Interferon Inhibition by the
NS1 protein - Enhanced
virulence/viral
pathogenesis by enabling the virus to disarm the host cell type IFN defense
system
Compiled by: Joseph M. Cummins, DVM,
PhD, Amarillo Biosciences, Inc. 800
West 9 th Avenue, Amarillo, Texas 79101-3206 Telephone:806-376-1741,
Fax: 806-376-9301 Email: jcummins@amarbio.com
(REFERENCE 1 OF 26)
Chien CY Xu Y Xiao R Aramini JM Sahasrabudhe PV
Krug RM Montelione GT
Biophysical characterization of the complex between double-stranded
RNA and the N-terminal domain of the NS1 protein from influenza A virus:
evidence for a novel RNA-binding mode.
In: Biochemistry (2004 Feb 24) 43(7):1950-62
The influenza virus nonstructural protein 1 encoded by influenza A
virus (NS1A protein) is a multifunctional protein involved in both protein-protein
and protein-RNA interactions. NS1A binds nonspecifically to double-stranded
RNA (dsRNA) and to specific protein targets, and regulates several
post-transcriptional processes. The N-terminal structural domain corresponding
to the first 73 amino acids of the NS1 protein from influenza A/Udorn/72
virus [NS1A(1-73)] possesses all of the dsRNA binding activities of
the full-length protein. Both NMR and X-ray crystallography of this domain
have demonstrated that it is a symmetric homodimer which forms a six-helix
chain fold, a unique structure that differs from that of the predominant
class of dsRNA-binding domains, termed dsRBDs, that are found in a
large number of eukaryotic and prokaryotic proteins. Here we describe
biophysical experiments on complexes containing NS1A(1-73) and a short
16 bp synthetic dsRNA duplex. From sedimentation equilibrium measurements,
we determined that the dimeric NS1A(1-73) binds to the dsRNA duplex
with a 1:1 stoichiometry, yielding a complex with an apparent dissociation
constant (K(d)) of approximately 1 microM. Circular dichroism and nuclear
magnetic resonance (NMR) data demonstrate that the conformations of both
NS1A(1-73) and dsRNA in the complex are similar to their free forms, indicating
little or no structural change in the protein or RNA upon complex formation.
NMR chemical shift perturbation experiments show that the dsRNA-binding epitope
of NS1A(1-73) is associated with helices 2 and 2'. Analytical gel
filtration and gel shift studies of the interaction between NS1A(1- 73) and
different double-stranded nucleic acids indicate that NS1A(1- 73) recognizes
canonical A-form dsRNA, but does not bind to dsDNA or dsRNA-DNA hybrids,
which feature B-type or A/B-type intermediate conformations, respectively.
On the basis of these results, we propose a three-dimensional model
of the complex in which NS1A(1-73) sits astride the minor groove of A-form
RNA with a few amino acids in the helix 2-helix 2' face forming an electrostatically
stabilized interaction with the phosphodiester backbone. This mode
of dsRNA binding differs from that observed for any other dsRNA-binding
protein.
Institutional address:
Center for Advanced Biotechnology and Medicine and Department
of Molecular Biology and Biochemistry
Rutgers University Piscataway New Jersey
08854-5638 USA.
(REFERENCE 2 OF 26)
Zhou Y Chan JH Chan AY Chak RK Wong EY Chye
ML Peiris JS Poon LL Lam E
Transgenic plant-derived siRNAs can suppress propagation of influenza virus
in mammalian cells.
In: FEBS Lett (2004 Nov 19) 577(3):345-50
As an example of the cost-effective large-scale generation of small-
interfering RNA (siRNAs), we have created transgenic tobacco plants that produce
siRNAs targeted to the mRNA of the non-structural protein NS1 from the influenza
A virus subtype H1N1. We have investigated if these siRNAs, specifically
targeted to the 5 [Formula: see text] -portion of the NS1 transcripts (5mNS1),
would suppress viral propagation in mammalian cells. Agroinfiltration of
transgenic tobacco with an Agrobacterium strain harboring a 5mNS1- expressing
binary vector caused a reduction in 5mNS1 transcripts in the siRNA-accumulating
transgenic plants. Further, H1N1 infection of siRNA-transfected mammalian
cells resulted in significant suppression of viral replication. These results
demonstrate that plant-derived siRNAs can inhibit viral propagation
through RNA interference and could potentially be applied in control of viral-borne
diseases.
Institutional address:
Department of Botany University of Hong Kong
Pokfulam Road Hong Kong Special Administrative Region PR China.
(REFERENCE 3 OF 26)
Muster T Rajtarova J Sachet M Unger H Fleischhacker
R Romirer I Grassauer A Url A Garcia-Sastre A
Wolff K Pehamberger H Bergmann M
Interferon resistance promotes oncolysis by influenza virus NS1- deletion
mutants.
