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J Virol. 2010 February; 84(4): 2122–2133.
Published online 2009 December 9. doi:  10.1128/JVI.01668-09
PMCID: PMC2812369

The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV [down-pointing small open triangle]

Abstract

A reassortant avian influenza virus (designated FPV NS GD), carrying the NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able to replicate more efficiently in different human cell lines and primary mouse epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and death in experimentally infected mice and was detected in mouse lungs; in contrast, rFPV was not able to replicate in mice effectively. These results indicated an altered host range and increased virulence. Furthermore FPV NS GD showed pronounced pathogenicity in chicken embryos. In an attempt to define the molecular basis for the apparent differences, we determined that NS1 proteins of the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable efficiencies in vitro. However, FPV NS GD infection resulted in (i) increased expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger beta interferon promoter inhibition than rFPV. Taken together, the results shed further light on the importance of the NS segment of an H5N1 strain for viral replication, molecular pathogenicity, and host range of HPAIVs and the possible consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.

Outbreaks of avian influenza (AI) are caused by influenza A viruses (IAVs) belonging to the family Orthomyxoviridae. IAVs are divided into different subtypes based on the antigenic nature of their hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Currently, 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) have been isolated from birds (59). So far, all isolates of highly pathogenic AIVs (HPAIVs) belong to H5 or H7 subtypes. These can cause up to 100% mortality in poultry. Seventeen major outbreaks of HPAIV were reported in different areas until the end of 20th century (1). Recent outbreaks with devastating consequences occurred in South and North America (H5N2, 1994 to 2003; H7N3, 2002, 2004; H5N2, 2004) (45), Europe (H7N1, 1999 to 2001; H7N7, 2003; H5N1, 2005 to 2007) (6, 58), Asia (H7N3, 1995 to 2003; H5N1, 1997, 2003 to 2007) (6, 58), and Africa (H5N1, 2006) (2, 58).

In addition to their high virulence in avian species, HPAIVs are also a potential threat to human health. In 1997, 18 people were infected by H5N1 AIV in Hong Kong, and of these, six patients died (50). An HPAIV H7N7 epidemic in the Netherlands in 2003 caused infection of more than 80 people, resulting in one fatal case (13). Recently, an H5N1 AI outbreak started in Asia and spread to Europe and Africa. Out of the 433 cases confirmed by WHO, 262 people died by June of 2009 (57).

The IAV genome includes eight viral RNA (vRNA) segments. The NS segment encodes both the nonstructural protein NS1, found only in infected cells, and the nuclear export protein (NS2/NEP), which supports nuclear export of the viral genome (36). NS1 is a multifunctional protein that plays a key role in the pathogenesis and virulence of IAVs (for a review, see reference 19). It targets the RNA helicase RIG-I, the major vertebrate sensor protein detecting influenza virus vRNA (14). Thereby, NS1 strongly inhibits the RIG-I-mediated activation of type I interferon (IFN) genes via the transcription factor IRF-3. In addition, NS1 decreases IFN-β promoter activation by blocking the Jun-N-terminal kinase-dependent activation of AP-1 transcription factors (30). Another important way by which NS1 prevents activation of the cellular innate immune response is thought to be mediated via double-stranded RNA (dsRNA) binding activity, thus preventing activation of the dsRNA-activated protein kinase (PKR). Activated PKR phosphorylates the initiation factor eIF2α, thereby inhibiting translation and viral replication. Inhibition of PKR may also suppress the activation of the transcription factor NF-κB and consequently induction of IFN expression (3, 20, 56). Deletion of the NS1 gene strongly attenuated IAV in IFN-competent hosts (16). Thus, the ability to suppress the innate type I IFN response of the host cell plays a major role in supporting efficient viral replication.

The NS1 proteins of certain IAV strains were also shown to inhibit host mRNA polyadenylation, host pre-mRNA splicing, and nuclear export of host mRNA, thereby suppressing the expression of antiviral genes. In this context NS1 binds and inhibits the function of the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) and the poly(A)-binding protein II (PABII) that are both required for the 3′-end processing of the cellular pre-mRNAs. To exert all these activities, the NS1 protein possesses two important functional domains: an RNA-binding domain near the amino-terminal end (amino acids [aa] 1 to 73) that can bind to various RNA species including poly(A) mRNA, U6 snRNA, and dsRNA and an effector domain in the carboxyl half of the molecule (aa 74 to 230) by which the NS1 protein inhibits nuclear poly(A)-mRNA export and splicing of pre-mRNA (for a review, see reference 19).

Furthermore, it was shown that the last four C-terminal amino acids of NS1 could be part of a PDZ domain binding motif that influences the activity of proteins containing a PDZ domain, which are frequently involved in cellular signal transduction pathways (35). Previously, the PDZ domain in the NS1 protein has been identified as a new virulence determinant of IAV (22). Moreover, the NS1 protein was recently found to activate the phosphatidylinositol-3 kinase (PI3K)/Akt-pathway, presumably as part of an antiapoptotic signaling response (11, 12, 17, 49).

The exchange of genome segments between different IAVs can lead to reassortant viruses with new characteristics (59). In light of the possibility that type H7 and H5 HPAIVs can reassort in nature, we intended to determine the impact that a NS segment encoding NS1 protein of a prototype H5 HPAIV would have on viral pathogenicity, replication, and transmission of a classical and strictly avian H7 HPAIV. Therefore, we generated a reassortant (FPV NS GD) between two classical model HPAIVs, namely, A/FPV/Rostock/34 (FPV; H7N1) and A/Goose/Guangdong/1/96 (GD; H5N1) by reverse genetics. FPV NS GD carries the NS-segment of GD in the genetic background of FPV. Strikingly, the reassortant showed a strongly enhanced replication, altered tissue tropism, and a new host range. To elucidate the molecular basis for the altered viral characteristics, we analyzed the capability of the NS1 proteins from GD and FPV to bind to CPSF or to suppress PKR and IFN promoter activation. Our data provide principal evidence that an H7 HPAIV acquiring H5 HPAIV genes in nature could gain new characteristics including the ability to replicate in mammalian hosts.

MATERIALS AND METHODS

Cells, mice, eggs, and infection.

HEK 293T cells (human embryonic kidney constitutively expressing the simian virus 40 [SV40] large T antigen), A549 cells (human alveolar epithelia cells [AECs]), and MDCK (Madin-Darby canine kidney) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics. BEAS cells (human bronchiolar epithelia) were maintained in bronchial epithelial cell basal medium (BEMB; Cambrex/Clonetics) and supplemented with BEGM SingleQuots (Cambrex/Clonetics). Cells were infected at the multiplicity of infection (MOI) indicated in the legend to Fig. Fig.11 and further incubated as previously described (38). Eight-week-old female C57BL/6 mice bred in the animal facility of the Friedrich Loeffler Institute in Tübingen, Germany, were used for infectivity experiments. Eleven-day-old embryonated specific-pathogen-free (SPF) chick eggs were used for viral infection into the allantoic cavity. Infected eggs were incubated at 37°C for the time indicated in the legend to Fig. Fig.11 and allantoic fluid was harvested for further analysis.

