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Journal of General Virology (2010), 91, 1996–2001
Short
Communication
DOI 10.1099/vir.0.021766-0
Inhibition of host innate immune responses and
pathogenicity of recombinant Newcastle disease
viruses expressing NS1 genes of influenza A
viruses
Shin-Hee Kim and Siba K. Samal
Correspondence
Siba K. Samal
Virginia–Maryland Regional College of Veterinary Medicine, University of Maryland,
8075 Greenmead Drive, College Park, MD 20742, USA
[email protected]
Received 09 March 2010
Accepted 19 April 2010
The NS1 protein has been associated with the virulence of influenza A viruses. To evaluate the
role of the NS1 protein in pathogenicity of pandemic H5N1 avian influenza and H1N1 2009
influenza viruses, recombinant Newcastle disease viruses (rNDVs) expressing NS1 proteins were
generated. Expression of the NS1 proteins resulted in inhibition of host innate immune responses
(beta interferon and protein kinase R production). In addition, the NS1 proteins were localized
predominantly in the nucleus of virus-infected cells. Consequently, expression of the NS1 protein
contributed to an increase in pathogenicity of rNDV in chickens. In particular, mutational analysis
of H5N1 NS1 protein indicated that both the RNA-binding and effector domains affect virus
pathogenicity synergistically. Our study also demonstrated that expression of H1N1/09 NS1
resulted in enhanced replication of rNDV in human cells, indicating that function of the NS1
proteins can be host-species-specific.
Influenza A viruses are associated with significant morbidity and mortality, causing worldwide epidemics yearly and
pandemics sporadically (Li et al., 2004). The genome of
influenza A virus consists of eight RNA segments that
encode nine structural proteins and two non-structural
proteins, NS1 and PB1-F2 (Palese & Shaw, 2007). The NS1
protein has been identified as a determinant of virulence of
influenza A viruses (Bergmann et al., 2000; Donelan et al.,
2003). A major function of the NS1 protein is to
antagonize host innate immune responses. However, the
mechanisms and targets for the NS1 protein vary among
influenza A viruses (Kochs et al., 2007). The length of the
NS1 protein is strain-specific. The NS1 protein is divided
into two distinct functional domains: an N-terminal RNAbinding domain and a C-terminal effector domain for
interactions with host cellular proteins. In particular, the
RNA-binding domain is highly conserved among influenza
A viruses, and three residues (Arg-35, Arg-38 and Lys-41)
in the domain are associated with binding double-stranded
RNA (dsRNA). The C-terminal 4 aa of NS1 has been
identified as a potential PDZ domain-binding motif that
might influence the activity of PDZ domain-containing
proteins, which are often involved in cellular signaltransduction pathways (Jackson et al., 2008).
Newcastle disease virus (NDV) is a member of the family
Paramyxoviridae and has a non-segmented, negative-sense
RNA genome consisting of six transcriptional units (39NP-P-M-F-HN-L-59) (Lamb & Parks, 2007). NDV causes a
highly contagious respiratory and neurological disease in
1996
chickens (Alexander, 1989). NDV strains are categorized
into three pathotypes in chickens: lentogenic (avirulent),
mesogenic (moderately virulent) and velogenic (highly
virulent) (Panda et al., 2004). In this study, we have used a
recombinant mesogenic NDV strain to evaluate the role of
pandemic influenza A virus NS1 protein in preventing
innate host defences and pathogenicity in chickens. The
NS1 genes of H5N1 highly pathogenic avian influenza and
pandemic H1N1 2009 influenza (H1N1/09) viruses were
inserted individually between the P and M genes of NDV
mesogenic strain Beaudette C (BC) (Fig. 1a). As the
functional role of the specific amino acid residues of the
NS1 proteins has been primarily studied with human
influenza virus strains, we attempted to identify critical
amino acids of the H5N1 NS1 protein responsible for virus
pathogenicity. The open reading frame (ORF) of the NS1
gene was altered by a single amino acid change, according
to previously published studies (Donelan et al., 2003; Jiao
et al., 2008). In addition, the C-terminal 4 aa (ESEV) of
H5N1 NS1 was replaced with that of H3N2 virus (RSKV)
to evaluate the effect of this potential PDZ domain-binding
motif on the function of the NS1 protein. We also
generated NS1 protein- and effector domain-deleted
viruses by introducing a stop codon at the N-terminal