In: Int J Cancer (2004 May 20) 110(1):15-21
NS1 protein of influenza virus is a virulence factor that counteracts Type
I interferon (IFN)-mediated antiviral response by the host. A recombinant
influenza A virus that lacks the NS1 protein only replicates efficiently
in systems that contain defective IFN pathways. We demonstrate that
the conditional replication properties of NS1-modified influenza A virus
mutants can be exploited for the virus-mediated oncolysis of IFN-resistant
tumor cells. IFN resistance in analyzed tumor cell lines correlated
with a reduced expression of STAT1. Addition of exogenous IFNalpha or supernatant
of virus- infected endothelial cells inhibited viral oncolysis in IFN-sensitive
but not in IFN-resistant cell lines. The oncolytic potential of NS1-
modified influenza A virus mutants could be exploited in vivo in a
SCID mouse model of a subcutaneously-implanted human IFN-resistant melanoma.
The data indicate that IFN-resistant tumors are a suitable target for oncolysis
induced by NS1-modified influenza virus mutants. STAT1 might serve as a marker
to identify these IFN-resistant tumors.
Institutional address: Department of
Dermatology University of Vienna Wahringer Gurtel 18-20
1090 Vienna Austria.
(REFERENCE 4 OF 26)
Lowy RJ
Influenza virus induction of apoptosis by intrinsic and extrinsic
mechanisms.
In: Int Rev Immunol (2003 Sep-Dec) 22(5-6):425-49
It is now firmly established that apoptosis is an important mechanism of
influenza virus-induced cell death both in vivo and in vitro. Data are predominantly
from experiments with influenza A virus and in vitro experimental systems.
Multiple influenza virus factors have been identified that can activate intrinsic
or extrinsic apoptotic induction pathways. Currently there is no evidence
for influenza virus directly accessing the apoptosis execution factors.
The best- studied influenza virus inducers of apoptosis are dsRNA, NS1, NA,
and a newly described gene product PB1-F2. PB1-F2 is the only influenza
virus factor to date identified to act intrinsically by localization
and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA
and NA have been shown to act via an extrinsic mechanism
involving proapoptotic host-defense molecules: PKR by induction of
Fas-Fas ligand and NA by activation of TGF-beta. PKR is capable of controlling
several important cell-signaling pathways and therefore may have multiple
effects; a predominant one is increased interferon (IFN) production
and activity. NS1 has been shown to be both proapoptotic and antiapoptotic.
Use of influenza virus NS1 deletion mutants has provided evidence for NS1
interference with apoptosis, IFN induction, and related cell-signaling pathways.
Influenza virus also has important exocrine paracrine effects, which are likely
mediated via TNF family ligands and oxygen, free radicals capable of
inducing apoptosis. Little is known about activation of inhibitors of
apoptosis such as inhibitory apoptotic proteins. Whether all these factors
always have a role in influenza virus-induced apoptosis is unknown.
The kinetics of synthesis of influenza virus factors affecting apoptosis
during the replication cycle may be an important aspect of apoptosis induction.
Institutional address: Armed Forces Radiobiology Research Institute
8901 Wisconsin Avenue Bethesda MD 20889-5603
USA. lowy@afrri.usuhs.mil
(REFERENCE 5 OF 26)
Delgadillo MO Saenz P Salvador B Garcia JA Simon-Mateo
C
Human influenza virus NS1 protein enhances viral pathogenicity and acts
as an RNA silencing suppressor in plants.
In: J Gen Virol (2004 Apr) 85(Pt 4):993-9
RNA silencing has a well-established function as an antiviral defence
mechanism in plants and insects. Using an Agrobacterium-mediated transient
assay, we report here that NS1 protein from human influenza A virus
suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein
of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor.
Moreover, we have shown that NS1 protein expression strongly enhances the
symptoms of Potato virus X in three different plant hosts, suggesting that
NS1 protein could be inhibiting defence mechanisms activated in the plant
on infection. These data provide further evidence that an RNA silencing pathway
could also be activated as a defence response in mammals.
Institutional address:
Centro Nacional de Biotecnologia (CSIC) Campus Universidad Autonoma
de Madrid 28049 Madrid Spain.
(REFERENCE 6 OF 26)
Bucher E Hemmes H de Haan P Goldbach R Prins M
The influenza A virus NS1 protein binds small interfering RNAs and suppresses
RNA silencing in plants.
In: J Gen Virol (2004 Apr) 85(Pt 4):983-91
RNA silencing comprises a set of sequence-specific RNA degradation pathways
that occur in a wide range of eukaryotes, including animals, fungi and plants.
A hallmark of RNA silencing is the presence of small interfering RNA molecules
(siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs
(dsRNAs) and provide the sequence specificity for degradation of cognate RNA
molecules. In plants, RNA silencing plays a key role in developmental
processes and in control of virus replication. It has been shown that many
plant viruses encode proteins, denoted RNA silencing suppressors, that interfere
with this antiviral response. Although RNA silencing has been shown to occur
in vertebrates, no relationship with inhibition of virus replication
has been demonstrated to date. Here we show that the NS1 protein of
human influenza A virus has an RNA silencing suppression activity in plants,
similar to established RNA silencing suppressor proteins of plant viruses.
In addition, NS1 was shown to be capable of binding siRNAs. The data presented
here fit with a potential role for NS1 in counteracting innate antiviral responses
in vertebrates by sequestering siRNAs.