FIG. 1.
Growth curves of rFPV and reassortant FPV NS GD. MDCK cells were infected at an MOI of 0.001 with rFPV ([filled lozenge]) or reassortant FPV NS GD ([filled triangle]). Supernatants were monitored in multicycle replication for HA titers (A) and for infectious particles ...

Isolation and culture of murine primary AEC II.

Type II AECs were isolated by the method developed by Corti et al. (7) with some modifications (21). Mice (C57BL/6N:PKR+/+ and C57BL/6J:PKR/−) (61) were sacrificed, and lung homogenates were prepared. Briefly, mouse primary AECs were isolated as previously described, but with some modifications. C57BL/6 mice were euthanized by an overdose of isofluorane (Abbott, Wiesbaden, Germany) and exsanguinated by cutting of the inferior vena cava. Lungs were then perfused with 20 ml of sterile Hanks balanced salt solution (HBSS; PAA) via the right ventricle until they were optically free of blood. A small incision was made into the exposed trachea to insert a shortened 21-gauge cannula, which was then firmly fixed. Sterile dispase (1.5 ml; BD Biosciences) followed by 500 μl of sterile 1% low-melting-point agarose in phosphate-buffered saline (PBS; Sigma Aldrich) was administered into the lungs. After 2 min of incubation, the lungs were removed and placed into a culture tube containing 2 ml of dispase for 40 min at room temperature. The lungs were then transferred into a culture dish containing DMEM-2.5% HEPES buffer-0.01% DNase (Serva), and the lung tissue was carefully dissected from the airways and large vessels. The cell suspension was successively filtered, resuspended in 10 ml of DMEM supplemented with 10% FCS and antibiotics, and incubated with biotinylated rat anti-mouse CD16/32, rat anti-mouse CD31, and rat anti-mouse CD45 monoclonal antibodies (MAbs; BD Pharmingen) for 30 min at 37°C. Cells were then washed and incubated with streptavidin-linked MagneSphere paramagnetic particles (Promega) for 30 min at room temperature with gentle rocking, followed by magnetic separation of contaminating leukocytes and endothelial cells for 15 min. The purity of freshly isolated AECs contained in the supernatant was always >95%, as assessed by immunofluorescence staining with rabbit anti-mouse wide-spectrum cytokeratin MAb (Dako), and viability was consistently >90% (analyzed by trypan blue staining). The cells were plated on 24-well cell culture plates at a density of 4 × 105 cells/well and grown to 90% confluence for 2 days with DMEM supplemented with 10% FCS and antibiotics. On day 2, the cells were washed and serum starved with 0.2% FCS and left until day 3, upon which they were submitted to virus infection, as described above.

Plasmid construction.

The NS gene of the strain A/Goose/Guangdong/1/96 (GD; H5N1) was PCR amplified from pCI-NS (containing cDNA of the GD NS segment) using the oligonucleotides 5′-GGCGAAGCTTGCTCTTCTGCCAGCAAAAGCAGGGTGACAAAGAC-3′ and 5′-GGCGGGGCCCGCTCTTCCATTAGTAGAAACAAGGGTGTTTTTTAT-3′ containing HindIII and SapI or ApaI and SapI sites, respectively. The PCR product was digested with HindIII/ApaI (NEB) and inserted into the corresponding sites of pcDNA3.1 (Invitrogen), resulting in the construct pcDNA3.1-NS. The insert was confirmed by sequencing. The construct was then digested by SapI (NEB), and the NS fragment was subcloned into the SapI site of pBD, resulting in the construct pBD-NS. The pBD vector contains a truncated version of the human polymerase I (Pol I) promoter and a hepatitis delta virus (HDV) ribozyme separated by SapI sites. This Pol I transcription unit (Pol I promoter and HDV ribozyme) is flanked by the Pol II promoter of the human cytomegalovirus and a polyadenylation signal of the gene encoding the bovine growth hormone (26).

The expression plasmids pHMG-PB1, -PB2, -PA, and -NP encode the subunits of the influenza virus A/PR/8/34 (PR8; H1N1) polymerase and the nucleoprotein (NP) (37). Plasmids pPol I-PA, -PB1, -PB2, -NP, -HA, -NA, -M, and -NS contain the viral segment of A/FPV/Rostock/34 (FPV; H7N1) cloned in between the human Pol I promoter and mouse Pol I terminator. Plasmid pIFN-β-Luci (10) contains the luciferase gene under the control of the IFN-β promoter.

Expression plasmids pcDNA3.0-NS1-GD and pcDNA3.0-NS1-FPV, encoding the NS1 proteins of GD and FPV, respectively, were constructed by PCR amplification of the NS1 fragments from pBD-NS and pPolI-NS using the oligonucleotide pair GD-NS1-Fw (5′-ATGGATTCCAACACGATAAC-3′) and GD-NS1-Bw (5′-TCAAACTTCTGACTCAACTC-3′) and the pair RS-NS1-Fw (5′-ATGGATTCCAACCATGTGTC-3′) and RS-NS1-Bw (5′-TCAAATTTCTGACTCAATTGTTC-3′), respectively, and cloned into pCR-BLUNT II-TOPO (Invitrogen). Subsequently, pCR-BLUNT II-TOPO constructs carrying the NS1 gene were digested with KpnI/XhoI (NEB), and the DNA fragment was cloned into the corresponding sites of pcDNA3.0. The correct gene sequences were confirmed by sequencing.

Rescue of reassortant viruses.

293T cells (106 cells/3.5-cm dish) were transfected with a mixture of 12 plasmids (5 μg of total DNA) using 7 μl of Lipofectamine and 10 μl of Plus reagent (Invitrogen). For the generation of recombinant wild-type FPV (rFPV), 1.2 μl (1 μg/μl) of the helper plasmids pHMG-PB1, -PB2, -PA, and -NP was used in a ratio of 1:1:0.2:1. Additionally, 3.8 μl (1 μg/μl) of plasmids pPolI-PB1, pPolI-PB2, pPolI-PA, pPolI-NP, pPolI-HA, pPolI-NA, pPolI-M, and pPolI-NS of the strain FPV (H7N1) was used in a ratio of 1:1:1:1:2:2:1:1.

To generate the reassortant virus (FPV NS GD), pPol I-NS (FPV) was replaced with pBD-NS (GD). The amount of DNA for transfection and the ratio among the plasmids were not changed. Forty-eight hours post transfection (p.t.), the supernatants (SN) from the transfected cells were harvested and used to infect confluent MDCK cells in 6-cm-diameter dishes to amplify progeny viruses. Rescued viruses were plaque purified three times. Purified virus was amplified and titrated on MDCK cells by standard plaque assay or focus assay and stored at −70°C.