region and the effector domain of the H5N1 NS1 protein,
respectively. Recombinant BCs (rBCs) were recovered by
using our standard protocol (Krishnamurthy et al., 2000)
and the expression of NS1 proteins by recovered viruses
was confirmed by Western blot analysis. Multicycle growth
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NDV expressing NS1 genes of influenza A viruses
Fig. 1. Generation and growth characterization of rBCs expressing the H5N1 NS1 protein
(including its mutants) and the H1N1/09 NS1
protein. (a) The NS1 gene was reversetranscribed and amplified with gene-specific
primers by RT-PCR from viral RNA of the
influenza virus A/Vietnam/1203/2004 (H5N1)
or A/California/04/09 (H1N1). Each NS1 gene
was flanked by a conserved gene start on its
upstream and a conserved gene end on its
downstream. The transcriptional unit was
inserted between the P and M genes in the
NDV genome. Infectious rBCs expressing NS1
proteins were generated by using a reversegenetics system. In addition, each H5N1 NS1
mutant virus was generated by altering a single
amino acid or introducing a stop codon into
the ORF of the NS1 gene (shown in bold). (b)
The growth characteristics of the rBCs were
examined by multicycle growth-curve analysis
in DF-1 cells infected at an m.o.i. of 0.01. Virus
titres were determined by plaque assay. (c)
Growth characteristics of rBC and rBCs
expressing NS1 proteins of H5N1 and H1N1
were examined by multicycle growth-curve
analysis in virus-infected HeLa cells at an
m.o.i. of 0.01.
kinetics of rBCs indicated that the expression of NS1
protein did not affect in vitro replication of rBCs in DF-1
cells [a chicken embryo fibroblast (CEF) cell line] (Fig. 1b).
Interestingly, in HeLa cells, rBC expressing H1N1/09 NS1
grew to 1 log10 higher titres than rBC expressing H5N1
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NS1 up to 24 h post-infection, indicating that H1N1/09
NS1 enhanced replication of rBC in human cells (Fig. 1c).
NS1 has been implicated in inhibition of the host antiviral
defence mediated by interferon (IFN) (Donelan et al.,
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1997
S.-H. Kim and S. K. Samal
2003). Point mutation, truncation or deletions of the NS1
gene of influenza A viruses resulted in enhanced IFNinducing capacity of the virus in vivo and in vitro (Donelan
et al., 2003; Ferko et al., 2004; Jiao et al., 2008; Z. Li
et al., 2006). In contrast, NDV has been known as a good
IFN-b inducer in infected cells (Poole et al., 2002) and the
NDV V protein can prevent IFN responses only in avian
cells, thus playing a role in host-range restriction (Park
et al., 2003). Therefore, we first determined whether rBCs
expressing NS1 proteins of avian and human strains
affected the levels of IFN-b mRNA in avian and human
cells differently (Fig. 2a). Our results showed that the
H5N1 NS1 protein inhibited IFN-b production efficiently
in both CEF and HeLa cells, whereas H1N1/09 NS1 protein
antagonized IFN-b synthesis more efficiently in HeLa cells
than in CEF cells, probably due to its interaction with
Fig. 2. RT-PCR analysis of IFN-b mRNA
levels in virus-infected CEF and HeLa cells
and PKR inhibition by NS1 protein in virusinfected HeLa cells. (a) CEF and HeLa cells
were infected at an m.o.i. of 2. Total RNAs
were extracted at 24 h post-infection and RTPCR was carried out using primers specific for
IFN-b. The levels of b-actin and GAPDH
mRNAs in CEF and HeLa cells, respectively,
were measured as loading controls. (b)
Activated PKR in virus-infected HeLa cells
was analysed by Western blotting. HeLa cells
in six-well plates were infected with rBCs at an
m.o.i. of 1. Total proteins were collected from
virus-infected cells at 24 h post-infection,
electrophoresed, transferred to a nitrocellulose
membrane and immunostained using polyclonal antibodies against phospho-PKR (top
panel) and NS1 (middle panel) or a monoclonal antibody against b-actin as a loading
control (bottom panel). Levels of activated
PKR were quantified from three independent
analyses; error bars indicate SEM. (c)
Localization of wild-type and mutated NS1
proteins was evaluated in virus-infected HeLa
cells infected with rBCs at an m.o.i. of 1.
Twenty-four hours post-infection, the cells
were fixed with 4 % paraformaldehyde, permeabilized with 0.2 % Triton X-100, stained
with a monoclonal antibody against the NS1
protein (N-terminal domain) followed by antiAlexa Fluor 488 conjugate and DAPI (49,6diamidino-2-phenylindole), and analysed by
confocal microscopy.
