (and a few possess
Arg). Lys is also found at PB2-627 in the 1997 H5N1
viruses that were highly pathogenic in mice. By
contrast, the 1997 H5N1 viruses that were of low
pathogenicity in mice contain Glu at this position,
which is also found in all avian viruses (with the
exception of the Qinghai Lake H5N1 viruses and
their descendants). Reverse genetics experiments
demonstrated that replacement of PB2-627Glu with
Lys renders viruses highly pathogenic, whereas the
reciprocal experiment generates a variant of low
pathogenicity in a mouse model [56]. PB2-627Lys
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Factors for influenza H5N1 virus pathogenicity
continues to be found in a substantial number (albeit
not all) of H5N1 viruses isolated from infected humans
[28,57–59], and has been found in H5N1 viruses
isolated from tigers in Thailand in 2004 and 2006 [60].
Interestingly, PB2-627Lys was also found in an H7N7
virus isolated from a fatal case of pneumonia in the
Netherlands in 2003 [61]. By contrast, virus isolates
from nonfatal cases and from chickens in this
outbreak contained Glu at this position. PB2-627Lys
thus appears to be selected during replication in
mammals. Proof for this concept comes from the finding
that chicken H5N1 viruses isolated from the brains of
infected mice all contained this substitution [62].
However, during the Qinghai Lake outbreak in China
from May to July 2005 [9,63], avian H5N1 viruses were
isolated that encoded PB2-627Lys. Descendants of the
Qinghai Lake viruses have maintained this mutation
and continue to circulate in northern China and to
cause outbreaks in Europe and Africa, attesting to
their biological fitness. For H5N1 viruses isolated
from infected individuals, both Lys and Glu have
been found at PB2-627. This might be explained by
differences in sampling time points.
How does PB2-627 affect viral pathogenicity?
Several studies have shown that the amino acid at PB2627 determines the replicative ability of the virus and
affects host range [64–67]. Viruses with PB2-627Lys
grow more efficiently in mouse, but not avian, cells
compared with those containing Glu at this position
[64]. Moreover, an artificial mini-replicon system
showed higher replication levels for PB2 proteins
encoding 627Lys than for those encoding 627Glu [67].
The amino acid at position 627 of PB2 does not,
however, affect the tissue tropism of the virus in mice
[64]. Collectively, these findings indicate that PB2627Lys supports virus replication in mammalian cells
and provide an explanation for its selection in
mammalian species. Viruses containing PB2-627Lys
probably replicate to high titres in mammals
resulting in efficient virus dissemination and the
ability to overwhelm the host defences.
The amino acid at position 627 of PB2 is now widely
recognized as a crucial determinant of H5N1 virus
pathogenicity. However, other amino acids in the viral
replication complex also affect viral replication and
pathogenicity. Two studies suggest an important role
for amino acid PB2-701 in viral pathogenicity [68,69].
An Asn-to-Asp replacement at this position attenuated
a duck H5N1 virus in mice, whereas the reciprocal
replacement enabled an otherwise non-pathogenic duck
H5N1 virus to replicate in mice [68]. Similar findings
were made with an H7N7 virus, in which PB2-701Asn
conferred superior replicative abilities relative to PB2701Asp [69]. The underlying molecular mechanism for
this effect is unknown.
Antiviral Therapy 12:4 Pt B
In another study [70], exchanging the HA and NA
genes of H5N1 viruses of high and low pathogenicity
did not alter their pathogenicity, yet pathogenicity in
mice and ferrets was determined by the origin of the
three polymerase genes [70]. In vitro assays revealed
higher replicative abilities for the replication complex
of the highly pathogenic variant compared with those
of the less pathogenic variant [70]. Thus, as observed
with viruses containing PB2-627Lys or PB2-627Glu,
increases in replicative ability can translate to increased
pathogenicity.
Collectively, these findings suggest that human-type
receptor binding specificity and efficient replication in
mammals (the latter mediated by mutations in the
replication complex) probably facilitate efficient viral
replication in humans. It was feared that the combination
of these two features would produce highly pathogenic
and potentially pandemic viruses. However a human
H5N1 virus isolated in 2006 in Turkey that contained
the PB2-627Lys mutation and a mutation in HA that
facilitated its binding to human-type receptors [71] did
not cause a pandemic, suggesting that yet other
mutations are needed for efficient human-to-human
transmission.