Immunohistochemical plaque staining (focus assay).

MDCK cells grown in 96-well plates to about 90% confluence were washed with PBS++ (PBS containing 1 mM MgCl2, 0.9 mM CaCl2) and infected with 50 μl of diluted virus. The inoculum was replaced by 150 μl of MC medium (1× DMEM, 1.5% methyl cellulose). At 48 h postinfection (p.i.), cells were fixed and permeabilized with 4% paraformaldehyde (PFA)-1% Triton X-100 in PBS ++. Cells were washed with PBS++ and 0.05% Tween 20 and incubated with 50 μl of anti-NP antibody (mouse anti-IAV nucleoprotein; Biozol) diluted (1:5,000) in PBS++ and 3% bovine serum albumin (BSA) for 1 h at room temperature. After the cells were washed, they were incubated with 50 μl of a second antibody (anti-mouse horseradish peroxidase [HRP] antibody; Santa Cruz) diluted (1:1,000) in PBS++ and 3% BSA for 1 h at room temperature. To detect foci, washed cells were incubated in 40 μl/well AEC staining solution (Sigma). For analysis the 96-well plates were scanned and analyzed using the Photoshop software package (Adobe).

RNA preparation, RT-PCR, and sequencing.

MDCK cells were infected with plaque-purified reassortant FPV NS GD or rFPV. Three days postinfection, SN were collected, and cellular debris was precipitated at 2,000 × g. Viruses were subsequently pelleted from the SN by ultracentrifugation at 100,000 × g for 1 h. RNA was isolated (Qiagen) and treated with DNase I (10 U/μl; Roche) at 37°C for 30 min, extracted with phenol-chloroform, and precipitated from the aqueous phase with isopropanol and glycogen. Finally, the RNA was ethanol washed, dried, and resuspended in RNase-free water. For restriction analysis the purified RNA was subjected to reverse transcription (RT) using Superscipt II (Invitrogen) and 1 pmol of Flu RT primer (5′-AGCAAAAGCAGG-3′). RT samples were amplified using Taq polymerase (Gibco) and 0.5 pmol of NS-specific primers (NS RT-Fw, 5′-CCAGCAAAAGCAGGGTGACA-3′; NS-RT Bw, 5′-TTAGTAGAAACAAGGGTGTT-3′). At the same time, direct PCR (control) was performed with half of the amount of the RNA used for RT. The RT products were digested with HaeIII, and cleavage products and direct PCR products were examined by electrophoresis in a 1% agarose gel. For sequencing the NS segments were amplified from the purified RNA by RT using 1.5 pmol of RT primer Bm-NS-1 (5′-TATTCGTCTCAGGGAGCAAAAGCAGGGG-3′). The cDNAs were PCR amplified from the RT samples using 0.5 pmol of Bm-NS-1 and Bm-NS-890 (5′-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3′). The RT-PCR products were gel purified and then directly cloned into PCR-XL-TOPO vector (Invitrogen). Plasmid DNA containing the NS segments isolated from three independent clones was sequenced and compared by the MegAlign program (Lasergene, version 6.0: DNASTAR) to the expected sequence.

Analysis of viral replication.

To analyze viral replication efficiency, aliquots of SN from cells infected in duplicates at the MOIs indicated in the legend to Fig. Fig.11 were collected at different time points. Hemagglutinating units (HAU ml−1) used to analyze virus particle formation were determined by standard HA assay using chicken red blood cells (0.5% in PBS) and were done in duplicates. The numbers of PFU (PFU ml−1) indicating infectious titers were determined by standard plaque assay or focus assay in duplicates.

Luciferase assay.

To determine virus-induced IFN-β promoter-dependent reporter gene activation, MDCK cells (106 cells/3.5-cm dish) were transfected with 0.1 μg of the IFN-ß promoter-dependent luciferase-expressing plasmid pIFN-β-Luci using 4 μl of Lipofectamine 2000 (Invitrogen). After 24 h, cells were either mock infected or infected with rFPV or FPV NS GD (MOI of 1). Cell extracts were prepared at 4 h p.i. in PLB cell lysis buffer (Promega) and assayed for luciferase activity. Data presented represent the means of four independent transfections and are given as the ratio of luciferase activity relative to activity in the mock-infected control. To determine poly(I·C)-induced IFN-ß-dependent reporter gene activation, MDCK cells were transfected with 0.1 μg of the IFN-ß promoter-dependent luciferase-expressing plasmid pIFN-ß-Luci and 0.2 μg of either empty vector (pcDNA 3.0) or pcDNA3.0-NS1-GD/pcDNA3.0-NS1-FPV using Opti-MEM medium (Invitrogen) and 2.5 μl of Lipofectamine 2000 (Invitrogen). After 24 h, cells were transfected with 5 μg of poly(I·C) (Sigma; 1 mg ml−1 in Ringer solution [Amersham]) using 16 μl of DOTAP [N(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methyl-sulfate; Roth]. Cell extracts were prepared at 4 h p.t. in 100 μl of PLB lysis buffer and assayed for luciferase activity. The difference in the relative luciferase activity between poly(I·C)- or mock-transfected cells of three independent experiments was calculated.

Western blotting and in-cell Western blotting (ICWB) analysis.

For Western blotting (WB), cells were lysed as previously described (38). To detect cellular eIF2α or the phosphorylated form P-eIF2α in cell lysates, the proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon FL transfer membranes; Millipore). Membranes were incubated with the phospho-specific rabbit anti P-eIF2α (1:1,000; Biosource) antiserum as a primary antibody diluted in blocking buffer (5% nonfat dry milk, TBS-Tween [20 mM Tris-HCl, pH 7.6, 140 mM NaCl, 0.05% Tween 20]). Protein was detected with goat anti-rabbit IRDye 680 incubated in the dark (Licor) (1:10,000 in blocking buffer plus 10% SDS), followed by detection and analysis using an Odyssey Infrared Imaging System and application software package (Licor). After the primary antibody was stripped (Rotifree stripping solution; Roth), blots were incubated with anti-eIF2α MAb (1:500, Biosource) and detected with goat anti-mouse IRDye 800 CW (1:10,000 in blocking buffer plus 10% SDS; Licor), followed by detection and analysis using the Odyssey Infrared Imaging System and application software package (Licor) to show equal loading.