1998
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Journal of General Virology 91
NDV expressing NS1 genes of influenza A viruses
unique sequences of specific host species (Melén et al.,
2007; Park et al., 2003). Furthermore, point mutations in
RNA-binding domains of H5N1 NS1 resulted in alteration
of IFN-b mRNA levels in CEF and HeLa cells. The basic
amino acids R38 and K41 are known to be associated
directly with RNA binding of NS1 (Geiss et al., 2002; Talon
et al., 2000). However, in our study, rBC/NS1 R38A
induced higher levels of IFN-b mRNA than did rBC/NS1
K41A, because mutation of R38A would be more effective
on complete abrogation of binding affinity of NS1 than
mutation of K41A (Wang et al., 1999). In influenza A/
WSN/33, mutations of NS1 protein (R38AK41AS42G)
resulted in increased IFN-b production and virus attenuation in mice, indicating that IFN-antagonist properties of
the NS1 protein can also be modulated by other amino acid
residues, such as S42, without direct association with RNA
binding (Donelan et al., 2003). Similarly, our study
suggested that, in the H5N1 NS1 protein, a certain amino
acid at position 42 would modulate IFN production in
virus-infected cells. In addition, point (E92A) and deletion
mutations in the effector domain of H5N1 NS1 resulted in
enhanced levels of IFN-b mRNA synthesis in virus-infected
cells, indicating that the effector domain also plays an
important role in antagonizing IFN-b synthesis. Indeed,
elimination of the effector domain has been shown to alter
the dsRNA-binding affinity of NS1 significantly (Li et al.,
2004). The effector domain is also known to bind to the
cellular proteins that are essential for the processing of
cellular pre-mRNA, including IFN-b mRNA (Noah et al.,
2003).
The NS1 protein can prevent activation of the IFNinducible dsRNA-dependent protein kinase R (PKR),
leading to stimulation of host protein synthesis in infected
cells (Donelan et al., 2003). Therefore, we further
characterized the function of the NS1 protein in inhibition
of PKR activation in virus-infected HeLa cells by Western
blotting (Fig. 2b). In general, levels of the mutated NS1
proteins were similar to that of the NS1 protein, indicating
that mutation of the NS1 protein did not affect levels of
protein expression. However, levels of activated PKR were
correlated with those of IFN-b mRNA in infected cells with
rBC and rBCs expressing various NS1 proteins. H5N1 and
H1N1/09 NS1 proteins inhibited PKR activation in
infected HeLa cells. In contrast, point mutations of R38A
and S42G and deletion of the effector domain in the H5N1
NS1 protein resulted in a slight increase in PKR activation
in infected HeLa cells. In A/Udorn/72, certain amino acid
residues in the effector domain of NS1 have been shown
to bind to a linker region in PKR, thus preventing a
conformational change required for release of PKR autoinhibition (S. Li et al., 2006; Min et al., 2007). Therefore, it
is possible that H5N1 NS1 inhibits PKR activation by
dsRNA binding- and direct binding-mediated mechanisms.
In infected cells, NS1 protein sequesters viral dsRNAs in
the nucleus, which leads to blocking the dsRNA activation
of PKR in the cytoplasm (Bergmann et al., 2000; Melén
et al., 2007). Consequently, we analysed the cellular
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localization of wild-type and mutated NS1 proteins in
virus-infected HeLa cells by confocal microscopy. Both
H5N1 and H1N1/09 NS1 expressed by rBCs were localized
predominantly in the nucleus (Fig. 2c). However, mutations of R38A and the effector domain (E92A and deletion)
resulted in localization of the NS1 protein in the cytoplasm
of virus-infected cells, indicating that these amino acids are
involved in the distribution of NS1 in the nucleus of
infected cells. In H1N1 NS1, the location of the NS1
protein can be facilitated by two nuclear-localization
signals (NLSs): NLS1 at residues 34–38 (Asp-Arg-LeuArg-Arg) and NLS2 at residues 216–221 (Pro-Lys-Gln-LysArg-Lys) (Greenspan et al., 1988). Our study also suggests
that the subcellular distribution of the H5N1 NS1 protein
can be affected by alteration of NLSs located in both the
RNA-binding and effector domains.