Institutional address:
Laboratory of Virology Wageningen University
Binnenhaven 11 6709 PD Wageningen The Netherlands.
(REFERENCE 7 OF 26)
Catchpole AP Mingay LJ Fodor E Brownlee GG
Alternative base pairs attenuate influenza A virus when introduced into
the duplex region of the conserved viral RNA promoter of either the NS or
the PA gene.
In: J Gen Virol (2003 Mar) 84(Pt 3):507-15
The development of plasmid-based rescue systems for influenza virus has
allowed previous studies of the neuraminidase (NA) virion RNA (vRNA)
promoter to be extended, in order to test the hypothesis that alternative
base pairs in the conserved influenza virus vRNA promoter cause attenuation
when introduced into other gene segments. Influenza A/WSN/33 viruses
with alternative base pairs in the duplex region of the vRNA promoter of
either the polymerase acidic (PA) or the NS (non- structural 1, NS1, and
nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK
cells demonstrated that one of the mutations, the D2 mutation (U-A replacing
G-C at nucleotide positions 12'-11), caused significant virus attenuation
when introduced into either the PA or the NS gene. The D2 mutation resulted
in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-
recombinant viruses, respectively. Since the D2 mutation attenuates influenza
virus when introduced into either the PA or the NS gene segments, or the
NA gene segment, as demonstrated previously, this suggests that this mutation
will lead to virus attenuation when introduced into any of the eight gene
segments. Such a mutation may be useful in the production of live-attenuated
viruses.
Institutional address: Sir William Dunn School of Pathology
University of Oxford Chemical Pathology Unit, South Parks Road
Oxford OX1 3RE UK.
(REFERENCE 8 OF 26)
Ferko B Stasakova J Romanova J Kittel C Sereinig
S Katinger H Egorov A
Immunogenicity and protection efficacy of replication-deficient influenza
A viruses with altered NS1 genes.
In: J Virol (2004 Dec) 78(23):13037-45
We explored the immunogenic properties of influenza A viruses with
altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1
proteins with an impaired RNA-binding function or insertion of a longer
foreign sequence did not replicate in murine lungs but still were capable
of inducing a Th1-type immune response resulting in significant titers of
virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with
lower titers of IgG1. In contrast, replicating viruses elicited high
titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient
NS1 mutant viruses induced a rapid local release of proinflammatory
cytokines such as interleukin-1beta (IL-1beta) and IL-6. Moreover, these viruses
also elicited markedly higher levels of IFN-alpha/beta in serum than the
wild-type virus. Comparable numbers of virus-specific primary CD8(+) T cells
were determined in all of the groups of immunized mice. The most rapid onset
of the recall CD8(+)-T-cell response upon the wild- type virus challenge
was detected in mice primed with NS1 mutant viruses eliciting high levels
of cytokines. It is noteworthy that there was one NS1 mutant virus
encoding NS1 protein with a deletion of 40 amino acids predominantly in the
RNA-binding domain that induced the highest levels of IFN-alpha/beta, IL-6
and IL-1beta after infection. Mice that were immunized with this virus were
completely protected from the challenge infection. These findings indicate
that a targeted modification of the RNA-binding domain of the NS1 protein
is a valuable technique to generate replication-deficient, but immunogenic
influenza virus vaccines.
Institutional address: Institute of Applied Microbiology
Muthgasse 18B A-1190 Vienna Austria. b.ferko@iam.boku.ac.at
(REFERENCE 9 OF 26)
Donelan NR Dauber B Wang X Basler CF Wolff T
Garcia-Sastre A
The N- and C-terminal domains of the NS1 protein of influenza B virus can
independently inhibit IRF-3 and beta interferon promoter activation.
In: J Virol (2004 Nov) 78(21):11574-82
The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins)
have only approximately 20% amino acid sequence identity. Nevertheless, these
proteins show several functional similarities, such as their ability
to bind to the same RNA targets and to inhibit the activation of protein
kinase R in vitro. A critical function of the A/NS1 protein is the inhibition
of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection.
Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta
synthesis in virus-infected cells. We have now found that the expression
of the B/NS1 protein complements the growth of an influenza A virus
with A/NS1 deleted. Expression of the full-length B/NS1 protein (281
amino acids), as well as either its N-terminal RNA-binding domain (amino
acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the
absence of any other influenza B virus proteins resulted in the inhibition
of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational
analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity
correlated with IFN-beta promoter inhibition. In addition, a recombinant
influenza B virus with NS1 deleted induces higher levels of IRF-3 activation,
as determined by its nuclear translocation, and of IFN-alpha/beta synthesis
than wild-type influenza B virus. Our results support the hypothesis that
the NS1 protein of influenza B virus plays an important role in antagonizing
the IRF-3- and IFN-induced antiviral host responses to virus infection.
Institutional address: Department of Microbiology Box 1124 Mount
Sinai School of Medicine 1 Gustave L. Levy Pl. New York
NY 10029 USA.
(REFERENCE 10 OF 26)
Falcon AM Marion RM Zurcher T Gomez P Portela
A Nieto A Ortin J
Defective RNA replication and late gene expression in temperature- sensitive
influenza viruses expressing deleted forms of the NS1 protein.