For ICWB, MDCK cells grown in 96-well plates were washed with PBS++ and fixed with PBS-3.7% PFA for 20 min, permeabilized by washing four times for 5 min each with PBS-0.1% Triton X-100, and blocked with PBS-5% nonfat dried milk. To detect NS1, NP, and extracellular signal-regulated kinase 2 (ERK2; loading control) cells were incubated for 2 h at room temperature on a shaker with a first antibody, which was either polyclonal rabbit anti-NS1 serum (anti-GST-NS1 9101 at 1:1,000; GST is glutathione S-transferase; T. Wolff), mouse anti-NP MAb (1:5,000; Abcam), mouse anti-ERK2 MAb (1:100; Santa Cruz), or polyclonal rabbit anti-ERK2 (1:100; Santa Cruz) serum diluted in PBS-5% nonfat dried milk-0.2% Tween-20. Thereafter, cells were washed four times for 5 min each with Tween washing solution (PBS-0.5% Tween-20) and incubated for 1 h at room temperature in the dark with a second antibody (goat anti-rabbit IRDye 680 or goat anti-mouse IRDye 800 CW [both from Licor]) in PBS-5% nonfat dried milk-0.2% Tween-20. Finally, plates were washed again as described above with PBS, dried, and analyzed using the Odyssey Infrared Imaging System and application software package (Licor). For each time point six wells were analyzed.

Glutathione-Sepharose binding analysis.

The F2F3 domain of CPSF30 fused to GST (F2F3-GST) was mixed with 35S-labeled NS1 protein (FPV or GD) and subjected to glutathione-Sepharose binding selection. Briefly, the plasmid pGEX3X-F2F3/GST, encoding the fragment F2F3 (aa 61 to 121) of CPSF30, fused in frame to GST in the pGEX3X vector, was used to express the F2F3-GST fusion protein in Escherichia coli strain Rosetta (DE3) pLysS (Novagen) and was purified as previously described (40). The glutathione-Sepharose 4B matrix (GS4B; GE Healthcare) was prepared according to the manufacturer's instructions. Instead of using PBS, a washing buffer (600 mM NaCl, 50 mM Tris-HCl, 0.5% NP-40) was used throughout the whole procedure. Finally, 75-μl aliquots of 50% GS4B were stored. To prepare the labeled NS1 proteins, NS1 proteins were in vitro translated using pcDNA3.0-NS1-GD, -NS1-FPV, or -NS1-PR8 as templates and radioactively labeled using a TNT-coupled reticulocyte lysate kit (Promega) with l-[35S]methionine (20 μCi; Amersham/GE Healthcare) according to the manufacturer's instructions. To analyze binding between NS1/F2F3-GST, 5 μl of TNT lysate containing the labeled NS1 protein, 6 μl of F2F3-GST (or 6 μl of H2O as a control), and 189 μl of H2O was incubated overnight at 4°C under vigorous shaking. Then, 75 μl of GS4B was added, and the mixture was incubated overnight at 4°C under vigorous shaking. The NS1/F2F3-GST/GS4B complexes were washed three times using 1 ml of washing buffer and precipitated at 500 × g for 5 min. The pellets were then redissolved in 10 μl of washing buffer and boiled for 5 min with 20 μl of Laemmli loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol [DTT], 4% SDS, 0.2% bromphenol blue, 20% glycerol) at 95°C, and 20 μl of sample was loaded onto a 10% SDS-PAGE gel. Five microliters of each TNT lysate (input) was mixed with 5 μl of H2O and 10 μl of Laemmli loading buffer and then boiled for 5 min at 95°C and loaded onto the gel. To visualize the amount of loaded F2F3-GST, the gel was stained with Coomassie (double-distilled H2O [ddH2O]-methanol at 1:1, 7% acetic acid, 0.1% Coomassie Brilliant blue R250) for 30 min, destained (40% methanol-10% acidic acid) for 1 h, fixed in fixing solution (10% ethanol-5% acetic acid) for 20 min, and dried prior to exposure to reporter screen (Fuji) and subsequent measurement with a Typhoon 9200 (Molecular Dynamics/GE Healthcare). The amount of bound NS1 was calculated and normalized to the amount of loaded F2F3-GST. Data presented represent the means of three independent experiments and are given as relative amounts of bound NS1.

PKR binding analysis.

For GST pulldown analysis, the kinase-inactive mutant PKR (K296R) was used since its expression and stability in bacteria are far superior to the wild type (5). The GST-PKR (K296R) fusion protein was expressed from pGEX-PKR (K296R) in E. coli BL26 and bound to glutathione-Sepharose beads (Amersham Biosciences AB) according to the manufacturer's protocol. NS1 proteins of GD and FPV were in vitro translated and labeled with [35S]methionine as described before and incubated with the GST-PKR (K296R)-Sepharose beads in binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.4% Triton X-100, 20% glycerol) for 2 h at 4°C. The beads were washed three times with the binding buffer, and the precipitated proteins were separated by SDS-gel electrophoresis and visualized by autoradiography.

Mouse infection experiments.

Infectivity experiments using recombinant influenza virus were performed in Giessen, Germany. Animals were weighed and anesthetized with 200 μl of 10% ketamine-rompun before intranasal infection with 2 × 105 PFU of either rFPV or FPV NS GD in a volume of 50 μl. Body weight and general health status were monitored daily. After onset of disease, general health status was examined twice per day. According to the German animal protection law, mice that developed the terminal stage of disease or lost 25% of their initial weight were sacrificed. To determine lung virus titers, mice were sacrificed, and lungs were collected in BSS buffer (0.137 mM NaCl, 5.3 mM KCl, 0.74 mM MgSO4·7H2O, 0.98 mM MgCl2·6H2O, 0.44 mM KH2PO4, 1.5 mM NaH2PO4·2H2O; Sigma). The samples were homogenized by a Branson Sonifier (10 pulses of 100 W followed a few minutes later by an additional 5 pulses) to obtain a 10% tissue homogenate. The samples were centrifuged at 3,000 rpm for 5 min, and SN were stored at −70°C for further analysis.

Confocal laser scanning microscopy.

MDCK cells grown on glass coverslips were infected and incubated in medium as indicated in Fig. S2 in the supplemental material; washed with PBS at 4 h, 6 h, 8 h, and 10 h p.i.; and fixed with 4% PFA overnight at 4°C to monitor HA surface expression. To analyze the RNP export, cells were further washed twice with PBS and permeabilized with 1% Triton X-100 (in ddH2O) for 30 min. The cells were then incubated with mouse anti-NP monoclonal IgG (clone F 1331; Biodesign International) or mouse anti-H7 HA monoclonal IgG (kind gift from H.-D. Klenk, Marburg, Germany) for 45 min. Cells were then washed twice with PBS and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Sigma) for 45 min in the dark. After additional washes, cells were incubated with DAPI ([4′,6′-diamidino-2-phenylindole] 10 mg ml−1 in PBS-3% BSA) for 10 min in the dark. All antibodies used were diluted 1:100 in PBS-3% BSA. Finally, cells were washed twice and fixed in Mowiol (Aldrich) in glycerol and H2O supplemented with 2.5% diazabicyclooctane (DABCO; Merck). Fluorescence was visualized using a TCS SP5 confocal laser scanning microscope (Leica).

Statistical analysis.

The results correspond to the mean ± standard error of the mean (SEM) of the indicated experiments. The statistical significance of differences between the indicated groups was tested using an unpaired Student's t test (see figure legends). Statistical analysis of survival time was performed by Kaplan-Meier tests.