Role of NS gene and NS1 protein in virus
pathogenicity
A marked cytokine imbalance occurs in individuals
infected with H5N1 viruses – particularly in those with
fatal outcome [28,72,73]. This imbalance is characterized
by high levels of interferon-induced protein 10,
monokine induced by interferon (IFN)-γ, monocyte
chemotactic protein 1, interleukin (IL)-8, IL-10, IL-6,
IFN-γ and tumor necrosis factor (TNF)-α. Cytokine
and chemokine imbalance was also found in in vitro in
H5N1-virus-infected primary macrophages [74–76]
and human primary alveolar and bronchial epithelial
cells [77]. In addition, mRNA upregulation of death
receptor ligands such as TNF-related apoptosisinducing ligand (TRAIL), but not Fas ligand, has been
documented in human monocyte-derived macrophages
infected with a highly pathogenic H5N1 virus [78].
This upregulation was accompanied by an increased
sensitivity of virus-infected cells to TRAIL-induced
apoptosis, which suggests a role in the cytotoxicity of
these viruses and thus their pathogenicity. Recent
studies also indicated that H5N1 viruses activate the
mitogen-activated protein kinase pathway [79];
interestingly, however, no difference was found in the
ability of H5N1 and H1N1 viruses to activate the
transcription factor NF-κB. Thus, a picture emerges in
which highly pathogenic H5N1 viruses are more potent
activators of signal transduction pathways than are
other influenza viruses, resulting in an upregulation of
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cytokines and chemokines and a possible explanation
for the unusual pathogenicity of these viruses.
A number of studies suggest a role for the influenza
viral NS gene in viral pathogenicity [76,80,81]. In pigs,
recombinant viruses containing the 1997 H5N1 NS
gene were more pathogenic than control viruses
containing a non-H5N1 NS gene [80,81]. The 1997
H5N1 NS gene induced high levels of chemokines and
conferred resistance to the antiviral effects of IFN.
Hence, highly pathogenic H5N1 viruses can resist IFN,
allowing them to continue to replicate and sustain the
upregulation of cytokines and chemokines.
The NS gene encodes two proteins, the interferon
antagonist NS1 and the nuclear export protein NEP
(also called NS2). Depending on the strain, NS1
encompasses 202–238 amino acids with an N-terminal
RNA-binding domain and a C-terminal effector
domain. The RNA-binding domain resides in the Nterminal 73 amino acids. The effector domain contains
binding sites for the cleavage and polyadenylation
specificity factor [82,83] and the polyA-binding
protein II (PABII) [84], both of which are crucial for
the functions of NS1.
The NS1 protein is an IFN antagonist that ensures
efficient virus replication in IFN-competent hosts. In
interferon-deficient systems, such as Vero cells or
STAT1–/– mice, influenza virus lacking NS1 replicates
efficiently [85–89]. To counteract the host cell defence
system, NS1 interferes with two major pathways: IFNβ production and the activation of IFN-induced
antiviral genes.
To inhibit IFN-β production, NS1 blocks the activation of transcription factors, such as NF-κB, IFN
regulatory factor 3 or activation protein 1 [90–92]. The
mechanism by which NS1 achieves this is not fully
understood; however, several studies indicate that the
RNA-binding activity of NS1 is crucial for this function
[93,94], probably because it sequesters the doublestranded RNA that is essential for the activation of the
IFN response. In addition to blocking transcription
factor activation, NS1 also directly interferes with the
activation of the IFN-β promoter.
As stated above, NS1 also interferes with IFN-βstimulated genes, such as protein kinase R (PKR)
[95–99]. PKR expression is stimulated by IFN-β and
the NS1-mediated inhibition of IFN-β also affects PKR
expression in infected cells. Moreover, NS1 has a direct
effect on PKR levels by sequestering dsRNA, a known
PKR activator, and, possibly, by directly interacting
with PKR [98]. In influenza-virus-infected cells the
cellular PKR inhibitor, P58IPK, is activated, suggesting
another means by which influenza virus interferes with
PKR expression. It is not known, however, whether
NS1 is directly involved in this mechanism. Recently, a
new concept has emerged in which dsRNA-binding by
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NS1 prevents the dsRNA-dependent activation of 2′-5′
oligosynthetase [100], which in turns activates RNase
L, a key player in the innate immune response. NS1
has also been shown to inhibit adaptive immunity by
suppressing dendritic cell maturation, migration and
T-cell-stimulatory activity [101].
The NS1 proteins of H5N1 viruses isolated from
infected individuals in 1997 in Hong Kong confer
resistance to the antiviral effects of IFN and induce
high levels of proinflammatory cytokines [76,80,81].