In: J Virol (2004 Apr) 78(8):3880-8
Influenza A virus mutants expressing C-terminally deleted forms ofthe NS1
protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses
were temperature sensitive and showed a small plaque size at the permissive
temperature. The accumulation of virion RNA in mutant virus-infected cells
was reduced at the restrictive temperature, while the accumulation of cRNA
or mRNA was not affected, indicating that the NS1 protein is involved in the
control of transcription versus replication processes in the infection. The
synthesis and accumulation of late virus proteins were reduced in NS1-
81 mutant-infected cells at the permissive temperature and were essentially
abolished for both viruses at the restrictive temperature, while synthesis
and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence,
the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive
temperature. These results indicate that the NS1 protein is essential for
nuclear and cytoplasmic steps during the virus cycle.
Institutional address: Centro Nacional de Biotecnologi
CSIC 28049 Madrid Spain.
(REFERENCE 11 OF 26)
Dauber B Heins G Wolff T
The influenza B virus nonstructural NS1 protein is essential for efficient
viral growth and antagonizes beta interferon induction.
In: J Virol (2004 Feb) 78(4):1865-72
We analyzed the functions of the influenza B virus nonstructural NS1-
B protein, both by utilizing a constructed mutant virus (Delta NS1-B) lacking
the NS1 gene and by testing the activities of the protein when expressed in
cells. The mutant virus replicated to intermediate levels in 6-day-old embryonated
chicken eggs that contain an immature interferon (IFN) system, whereas older
eggs did not support viral propagation to a significant extent. The Delta
NS1-B virus was substantially stronger inducer of beta IFN (IFN-beta)
transcripts in human lung epithelial cells than the wild type, and furthermore,
transiently expressed NS1-B protein efficiently inhibited virus- dependent
activation of the IFN-beta promoter. Interestingly, replication of the Delta
NS1-B knockout virus was attenuated by more than 4 orders of magnitude in
tissue culture cells containing or lacking functional IFN-alpha/beta
genes. These findings show that the NS1-B protein functions as a viral IFN
antagonist and indicate a further requirement of this protein for efficient
viral replication that is unrelated to blocking IFN effects.
Institutional address: Robert Koch-Institut
13353 Berlin Germany.
(REFERENCE 12 OF 26)
Donelan NR Basler CF Garcia-Sastre A
A recombinant influenza A virus expressing an RNA-binding-defective NS1
protein induces high levels of beta interferon and is attenuated in mice.
In: J Virol (2003 Dec) 77(24):13257-66
Previously we found that the amino-terminal region of the NS1 protein
of influenza A virus plays a key role in preventing the induction of beta
interferon (IFN-beta) in virus-infected cells. This region is characterized
by its ability to bind to different RNA species, including double-stranded
RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether
the NS1 RNA-binding activity is required for its IFN antagonist properties,
we have generated a recombinant influenza A virus which expresses a
mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted
alanines for two basic amino acids within NS1 (R38 and K41) that were
previously found to be required for RNA binding. Cells infected with
the resulting recombinant virus showed increased IFN-beta production, demonstrating
that these two amino acids play a critical role in the inhibition of IFN production
by the NS1 protein during viral infection. In addition, this virus
grew to lower titers than wild-
type virus in MDCK cells, and it was attenuated in mice. Interestingly,
passaging in MDCK cells resulted in the selection of a mutant virus containing
a third mutation at amino acid residue 42 of the NS1 protein (S42G). This
mutation did not result in a gain in dsRNA-binding activity by the NS1
protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G
mutant virus was able to replicate in MDCK cells to titers close to
those of wild-type virus. This mutant virus had intermediate virulence in
mice, between those of the wild-type and parental NS1 R38AK41A viruses. These
results suggest not only that the IFN antagonist properties of the NS1 protein
depend on its ability to bind dsRNA but also that they can be modulated by
amino acid residues not involved in RNA binding.
Institutional address: Department of Microbiology. Microbiology
Graduate School Training Program, Mount Sinai School of Medicine
New York New York 10029 USA.
(REFERENCE 13 OF 26)
Basler CF Mikulasova A Martinez-Sobrido L Paragas J
Muhlberger E Bray M Klenk HD Palese P Garcia-Sastre
A
The Ebola virus VP35 protein inhibits activation of interferon regulatory
factor 3.
In: J Virol (2003 Jul) 77(14):7945-56
The Ebola virus VP35 protein was previously found to act as an interferon
(IFN) antagonist which could complement growth of
influenza delNS1 virus, a mutant influenza virus lacking the influenza
virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent
the virus- or double-stranded RNA-mediated transcriptional activation
of both the beta IFN (IFN-beta) promoter and the IFN-stimulated ISG54
promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000).
We now show that VP35 inhibits virus infection-induced transcriptional activation
of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and
that VP35 does not block signaling from the IFN-alpha/beta receptor. The ability
of VP35 to inhibit this virus-induced transcription correlates with
its ability to block activation of IRF-3, a cellular transcription factor
of central importance in initiating the host cell IFN response. We demonstrate
that VP35 blocks the Sendai virus-induced activation of two promoters
which can be directly activated by IRF-3, namely, the ISG54 promoter
and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent
activation of the IFN-alpha4 promoter in response to viral infection.