RESULTS

Rescue and identification of recombinant HPAIVs.

The FPV NS GD reassortant virus carrying the NS gene of GD in the background of the FPV genome and the corresponding wild-type FPV (rFPV) were rescued and plaque purified. To confirm the origin of the NS segment of FPV NS GD, viral RNA was used for reverse transcription-PCR (RT-PCR) and for direct PCR (control) with a pair of NS-specific primers. The generated PCR products of about 890 nucleotides (nt) were characterized by restriction analysis with HaeIII showing the expected fragments of 523 nt and 367 nt for the reassortant virus, whereas no cleavage products occurred using the RT-PCR product of rFPV (data not shown). Additionally, full-length RT-PCR products of the NS segments obtained from both viruses after several passages on MDCK cells were cloned and sequenced. Analysis of plasmid DNA isolated from each of three independent bacterial clones showed no changes to the expected sequences (data not shown).

Growth characteristics of the reassortant FPV NS GD.

The replication characteristics of both viruses (rFPV and FPV NS GD) were investigated by infection of MDCK cells at MOIs of 1 and 0.001 to allow single- as well as multicycle replication. Supernatant taken at 8 h p.i. for single-cycle replication and at 12 h, 24 h, 36 h, and 48 h p.i. for multicycle replication were monitored for infectious progeny viruses (PFU) and virus particle formation (HAU), respectively. At 8 h p.i. FPV NS GD showed an increase of more than 1 log10 in the titer of infectious virus (Fig. (Fig.1E).1E). Also, in the multicycle replication the HA titers demonstrated that the reassortant FPV NS GD replicated faster and produced higher particle numbers than rFPV (Fig. (Fig.1A).1A). This is also reflected in the infectious titer of the reassortant FPV NS GD, which increased more rapidly and was about 1 order of magnitude higher than that of rFPV (Fig. (Fig.1B).1B). Furthermore, we observed a difference in plaque sizes formed by the reassortant FPV NS GD, which formed larger plaques than those formed by rFPV (Fig. (Fig.1F),1F), supporting the idea that FPV NS GD replicates more efficiently in MDCK cell culture.

In order to assess whether enhanced replication of the reassortant virus also resulted in altered pathogenicity, we infected 11-day-old embryonated chicken eggs with different amounts of each virus (5 eggs per dose). As shown in Fig. 1C and D, titers for both viruses analyzed at 36 h p.i. are comparable in eggs infected with a calculated amount of 0.01 to 100 PFU, whereas infection with 1,000 PFU/egg results in much lower titers. Even though the HA and PFU titer differences between both viruses were less apparent than replication in cell culture, rFPV almost never reached the maximum titer of FPV NS GD. Infections with both viruses at doses of 0.1 PFU/egg or higher were lethal. Interestingly, chicken embryos infected with rFPV at 0.001 or 0.01 PFU survived, whereas when embryos were infected with the same doses of the reassortant FPV NS GD virus, only three or one, respectively, remained alive (Table (Table1).1). Additionally, we observed that most embryos of FPV NS GD-infected eggs were smaller and less developed than embryos of the rFPV-infected eggs. These results demonstrate stronger pathogenicity of the reassortant virus for chicken embryos.

TABLE 1.
Survival of chicken embryos at 36 h p.i.

Recombinant wild-type FPV infection leads to a stronger IFN-ß induction.

IFN-β is one of the first antiviral cytokines to be expressed upon virus infection, initiating an auto-amplification loop that causes an efficient and strong type I IFN response (53). IFN-β synthesis is therefore a hallmark of the early innate immune response of the host against virus infection. Previous studies have shown that an IAV with a deletion of the NS1 gene, the delNS1 virus, can replicate efficiently only in a type I IFN-deficient cell system, indicating that NS1 suppresses the type I IFN response of the host (16). The reassortant virus that replicates to significantly higher titers differs from the rFPV only in the NS segment encoding the NS1 protein. Consequently, we investigated the capacity of both viruses to suppress induction of IFN-β.

To this end, we transfected MDCK cells with a plasmid expressing an IFN-β promoter-dependent reporter gene (luciferase) and subsequently infected these cells with either rFPV or the reassortant FPV NS GD virus (MOI of 1). The results showed that reporter gene induction by rFPV was slightly higher (about 1.75-fold compared to induction in uninfected cells, which was set to 1-fold) than by the reassortant FPV NS GD (about 1.49-fold; P value, 0.046) (Fig. (Fig.2).2). The stronger induction of the IFN-β promoter activity suggests that an enhanced IFN response might contribute to the reduced replication efficiency of rFPV compared to FPV NS GD. In order to exclude effects based on virus replication and/or MOI, we cotransfected MDCK cells with the IFN-promoter/reporter plasmid and either empty vector or plasmid expressing FPV NS1 or GD NS1. Subsequently, the cells were stimulated by transfection of the dsRNA analog poly(I·C) to induce IFN promoter activation. The results shown in Fig. Fig.2B2B demonstrate that the GD NS1 suppresses IFN promoter activity more efficiently than FPV NS1 relative to cells transfected with empty vector. Transfection efficiencies were controlled by green fluorescent protein (GFP) expression from a control vector (data not shown).

FIG. 2.
Induction of the IFN-β promoter. (A) MDCK cells were transfected with the IFN-ß promoter luciferase reporter plasmid (pIFN-Luci). At 24 h p.t., cells were either mock infected or infected with the rFPV or the reassortant FPV NS GD (MOI ...

The reassortant HPAIV shows a wider range in cell tropism than the wild-type HPAIV.

The receptor specificity and the nature of the HA cleavage site as well as the characteristics of other structural components like the subunits of the RNA-dependent RNA polymerase ([RdRp] PB1, PB2, and PA), NP, and the M2 protein have been implicated in the ability of influenza viruses to replicate productively in different hosts (33). FPV is an AIV that propagates efficiently in primary chicken embryo fibroblasts. Except for MDCK cells, FPV does not replicate well in mammalian cell lines, such as human cell cultures, and does not propagate in mice or mouse-derived cells (4, 42). FPV NS GD differs from the rFPV only by the exchange of the NS segment. Therefore, we aimed to assess whether the change in the genome composition would affect the ability of the reassortant to infect and replicate in human cell cultures.

Accordingly, the human cell lines A549 (alveolar epithelia), BEAS (bronchiolar epithelia), and 293T (embryonic kidney cells) were infected with each virus (Table (Table2)2) at a low MOI (0.01) to allow multicycle replication. rFPV replicated only in BEAS cells (4.4 × 105 ml−1; 48 h p.i.). In contrast, FPV NS GD grew to titers of 6 to 7 orders of magnitude in 293T and A549 cells and reached titers on BEAS cells which were 2 logs higher than those of rFPV. This result indicates that the GD NS segment altered cell tropism of FPV.

TABLE 2.
Replication abilities of rFPV and FPV NS GD in different human cell lines

Infection of mice with rFPV or FPV NS GD.