The resultant cytokine imbalance, which has also been
observed in victims of H5N1 virus infections [73,102],
might be crucial to the pathogenicity of these viruses.
Likewise, the 2003 H5N1 viruses similarly induce high
levels of certain cytokines in patients and cell culture
[72,75], although the NS genes of these two groups of
viruses differ.
The NS1 proteins of highly pathogenic H5N1
viruses isolated in the 1997 outbreak differ from other
NS1 proteins by an aspartic-acid-to-glutamic-acid
change at amino acid 92, a substitution that is critical
for the pathogenicity and/or cytokine resistance of
these viruses in pig and mouse models [80,81,103]. In
addition, a study of reassortant and mutant H5N1
geese viruses in chickens found a crucial role for amino
acid 149 of NS1 [104]. How these mutations affect the
biological properties of NS1 is unknown; however, the
recently solved crystal structure of the NS1 effector
domain [105] might provide clues as to the molecular
mechanisms by which NS1 modulates host cell
responses.
Large-scale genome analyses to identify
amino acid changes that affect pathogenicity
and/or transmissibility
Since the first human infections with highly pathogenic H5N1 viruses in Hong Kong in 1997,
researchers have searched for key amino acids that
predict the pathogenicity and/or transmissibility of
these viruses, in order to predict the outcome of human
infections. Bioinformatics approaches were initially
hampered by the low number of available H5N1 virus
genomic sequences; in fact, until recently, even the
number of complete genomic sequences for non-H5N1
influenza viruses was surprisingly small. However, over
the last few years, tremendous efforts have been made
to sequence complete influenza viral genomes. Data
from a large-scale sequence analysis of avian
influenza viruses were published recently [106] and
revealed a previously unrecognized PDZ domain
ligand (PL) at the C-terminus of NS1. PDZ domains
are modular protein interaction domains that are
found in proteins involved in signalling pathways.
Human and avian influenza virus NS1 proteins differ
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Factors for influenza H5N1 virus pathogenicity
in their PL motifs, resulting in different binding
patterns to human PDZ domains [106]. Human
H5N1 viruses isolated during the outbreaks in 1997
and 2003–2004 are characterized by ‘avian-type’ PL
domains [106], which might interfere more severely
with signal transduction in human cells than do their
human counterparts. Although hypothetical at this
point, this scenario demonstrates the potential of largescale genome analyses for the identification of amino
acids that affect pathogenicity.
Comparisons of avian and human influenza virus
sequences have identified ‘avian-like’ and ‘human-like’
signature amino acids (reviewed in [107,108]). The
conservation of these amino acids in avian or human
virus isolates, respectively, suggests a biological
function; however, direct experimental evidence for an
involvement in host range restriction, virulence and/or
pathogenicity is lacking for most of these signature
amino acids.
An alternative approach to identifying H5N1 virus
amino acids critical for viral pathogenicity involved
comparing genomic sequences of viruses isolated from
fatal and non-fatal human cases [28]. This approach,
however, yielded no changes that correlated with
outcome, suggesting that other factors, such as the
immune status and/or genetic factors of the infected
individual, have a crucial role in the disease course.
Summary and outlook
Molecular studies of H5N1 influenza viruses have
identified amino acids that contribute crucially to
pathogenicity. However, none of the viruses containing
these key amino acids has acquired the ability to spread
efficiently among humans; in fact, high pathogenicity
might not be a requirement for a pandemic virus.
Efficient human-to-human transmission will probably
require a combination of several identified and as yet
unidentified key changes in the viral genome that
facilitate efficient binding to and replication in
epithelial cells of the upper respiratory tract, as well
as efficient suppression of host immune responses.
With the advent of bioinformatics approaches and the
growing number of genomic influenza virus sequences
available, additional determinants of pathogenicity will
probably be identified and tested for their biological
significance. The combination of experimental and
bioinformatics approaches should generate valuable
data on influenza virus pathogenicity, virulence and
transmissibility. It should be borne in mind, however,
that a defined set of mutations that determines pathogenicity and transmissibility might not exist; rather,
different combinations of mutations may render a
virus pathogenic and/or pandemic, depending on the
viral background.
Antiviral Therapy 12:4 Pt B
Acknowledgements
We thank Susan Watson for editing the manuscript. We
also thank those in our laboratories who contributed to
the data cited in this review. Our original research was
supported by National Institute of Allergy and Infectious
Diseases Public Health Service research grants; by
CREST (Japan Science and Technology Agency), and by
grants-in-aid from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
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