Pyres: 2001 UK FMD Outbreak - Photo: Murdo Macleod.  Slides L-R: Smallpox, SARS Coronavirus , Foot and Mouth Disease, West Nile Virus.
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:


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.

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.
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.
 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

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.

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.

 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.
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.
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.

 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.

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.
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.
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.
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.

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.
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.
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.
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.

  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.

 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.

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.
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.


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.
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.

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.
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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