The AIV A/FPV/Rostock/34 (H7N1) is known to be apathogenic for mice (4, 42). We therefore investigated whether the reassortant FPV NS GD differs in this respect. In the first set of experiments, 8-week-old female C57BL/6 mice were intranasally infected with either virus at various doses in a range of 102 to 106 PFU. As expected, none of the animals infected with rFPV developed influenza-specific disease symptoms (e.g., weight loss or apathetic behavior). In contrast, mice infected with FPV NS GD at concentrations of 104, 105, or 106 PFU developed disease, and 10 to 30% of the mice died (data not shown). In the second set of experiments, 10 female C57BL/6 mice were infected with 105 PFU of either rFPV or FPV NS GD. The general health status and weight were monitored on a daily basis. One out of 9 mice infected with rFPV showed mild disease symptoms for 2 days, while 7 out of 10 mice infected with FPV NS GD developed severe disease symptoms and died at 6 to 9 days p.i. (Table (Table3;3; Fig. Fig.3B).3B). Three mice that survived showed weight loss at 7 to 10 days p.i. (Fig. (Fig.3A).3A). To further investigate virus replication after infection of mice with rFPV or FPV NS GD, five animals were infected with 105 PFU of either virus and sacrificed at day 6 p.i. No infectious virus was detectable in the lungs of rFPV-infected animals. In contrast, 4 of 5 mice infected with FPV NS GD presented virus in the lung, with a mean titer of 9.3 × 104± 2.5 × 104 PFU/ml (Table (Table3).3). Taken together, these data indicate that the introduction of the NS segment from the H5N1 GD strain into FPV conferred the ability to propagate in mice without previous adaptation, showing pathogenicity in a mammalian host.

FIG. 3.
Survival rate of C57BL/6 mice after infection with rFPV or FPV NS GD. Female C57BL/6 mice were intranasally infected with 105 PFU of either rFPV (n = 9) or FPV NS GD (n = 10). (A) The average weight of the surviving mice infected with ...
TABLE 3.
Development of disease, death, and virus titer in the lungs of mice infected with rFPV or FPV NS GD

The GD NS1 protein enhances replication of rFPV.

The results obtained so far suggested an important role for the NS1 protein in enhancing the replication efficiency of FPV NS GD. To further elucidate the role of the GD NS1 on a molecular level, we investigated whether transient expression of GD NS1 protein would also affect rFPV replication. Accordingly, MDCK cells were transfected with either an empty vector or a plasmid expressing the FPV or the GD NS1 protein 24 h prior to infection with rFPV (MOI of 1). The virus titers at 8 h p.i. show that the expression of the FPV NS1 protein itself enhances rFPV replication significantly by about 8-fold (Fig. (Fig.4).4). However, transiently expressed GD NS1 increased rFPV replication up to 14-fold (P value, 0.008). This result suggests that expression of the NS1 protein encoded by the GD NS segment might play a role in promoting FPV NS GD replication.

FIG. 4.
Transient expression of the GD NS1 enhances the rFPV titers. MDCK cells were transfected with either an empty vector or a plasmid expressing the FPV or GD NS1 protein. At 24 h p.t., cells were infected with rFPV (MOI of 1), and virus titers were determined ...

The increased NS1 protein expression level of FPV NS GD correlates with stronger inhibition of PKR activity in infected cells.

NS1 is important to promote IAV replication, and our results obtained from transient NS1 expression suggest that the GD NS1 increases rFPV replication. We therefore investigated the NS1 expression levels of both viruses in infected cells. In-cell Western blot analyses shown in Fig. Fig.5A5A demonstrate that the NS1 amount in rFPV-infected cells slowly increases, whereas NS1 in FPV NS GD-infected cells accumulates earlier and to higher levels than in rFPV-infected cells. Additionally, we analyzed NP production. The increased NP production indicates that the overall rate of viral protein expression is enhanced, which coincides with the higher virus titers.

FIG. 5.
NS1 protein expression, inhibition of PKR activity, and PKR binding capacity. (A) Expression levels of NS1 and NP protein in cells infected with either rFPV ([filled lozenge]) or FPV NS GD ([filled triangle]) (MOI of 1). The relative NS1 and NP amounts from six independent ...

One of the prominent functions of NS1 is to inhibit PKR. Normally, accumulation of dsRNA generated during viral propagation will activate PKR, which in turn phosphorylates eIF2α and blocks the translation process. To analyze the ability of both viruses to downregulate PKR activity, we investigated the extent of eIF2α phosphorylation as an indicator of PKR activity. The results showed that the activation of PKR was less prominent in FPV NS GD-infected cells than in rFPV-infected cells (Fig. (Fig.5B),5B), which correlated well to the differential expression levels of NS1 proteins (Fig. (Fig.5A)5A) and IFN promoter activation by the two viruses (Fig. (Fig.22).

In order to support these results we infected primary mouse alveolar epithelia cells (AECs) from PKR+/+ and PKR−/− mice (61) with both viruses at different MOIs (0.001, 0.01, and 1) and monitored virus production during multicycle replication for 48 h. As shown in Table Table4,4, rFPV replication on both AEC types was detectable upon infection only at MOIs of 0.01 and 1, whereas FPV NS GD was also able to replicate after infection even at an MOI of 0.001. In general FPV NS GD replicated to higher titers than rFPV. This correlates with the previous results obtained from infections of mice (Fig. (Fig.4)4) and different human cell lines (Table (Table2).2). Interestingly the titer difference between rFPV and FPV NS GD on PKR−/− cells was less pronounced than the titer difference obtained from PKR+/+ AECs. Accordingly, the ability of GD NS1 to suppress PKR more efficiently (Fig. (Fig.5B)5B) could partly account for the replication advantage of FPV NS GD.

TABLE 4.
Replication of rFPV and FPV NS GD on PKR+/+ or PKR−/− AECs

Previous studies indicated that the NS1 dsRNA-binding ability and an amino acid stretch covering positions 123 to 127 are important for inhibition of human PKR (20, 31). The sequences corresponding to the RNA-binding domain differ substantially between the NS1 proteins of FPV NS GD and rFPV, and the domain previously defined for direct PKR binding (31) shows two amino acid variations at positions 125 and 127 (see Fig. S1A in the supplemental material). Nevertheless, in vitro both NS1 proteins bind to PKR with similar capacities (Fig. (Fig.5C),5C), indicating that the different PKR-inhibiting activities might in part be due to a difference in the expression rate of NS1 proteins of the respective viruses.

Both NS1 proteins exhibit similar CPSF30 F2F3 fragment-binding capacities.