The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation.
VP35 blocks virus- induced IRF-3 phosphorylation and subsequent IRF-3 dimerization
and nuclear translocation. Consistent with these observations, Ebola
virus infection of Vero cells activated neither transcription from the
ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that
in Ebola virus-infected cells, VP35 inhibits the induction of antiviral
genes, including the IFN-beta gene, by blocking IRF-3 activation.
Institutional address: Department of Microbiology
Mount Sinai School of Medicine, 1 Gustave L. Levy Place New York
NY 10029 USA. chris.basler@mssm.edu
(REFERENCE 14 OF 26)
Park MS Shaw ML Munoz-Jordan J Cros JF Nakaya T
Bouvier N Palese P Garcia-Sastre A Basler CF
Newcastle disease virus (NDV)-based assay demonstrates interferon- antagonist
activity for the NDV V protein and the Nipah virus V, W, and C proteins.
In: J Virol (2003 Jan) 77(2):1501-11
We have generated a recombinant Newcastle disease virus (NDV) that
expresses the green fluorescence protein (GFP) in infected chicken embryo
fibroblasts (CEFs). This virus is interferon (IFN) sensitive, and pretreatment
of cells with chicken alpha/beta IFN (IFN- alpha/beta) completely blocks
viral GFP expression. Prior transfection of plasmid DNA induces an IFN response
in CEFs and blocks NDV-GFP replication. However, transfection of known inhibitors
of the IFN-alpha/beta system, including the influenza A virus NS1 protein
and the Ebola virus VP35 protein, restores NDV-GFP replication. We therefore
conclude that the NDV-GFP virus could be used to screen proteins expressed
from plasmids for the ability to counteract the host cell IFN response.
Using this system, we show that expression of the NDV V protein or the Nipah
virus V, W, or C proteins rescues NDV-GFP replication in the face of the
transfection- induced IFN response. The V and W proteins of Nipah virus,
a highly lethal pathogen in humans, also block activation of an IFN-inducible
promoter in primate cells. Interestingly, the amino-terminal region of the
Nipah virus V protein, which is identical to the amino terminus of
Nipah virus W, is sufficient to exert the IFN-antagonist activity.
In contrast, the anti-IFN activity of the NDV V protein appears to
be located in the carboxy-terminal region of the protein, a region implicated
in the IFN-antagonist activity exhibited by the V proteins of mumps virus
and human parainfluenza virus type 2.
Institutional address: Department of Microbiology
Mount Sinai School of Medicine New York New York 10029
USA.
(REFERENCE 15 OF 26)
Diebold SS Montoya M Unger H Alexopoulou L Roy P
Haswell LE Al-Shamkhani A Flavell R Borrow P Reis
e Sousa C
Viral infection switches non-plasmacytoid dendritic cells into high interferon
producers.
In: Nature (2003 Jul 17) 424(6946):324-8
Type I interferons (IFN-I) are important cytokines linking innate and
adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I
in response to viral infection and are thought to be the major source of the
cytokines in vivo. Here, we show that conventional non- plasmacytoid dendritic
cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic
choriomeningitis virus make similarly high levels of IFN-I on subsequent
culture. Similarly, non-plasmacytoid dendritic cells secrete high levels
of IFN-I in response to double-stranded RNA (dsRNA), a major viral
signature, when the latter is introduced into the cytoplasm to mimic
direct viral infection. This response is partially dependent on the cytosolic
dsRNA-binding enzyme protein kinase R and does not require signalling through
toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we
show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains
the inability of conventional dendritic cells to produce IFN-I on infection
with influenza. Our results suggest that multiple dendritic cell types,
not just plasmacytoid cells, can act as specialized interferon-producing
cells in certain viral infections, and reveal the existence of a TLR-independent
pathway for dendritic cell activation that can be the target of viral interference.
Institutional address: Immunobiology Laboratory
Cancer Research UK London Research Institute London WC2A 3PX
UK.
(REFERENCE 16 OF 26)
Taubenberger JK Reid AH Janczewski TA Fanning TG
Integrating historical, clinical and molecular genetic data in order to
explain the origin and virulence of the 1918 Spanish influenza virus.
In: Philos Trans R Soc Lond B Biol Sci (2001 Dec 29) 356(1416):1829-39
The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30%
of the world's population and resulted in the death of 40 million people.
The complete genomic sequence of the 1918 influenza virus will be deduced
using fixed and frozen tissues of 1918 influenza victims. Sequence and
phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase
(NA) genes show them to be the most avian-like of mammalian sequences
and support the hypothesis
that the pandemic virus contained surface protein-encoding genes derived
from an avian influenza strain and that the 1918 virus is very similar
to the common ancestor of human and classical swine H1N1 influenza strains.