We then evaluated the contribution of the NS1 protein to
pathogenicity of rBCs in embryonated chicken eggs and in
chickens (Table 1). The pathogenicity of rBCs was
determined by mean death time (MDT) test in 9-day-old
embryonated specific-pathogen-free (SPF) chicken eggs
and by intracerebral pathogenicity index (ICPI) test in 1day-old SPF chicks (Alexander, 1989). The scale of the ICPI
value in evaluating the virulence of NDV strains is from
0.00 (avirulent strains) to 2.00 (highly virulent NDV
strains). In general, MDT and ICPI values were correlated
in evaluating pathogenicity of rBCs expressing wild-type
and mutated NS1 proteins. Expression of H5N1 and
Table 1. Pathogenicity of the rBCs expressing wild-type and
mutated NS1 proteins of influenza A viruses in embryonated
chicken eggs and chickens
Virus
rBC
rBC/NS1
rBC/DNS1
rBC/NS1 R38A
rBC/NS1 K41A
rBC/NS1 S42P
rBC/NS1 S42G
rBC/NS1 DED
rBC/NS1 E92A
rBC/NS1 RSKV
rBC/H1N1/09 NS1
MDT (h)*
ICPID
57
44
56
48
44
44
52
49
50
51
45
1.51
1.75
1.48
1.43
1.78
1.76
1.45
1.55
1.54
1.54
1.73
*Mean time (h) for the minimum lethal dose of virus to kill all of the
inoculated embryos. Five eggs for each diluent of allantoic fluid of
infectious virus were tested. The criteria for classifying the virulence
of NDV strains are: taking ,60 h to kill embryos for virulent strains;
taking 60–90 h to kill embryos for moderately virulent strains; and
taking .90 h to kill embryos for avirulent strains.
DPathogenicity of rBCs in 1-day-old SPF chicks (10 chicks for each
virus) was evaluated by the ICPI test: virulent strains, 1.50–2.00;
moderately virulent strains, 0.70–1.50; and avirulent strains, 0.00–
0.70.
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1999
S.-H. Kim and S. K. Samal
H1N1/09 NS1 proteins resulted in increased pathogenicity
of rBCs (ICPI: 1.75 and 1.73, respectively) compared
with those of rBC and rBC/DNS1 (ICPI: 1.51 and 1.48,
respectively) (P,0.05). We also confirmed the contribution of the NS1 protein to increased virus replication
in 2-week-old chickens. The rBC expressing H5N1 NS1
replicated to 2–3 log10 higher titres than rBC in various
tissues at 3 days post-infection. The titres of rBC/NS1
and rBC were 4.5±0.10 and 2.2±0.15 log10(p.f.u. g21),
respectively, in brain; 7.2±0.33 and 5.4±1.04 log10(p.f.u.
g21), respectively, in trachea; 6.2±0.54 and 3.6±
0.11 log10(p.f.u. g21), respectively, in lung; and 5.5±0.15
and 3.7±0.44 log10(p.f.u. g21), respectively, in spleen.
However, ICPI results indicated that expression of NS1
with point mutations in the RNA-binding domain (R38A
and S42G) did not alter the pathogenicity of rBC (P.0.05).
Deletion of the effector domain and alteration of the Cterminal 4 aa also did not affect the pathogenicity of rBC.
Furthermore, our ICPI results suggest that the uniquely present glutamic acid residue at position 92 in H5N1 NS1
(Talon et al., 2000) plays an important role in virus pathogenicity (P,0.05; one-way ANOVA, SPSS 13.0 for Windows).
In summary, we have evaluated the function of the NS1
protein of H5N1 highly pathogenic avian influenza and
pandemic H1N1 2009 influenza (H1N1/09) viruses in
pathogenicity. Expression of H5N1 NS1 protein led to
alteration of host innate immune response in avian and
human cells and enhancement of the replication of rNDV in
chickens. Mutational analysis of H5N1 NS1 suggests that
amino acids R38 and S42 in the RNA-binding domain and
effector domain synergistically affect inhibition of host
innate immune system and, consequently, virus pathogenicity. Particularly, the expression of H1N1/09 NS1 enabled
rNDV to circumvent host innate immune responses and
replicate well in human cells, indicating that the function of
NS1 proteins can be host-species-specific.
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Acknowledgements
We thank Daniel Rockemann, Flavia Dias, Yunsheng Wang and our
laboratory members for excellent technical assistance and LaShae
Green for proofreading of the manuscript.
Li, Z., Jiang, Y., Jiao, P., Wang, A., Zhao, F., Tian, G., Wang, X., Yu, K.,
Bu, Z. & Chen, H. (2006). The NS1 gene contributes to the virulence
of H5N1 avian influenza viruses. J Virol 80, 11115–11123.
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