It was previously suggested that the NS1 proteins of certain virus strains block the innate immune response by impairing posttranscriptional pre-mRNA processing due to the ability to bind to CPSF30 (24, 34). Therefore, we analyzed the in vitro binding activity of both NS1 proteins to the F2F3 fragment of CPSF30 (55). As shown in Fig. Fig.6,6, the two NS1 proteins were precipitated in comparable amounts, indicating similar binding capacity to the F2F3-GST protein. This reflects the sequence similarity of their CPSF30-binding regions (see Fig. S1A in the supplemental material) except for position 103, where F was shown to be critical for CSPF30 binding, and a tryptophan is found in NS1 of FPV NS GD instead. The NS1 protein expressed by the IAV strain A/PR/8/34 (H1N1; control) bound to F2F3-GST only to a minor extent, which coincides with the findings of a recent report (24).

FIG. 6.
The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and 35S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H2O as a control and precipitated using glutathione-Sepharose ...

DISCUSSION

The NS segment-encoded NS1 protein is considered a virulence factor of IAV because it counteracts the host cell IFN and tumor necrosis factor (TNF) response (15, 25, 46), the first line of the host defense against viruses. In our study, the reassortant FPV NS GD, which differs from the rFPV only by the NS segment, replicated to higher titers in embryonated chicken eggs, and it seemed to be more pathogenic. Interestingly, it was able to replicate more efficiently in human cell lines, in primary mouse AECs, and in mice without previous adaptation. Even though a general conclusion about possible viral characteristics that occur as a result of a natural NS reassortant between type H7 and H5 IAVs cannot be drawn, these remarkable changes in tissue tropism and host range of an AIV toward mammalian species are surprising. In another study the NS segments of A/Viet Nam/1203/04 (H5N1) or A/Hong Kong/483/97 (H5N1) in the background of the human IAV A/Udorn/72 (H3N2) either had no significant effect or strongly reduced virus titers, plaque morphology, and increased IFN mRNA, which correlated to the CSPF30 binding ability of the NS1 proteins (54). Based on the data presented in this study, the reassortant virus FPV GD NS demonstrates increased replication efficiency in IFN-competent cells and reduced IFN-ß promoter induction as well as a stronger reduction of PKR activity. The relevance of PKR inhibition for increased FPV NS GD propagation could be demonstrated in primary mouse AECs of PKR−/− mice as differences in virus titers here were less obvious. In relation to FPV NS GD, rFPV replication is improved from 22% to 40% and from 3% to 16% in PKR−/− compared to PKR+/+ AECs at MOIs of 1 and 0.01, respectively. Therefore, the GD NS1 protein seems to have a greater potential to counteract a PKR-mediated cellular response to virus infection than rFPV NS1. In part, this could be the basis for higher virus titers for FPV NS GD. However, the remaining titer differences between the two viruses in PKR−/− AECs might reflect additional effects by NS1 or NS2. Interestingly, at an MOI of 0.001, no replication of rFPV was detectable on either type of AECs, whereas FPV NS GD replication was detected, showing that the AECs are more susceptible for the reassortant. The fact that the NS1 protein in FPV NS GD-infected cells is expressed at higher levels than the isogenic NS1 protein of rFPV, which is also reflected by the increased NP expression, further suggests a quantitative effect.

The length of both NS segments is 890 nt, encoding the 230-aa NS1 protein and the 121-aa NS2/NEP protein. The NS1 of FPV is only 69.3% identical to the corresponding GD gene on the amino acid level (Fig. (Fig.6A).6A). This correlates with the fact that the NS segment of FPV belongs to the A allele (predominantly found in IAV replicating in birds and mammals) while the GD NS is grouped with the B allele (found exclusively in IAV replicating in birds) (29). Nevertheless, the GD NS segment allowed FPV to replicate efficiently in human cell lines and mice without prior adaptation. The RNA-binding domain (aa 1 to 73) (25) of the FPV NS1 shows seven continuous amino acid differences (21RFADQEMG28 to 21LLSMRDMC28, indicated by boldface type) from the GD NS1 (see Fig. S1A in the supplemental material). Currently, it is unknown how this might affect the replication activity of FPV NS GD. Within the effector domain (aa 74 to 230) (25) of both NS1 proteins, there is a difference of 8 aa (134 to 161) (Fig. S1A in the supplemental material). Previous studies have suggested that conserved amino acids L144 and L146 (Fig. S1A, filled triangle) in the effector domain are crucial for NS1-mediated regulation of nucleocytoplasmic transport of unspliced and partially spliced viral and host mRNAs (39, 44). It was shown that the mutation L146A in the NS1 effector domain resulted in an NS1 redistribution throughout the cell, unlike the wild-type NS1 protein, which is localized predominantly in the nucleus (39). Instead of L146, S146 is found in the GD NS1. Analysis of the intracellular NS1 distribution in cells infected with each virus revealed that the FPV NS1 is found predominantly in the nucleus while the GD NS1 additionally shows distinct cytoplasmic localization (see Fig. S2 in the supplemental material). We do not think that the differences are caused by the different expression levels (Fig. (Fig.5A)5A) as the nuclear localization of GD NS1 does not increase over time. Furthermore, an effect of the K221Y difference in the second NLS on intracellular NS1 distribution cannot be excluded. Whether these differences affect nuclear mRNA export is currently under investigation. NS1 is a multifunctional protein that has numerous interaction partners. It would be surprising if such a small protein would bind multiple cellular proteins simultaneously. Therefore, its intracellular localization could define the interaction with different cellular proteins. It is likely that differences in the NS1 interaction with cellular proteins that result from different intracellular NS1 distributions between rFPV- and FPV NS GD-infected cells might affect virus propagation.

NS1 was reported to bind to the important antiviral molecule PKR via a PKR-binding domain, including aa 123 to 127 (31), and to inhibit kinase activation by either PACT or dsRNA (27). Our study showed that activation of PKR occurred to a lesser extent in FPV NS GD-infected cells (Fig. (Fig.5B),5B), correlating to the higher NS1 expression levels (see Fig. Fig.5A)5A) and stronger IFN-β promoter inhibition (Fig. (Fig.2).2). Interestingly in PKR−/− AECs the viruses grew to similar titers. Although the PKR-binding domains of the two NS1 proteins shows differences, PKR binding of the NS1 proteins was comparable. Nevertheless, it cannot be excluded that higher cytoplasmic GD NS1 concentrations in the early replication phase (see Fig. S2) lead to a more efficient PKR inhibition. Our data showing increased rFPV titers after infection of cells that express GD NS1 would support such a conclusion.

It has been reported that the NS1 proteins of most human IAV and human H5N1 isolates after 1998 contain amino acids F103 and M106 (in the supplemental material see Fig. S1A, filled square) in the effector domain which are critical for CPSF30-binding and enhance virus replication in mammalian cells (8, 24, 34). It is believed that the binding of NS1 to CPSF30 will also inhibit IFN-β mRNA processing. It must be noted that the GD NS1, which contains Y103 and several other variations among the amino acids that comprise the interaction to CPSF30, shows slightly less binding capacity to the F2F3 fragment of CPSF30 (Fig. (Fig.6).6). Both NS1 proteins contain almost identical PDZ binding domains (see Fig. S1A). Even though it was shown that a 1-aa difference can significantly increase virulence (22), it remains to be determined whether this difference as well as the differences in the overlapping PABII-binding site might contribute to the growth characteristics of both viruses.