Neither the 1918 HA genes nor the NA genes possessed mutations that are known
to increase tissue tropicity, which accounts for the virulence of other
influenza strains such as A/WSN/33 or fowl plague viruses. The complete
sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced
and tested for the hypothesis that the enhanced virulence in 1918 could
have been due to type I interferon inhibition by the NS1 protein. The
results from these experiments were inconclusive. Sequence analysis of the
1918 pandemic influenza virus is allowing us to test hypotheses as to the
origin and virulence of this strain. This information should help to elucidate
how pandemic influenza strains emerge and what genetic features contribute
to their virulence.
Institutional address: Department of Cellular Pathology
and Genetics Armed Forces Institute of Pathology Room 1057D
Building 101 1413 Research Boulevard Rockville MD 20850-3125
USA. taubenbe@afip.osd.mil
(REFERENCE 17 OF 26)
Li WX Li H Lu R Li F Dus M Atkinson P
Brydon EW Johnson KL Garcia-Sastre A Ball LA
Palese P Ding SW
Interferon antagonist proteins of influenza and vaccinia viruses are
suppressors of RNA silencing.
In: Proc Natl Acad Sci U S A (2004 Feb 3) 101(5):1350-5
Homology-dependent RNA silencing occurs in many eukaryotic cells. We
reported recently that nodaviral infection triggers an RNA silencing- based
antiviral response (RSAR) in Drosophila, which is capable of a rapid virus
clearance in the absence of expression of a virus-encoded suppressor. Here,
we present further evidence to show that the Drosophila RSAR is mediated by
the RNA interference (RNAi) pathway, as the viral suppressor of RSAR
inhibits experimental RNAi initiated by exogenous double-stranded RNA
and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions
as a natural antiviral immunity in mosquito cells. We further show that vaccinia
virus and human influenza A, B, and C viruses each encode an essential
protein that suppresses RSAR in Drosophila. The vaccinia and influenza
viral suppressors, E3L and NS1, are distinct double- stranded RNA-binding
proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated
innate antiviral response. We found that the double-stranded RNA-binding
domain of NS1, implicated in innate immunity suppression, is both essential
and sufficient for RSAR suppression. These findings provide evidence that
mammalian virus proteins can inhibit RNA silencing, implicating this
mechanism as a nucleic acid-based antiviral immunity in mammalian cells.
Institutional address: Departments of Plant Pathology
and Entomology and Microbiology Program University of California
Riverside CA 92521 USA.
(REFERENCE 18 OF 26)
Geiss GK Salvatore M Tumpey TM Carter VS Wang X
Basler CF Taubenberger JK Bumgarner RE Palese P Katze
MG Garcia-Sastre A
Cellular transcriptional profiling in influenza A virus-infected lung epithelial
cells: the role of the nonstructural NS1 protein in the evasion of the
host innate defense and its potential contribution to pandemic influenza.
In: Proc Natl Acad Sci U S A (2002 Aug 6) 99(16):10736-41
The NS1 protein of influenza A virus contributes to viral pathogenesis,
primarily by enabling the virus to disarm the host cell
type IFN defense system. We examined the downstream effects of NS1
protein expression during influenza A virus infection on global cellular mRNA
levels by measuring expression of over 13,000 cellular genes in response to
infection with wild-type and mutant viruses in human lung epithelial
cells. Influenza A/PR/8/34 virus infection resulted in a significant
induction of genes involved in the IFN pathway. Deletion of the viral
NS1 gene increased the number and magnitude of expression of cellular
genes implicated in the IFN, NF- kappaB, and other antiviral pathways.
Interestingly, different IFN- induced genes showed different sensitivities
to NS1-mediated inhibition of their expression. A recombinant virus
with a C-terminal deletion in its NS1 gene induced an intermediate cellular
mRNA expression pattern between wild-type and NS1 knockout viruses.
Most
significantly, a virus containing the 1918 pandemic NS1 gene was more
efficient at blocking the expression of IFN-regulated genes than its parental
influenza A/WSN/33 virus. Taken together, our results suggest that the cellular
response to influenza A virus infection in human lung cells is significantly
influenced by the sequence of the NS1 gene, demonstrating the importance of
the NS1 protein in regulating the host cell response triggered by virus
infection.
Institutional address: Department of Microbiology
School of Medicine University of Washington Seattle WA 98195
USA.
(REFERENCE 19 OF 26)
Basler CF Reid AH Dybing JK Janczewski TA
Fanning TG Zheng H Salvatore M Perdue ML Swayne
DE Garcia-Sastre A Palese P Taubenberger JK
Sequence of the 1918 pandemic influenza virus nonstructural gene (NS)
segment and characterization of recombinant viruses bearing the 1918
NS genes.
In: Proc Natl Acad Sci U S A (2001 Feb 27) 98(5):2746-51
The influenza A virus pandemic of 1918-1919 resulted in an estimated
20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences
of the 1918 virus were previously determined. We here report the sequence
of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding
two proteins, NS1 and nuclear export protein. Phylogenetically, these genes
appear to be close to the common ancestor of subsequent human and classical
swine strain NS genes. Recently, the influenza A virus NS1 protein was
shown to be a type I IFN antagonist that plays an important role in viral
pathogenesis. By using the recently developed technique of generating influenza
A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus
NS1 gene played a role in virulence was tested in a mouse model. In
a BSL3+ laboratory, viruses were generated that possessed either the
1918 NS1 gene alone or the entire 1918 NS segment in a background of
influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human
influenza strain first isolated in 1933. These 1918 NS viruses replicated
well in tissue culture but were attenuated in mice as compared with
the isogenic control viruses. This attenuation in mice may be related
to the human origin of the 1918 NS1 gene. These results suggest that
interaction of the NS1 protein with host-cell factors plays a significant
role in viral pathogenesis.