The NS gene of the lethal H5N1 IAV from the 1997 H5N1 epidemic in Hong Kong contributes to the virus's ability to escape host antiviral cytokine responses, leading to its lethal potential. This seems to require the presence of E92 in the NS1 (47). However, GD NS1 and FPV NS1 have a conserved D92 (see Fig. S1A, filled diamond) as found in other IAVs, indicating that this position does not contribute to the pathogenic phenotype of FPV NS GD. GD H5N1 was the first documented HPAIV in China, which in 1996 caused an outbreak with 40% morbidity in geese and was able to cause illness and death in chickens (52, 60). This virus is thought to be one of the gene donors in the 1997 epidemic in Hong Kong. However, from the conserved amino acids (T202, Q218, and F223) (Fig. (Fig.6A,6A, filled circle) in NS1 proteins of H5N1 AIVs which transmitted to humans, only Q218 is found in the FPV and GD NS1 proteins. The other two corresponding amino acids of both NS1 proteins are similar to those of the human H3N2 virus (48).

The results suggest that the expression of the GD NS segment allows FPV NS GD to propagate more efficiently. Unlike rFPV (4, 42), the reassortant FPV NS GD virus also replicated efficiently in three human cell lines and in mice, suggesting that the GD NS1 could be responsible for the changes in host tropism. Nevertheless, both the wild-type FPV and rFPV are highly pathogenic for chicken and show no signs of attenuation even though they are laboratory strains (data not shown).

The importance of NS1 for pathogenicity and virulence has been demonstrated in recent studies of several IAV subtypes in mice and pigs (41, 51). A naturally occurring deletion in the NS1 protein (aa 191 to 195) contributed to the attenuation of a porcine H5N1 IAV in chickens and affected the ability of the virus to antagonize IFN induction in host cells (62). S42 (in the supplemental material see Fig. S1A, open circle) contributes to the virulence of a duck H5N1 virus in mice (23). However, no deletion was present at amino acid positions 191 to 195 in the NS1 proteins of FPV and GD. A possible impact of the S42A difference between the FPV NS1 and GD NS1 on virulence is currently unknown, but it should be noted that this position has an effect on the IFN-antagonistic properties of NS1 (9).

The NS2/NEP gene of FPV is only 82% identical to that of GD at the amino acid level. NS2/NEP mediates the nuclear export of the viral RNPs (36). Two amino acid positions (M14Q and G22E, FPV sequence relative to GD) are different within the nuclear export signals (NES) of the proteins (see Fig. S1B in the supplemental material). However, positions M16, M19, and L21 in the NS2/NEP-NES, which are crucial for viral replication and for RNP nucleocytoplasmic transport (32), are conserved in both NS2/NEP proteins. In addition, the recently described T215 that can be phosphorylated via cyclin-dependent kinase (CDK)/ERK and might play a role in early viral propagation is conserved in both proteins (18). In a plasmid-based replication system expressing the RNA-dependent RNA polymerase (RdRp), vRNA, and NS2/NEP, it was recently revealed that the NS2/NEP protein affected viral RNA levels by reducing the accumulation of transcription products and increasing the amount of replication products (43). The authors assume that the NS2/NEP protein changes RNA levels by specific alteration of the viral transcription and replication machinery rather than through an indirect effect on the host cell. Deletion of the NES did not affect the ability of the protein to regulate RNA levels. To what extent NS2/NEP of FPV and GD affect RdRp activity of FPV will have to be investigated. Our own preliminary results indicated no change in the ratio of transcription and replication products in cells infected with either virus (data not shown). The NS2/NEP structure is characterized by a high degree of plasticity, suggesting that NS2/NEP may indeed exist in multiple conformations in vivo (28) to mediate nuclear RNP export. Since there are several amino acid differences between the two NS2/NEP proteins, it remains to be analyzed whether and to what extent they contribute to the replication characteristics of FPV NS GD. In order to rule out the possibility that the differences between both NS2/NEPs affect viral replication, it would be necessary to analyze these separately in the respective viruses. This, however, would also alter the NS1 sequence. Therefore, we made use of transiently expressed NS1 proteins in parts of our experiments.

The virulence of IAVs seems to be a multigenic trait although several studies demonstrated that the NS gene directly contributes to the virulence/pathogenesis of IAVs. As we have analyzed the effect of only one specific NS segment reassortment, the analysis of further NS reassortments is necessary before a more general conclusion can be reached. Nevertheless, the present work sheds light on the possible impact of a specific NS reassortment on virulence and host range of a particular H7 HPAIV. Future work should identify the specific NS1 and/or NS2/NEP residues that mediate these characteristics to gain further information on the molecular nature of the NS-based viral pathogenicity mechanisms.

Biosafety.

All experiments with infectious virus were performed according to German regulations for the propagation of influenza A viruses. All experiments involving highly pathogenic influenza A viruses were performed in a biosafety level 3 (BSL3) containment laboratory approved for such use by the local authorities (Regional Council, Giessen).

Supplementary Material

[Supplemental material]

Acknowledgments

We thank J. Pavlovic (Institute of Virology, Zurich, Switzerland) for providing the plasmids pHMG-PB1, -PB2, -PA, and -NP and mice and N. Cox and K. Subbarao (CDC, Atlanta, GA) for providing the plasmid pBD. We also thank R. M. Krug (Institute of Cellular and Molecular Biology, Austin, TX) for the plasmid pGEX3X-F2F3/GST. In addition, we thank J. Lampe, K. Oesterle, K. Schultheiss, E. Lenz, and M. Stein for their excellent technical assistance.

This work was supported in part by grants of the DAAD (fellowship to W.M.) and the DFG (GRK1384 fellowship to Z.W.) and in part by grants of the European Specific Targeted Research Project, EuroFlu—Molecular Factors and Mechanisms of Transmission and Pathogenicity of Highly Pathogenic Avian Influenza Virus, funded by the 6th Framework Program (FP6) of the EU (SP5B-CT-2007-044098, to S.P., S.L., and O.P.); the FluResearchNet—Molecular Signatures Determining Pathogenicity and Species Transmission of Influenza A Viruses (01 KI 07136 to S.P.); the DFG Graduate School (GRK1409) and the interdisciplinary Clinical Research Centre (IZKF) of the University of Münster (Lud27032/06 to S.L.); the Influenza Research Program FSI of the Federal German Government (to T.W. and O.P.); and NIH funding (contract HHSN266200700005C to J.A.R.). Furthermore, this work is a part of the activities of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a grant (LSHMCT-2004-503359) from the Priority 1 Life Sciences, Genomics and Biotechnology for Health program (FP6) (to S.P. S.L., and O.P.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 9 December 2009.

Supplemental material for this article may be found at http://jvi.asm.org/.

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