Comment in: Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2115-6
Institutional address: Department of Microbiology
Mount Sinai School of Medicine New York NY 10029 USA.
(REFERENCE 20 OF 26)
Vodeiko GM McInnis J Chizhikov V Levandowski RA
Genetic and phenotypic analysis of reassortants of high growth and
low growth strains of influenza B virus.
In: Vaccine (2003 Sep 8) 21(25-26):3867-74
The yield of influenza virus in eggs is critical to influenza vaccine production
and availability, but the contribution of specific genes to the growth
properties of influenza B viruses is not well understood. Influenza B/Beijing/184/93
and B/Shangdong/7/97 were chosen for study because B/Shangdong/7/97 replicated
to several fold higher titers in eggs than B/Beijing/184/93 as demonstrated
by hemagglutination titers and EID50. A reassortant with the HA, NP
and PB2 genes from B/Beijing/184/93 and all other genes from B/Shangdong/7/97
had the high growth phenotype of B/Shangdong/7/97 in eggs, which suggests
that NS, M, NA, PB1 or PA, or a combination of these genes derived from B/Shangdong/7/97
were needed for the high growth phenotype of the reassortants. A high
degree of homology was found among the genetic sequences of B/Beijing/184/93,
B/Shangdong/7/97, and other influenza B viruses. However, differences potentially
related to growth characteristics were suggested by analysis of the
deduced amino acid (AA) sequences of four genes: NS (NS1, NS2), M (BM2), NA
(NA, NB) and PB1. The studies identify multiple genes that may affect growth
of influenza B viruses in eggs.
Institutional address: Laboratory of Pediatric and Respiratory
Viral Diseases, Division of Viral Products, Office of Vaccines
Research and Review, Center for Biologics and Evaluation
and Research, Food and Drug Administration, Bethesda
MD 20892 USA. vodeiko@cber.fda.gov
(REFERENCE 21 OF 26)
Chesler DA Munoz-Jordan JL Donelan N Garcia-Sastre A
Reiss CS
PKR is not required for interferon-gamma inhibition of VSV replication
in neurons.
In: Viral Immunol (2003) 16(1):87-96
In this report, the contribution of PKR to the IFN-gamma mediated
inhibition of VSV replication in neurons was examined. IFN-gamma treatment
of NB41A3 murine neuroblastoma cells resulted in the reduced expression of
VSV protein during infection. PKR was found to be modestly upregulated
in NB41A3 cells following IFN-gamma treatment. The phosphorylation state
of PKR and its downstream target, eIF2alpha, were unaffected by either IFN-gamma
or VSV infection. Inhibition of PKR through the use of 2-aminopurine or the
expression of the Influenza A NS1 gene had no effect on the ability
of IFN-gamma to inhibit the replication of VSV in vitro. These data indicate
that endogenously expressed PKR is not required for the IFN- gamma mediated
inhibition of VSV replication in NB41A3 neuroblastoma cells.
Institutional address: Department of Biology
New York University New York New York 10003
USA.
(REFERENCE 22 OF 26)
Hartman AL Towner JS Nichol ST
A C-terminal basic amino acid motif of Zaire ebolavirus VP35 is essential
for type I interferon antagonism and displays high identity with the RNA-binding
domain of another interferon antagonist, the NS1 protein of influenza A virus.
In: Virology (2004 Oct 25) 328(2):177-84
The ebolavirus VP35 protein antagonizes the cellular type I interferon response
by blocking phosphorylation of IRF-3, a transcription factor that turns on
the expression of a large number of antiviral genes. To identify the
domain of VP35 responsible for interferon antagonism, we generated mutations
within the VP35 gene and found that a C-terminal basic amino acid motif
is required for inhibition of ISG56 reporter gene expression as well
as IFN-beta production. Remarkably, this basic amino acid motif displayed
high sequence identity with part of the N-terminal RNA-binding domain
of another interferon-antagonist, the NS1 protein of influenza A virus.
Institutional address: Special Pathogens Branch
Division of Viral and Rickettsial Diseases National Center for
Infectious Diseases Centers for Disease Control and Prevention
1600 Clifton Road MS G-14 Atlanta GA 30329 USA. biq7@cdc.gov
(REFERENCE 23 OF 26)
Kittel C Sereinig S Ferko B Stasakova J Romanova
J Wolkerstorfer A Katinger H Egorov A
Rescue of influenza virus expressing GFP from the NS1 reading frame.
In: Virology (2004 Jun 20) 324(1):67-73
In this study, several influenza NS1 mutants were examined for their growth
ability in interferon (IFN)-deficient Vero cells treated with human
interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed
similar growth properties as the wild-type virus, whereas viruses carrying
an impaired RNA binding domain were dramatically attenuated. Relying on the
ability of the first half of the NS1 protein to antagonize the IFN action,
we established a rescue system for the NS gene based on the transfection
of one plasmid expressing recombinant NS vRNA and subsequent coinfection
with an IFN sensitive helper virus followed by adding of human IFN-alpha
as a selection drug. Using this method, a recombinant influenza A virus
expressing green fluorescence protein (GFP) from the NS1 reading frame was
rescued. To ensure the posttranslational cleavage of GFP from the N-terminal
125 amino acids (aa) of NS1 protein, a peptide sequence comprising a
caspase recognition site (CRS) was inserted upstream the GFP protein. Although
a rather long sequence of 275 aa was inserted into the NS1 reading frame,
the rescued recombinant vector appeared to be genetically stable while passaging
in Vero cells and was able to replicate in PKR knockout mice.
Institutional address: Institute of Applied Microbiology
University of Natural Resources and Applied Life Sciences
A 1190 Vienna Austria.
(REFERENCE 24 OF 26)
Sato Y Yoshioka K Suzuki C Awashima S Hosaka Y
Yewdell J Kuroda K
Localization of influenza virus proteins to nuclear dot 10 structures in
influenza virus-infected cells.
In: Virology (2003 May 25) 310(1):29-40
We studied influenza virus M1 protein by generating HeLa and MDCK
cell lines that express M1 genetically fused to green fluorescent protein
(GFP). GFP-M1 was incorporated into virions produced by influenza virus infected
MDCK cells expressing the fusion protein indicating that the fusion
protein is at least partially functional. Following infection of either
HeLa or MDCK cells with influenza A virus (but not influenza B virus),
GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in
nuclear dots. Immunofluorescence revealed that the nuclear dots represent
nuclear dot 10 (ND10) structures. The colocalization of authentic M1,
as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence
following in situ isolation of ND10. These findings demonstrate a previously
unappreciated involvement of influenza virus with ND10, a structure involved
in cellular responses to immune cytokines as well as the replication of a
rapidly increasing list of viruses.
Institutional address: Department of Virology and Immunology
Osaka University of Pharmaceutical Sciences 4-20-1 Nasahara Takatsuki
Osaka 569-1094 Japan.
(REFERENCE 25 OF 26)
Noah DL Twu KY Krug RM
Cellular antiviral responses against influenza A virus are countered
at the posttranscriptional level by the viral NS1A protein via its binding
to a cellular protein required for the 3' end processing of cellular pre-mRNAS.
In: Virology (2003 Mar 15) 307(2):386-95
The influenza A virus NS1 protein (NS1A protein) binds and inhibits
the function of the 30-kDa subunit of CPSF, a cellular factor that is required
for the 3'-end processing of cellular pre-mRNAs. Here we generate a
recombinant influenza A/Udorn/72 virus that encodes an NS1A protein
containing a mutated binding site for the 30-kDa subunit of CPSF.
This mutant virus is substantially attenuated, indicating that this binding
site in the NS1A protein is required for efficient virus replication.
Using this mutant virus, we show that NS1A binding to CPSF mediates the viral
posttranscriptional countermeasure against the initial cellular antiviral
response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation
of the transcription of cellular antiviral genes, which requires the
interferon regulatory factor-3 (IRF-3) transcription factor that is activated
by virus infection. Whereas the posttranscriptional processing of these
cellular antiviral pre-mRNAs is inhibited in cells infected by wild-
type influenza A virus, functional antiviral mRNAs are produced in cells
infected by the mutant virus. These results establish that the binding
of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional
inhibition of the processing of these cellular antiviral pre-mRNAs.
Mutation of this binding site in the NS1A protein also affects a second
cellular antiviral response: in cells infected by the mutant virus, IFN-beta
mRNA is produced earlier and in larger amounts.
Institutional address: Institute for Cellular and
Molecular Biology Section of Molecular Genetics and Microbiology
University of Texas at Austin 78712 USA.
(REFERENCE 26 OF 26)
Seo SH Hoffmann E Webster RG
The NS1 gene of H5N1 influenza viruses circumvents the host anti- viral
cytokine responses.
In: Virus Res (2004 Jul) 103(1-2):107-13
The H5N1 influenza viruses transmitted to humans in 1997 were highly
virulent, but the mechanism of their virulence in humans is largely unknown.
Here we show that lethal H5N1 influenza viruses, unlike other human,
avian, and swine influenza viruses, are resistant to the anti-viral effects
of interferons and tumor necrosis factor alpha The nonstructural (NS) gene
of H5N1 viruses is associated with this resistance. Pigs infected with recombinant
human H1N1 influenza virus that carried the H5N1 NS gene experienced
significantly greater and more prolonged viremia, fever, and weight loss than
did pigs infected with wild-type human H1N1 influenza virus. These effects
required the presence of glutamic acid at position 92 of the NS1 molecule.
These findings may explain the mechanism of the high virulence of H5N1
influenza viruses in humans and provide insight into the virulence of
1918 Spanish influenza.
Institutional address: Division of Virology
Department of Infectious Diseases St. Jude Children's Research Hospital
332 North Lauderdale Memphis TN 38105-2794 USA.
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