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. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Virology. 2017 Jan 29;504:25–35. doi: 10.1016/j.virol.2017.01.015

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

Jinhwa Lee 1, Hai Yu 1,2, Yonghai Li 1, Jingjiao Ma 1, Yuekun Lang 1, Michael Duff 1, Jamie Henningson 1, Qinfang Liu 1,2, Yuhao Li 1, Abdou Nagy 1,#, Bhupinder Bawa 1, Zejun Li 2, Guangzhi Tong 2, Juergen A Richt 1, Wenjun Ma 1,*
PMCID: PMC5651993  NIHMSID: NIHMS849737  PMID: 28142079

Abstract

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses.

Keywords: Influenza A virus, PA-X, virus replication, host immune response

Introduction

Influenza A virus (IAV) is a single-stranded negative sense RNA virus that belongs to the Orthomyxoviridae family. It contains eight segmented viral genomes and was thought to encode 10 viral proteins (PB2, PB1, PA, HA, NP, NA, M1, M2, NS1 and NS2). Recently, several new influenza viral proteins have been discovered, such as PB1-F2 (Chen et al., 2001), PB1-N40 (Wise et al., 2009), PA-X (Jagger et al., 2012), NS3 (Selman et al., 2012), PA-N155, PA-N182 (Muramoto et al., 2013), M42 (Wise et al., 2012), and PB2-S1 (Yamayoshi et al., 2016). Among these, the PA-X protein is translated through a +1 ribosomal frameshifting event in segment 3, the PA gene. This PA-X protein has the identical N-terminal 191 amino acids (aa) to PA protein and a PA-X unique C-terminal domain of 41 or 61 aa by alternative translation (Jagger et al., 2012). Expression of PA-X protein in most IAVs is the full-length form with 61 aa in the C-terminal domain, while both the 2009 pandemic H1N1 (pH1N1) virus and triple-reassortant swine influenza viruses circulating in North American swine herds express a truncated PA-X protein with only 41 aa due to a stop codon at position 42 in the C-terminal domain (Jagger et al., 2012; Shi et al., 2012). Since PA-X shares the N-terminal endonuclease domain with PA, it provides the host shutoff activity for PA-X to destroy the host mRNA and suppress host protein synthesis (Desmet et al., 2013; Jagger et al., 2012). Although PA and PA-X have the same N-terminal domain, PA-X has been shown to have a stronger endonucleolytic activity than PA, indicating that the unique C-terminal part of PA-X is also responsible for shutoff activity (Bavagnoli et al., 2015; Desmet et al., 2013; Hayashi et al., 2016; Oishi, Yamayoshi, and Kawaoka, 2015).

Recently, many studies have described the effects of PA-X on viral replication and pathogenicity. Jagger et al. reported that PA-X expression reduced the viral pathogenicity of the1918 pandemic H1N1 virus, even if PA-X had no effect on viral replication (Jagger et al., 2012). Two other reports also showed that loss of PA-X expression increased viral replication and pathogenicity of 2009 pH1N1 virus and H5N1 highly pathogenic avian influenza virus (HPAIV) in vitro and in vivo when compared to the wild type viruses that express PA-X (Gao et al., 2015b; Hu et al., 2015). In contrast to previous reports, recent studies showed that PA-X deficient 2009 pH1N1 and H9N2 viruses attenuated viral pathogenicity in mice compared to the wild type virus (Gao et al., 2015c; Hayashi, MacDonald, and Takimoto, 2015). Other studies also investigated the contribution of 20aa at the C-terminal end of PA-X to viral replication and virulence by comparing PA-X full-length form with the truncated form. Gao et al. demonstrated that three different IAVs (2009 pH1N1, H5N1 HPAIV and H9N2 AIV) increased viral replication and pathogenicity when the viruses express the full-length PA-X protein with 61aa in the C-terminal domain compared to those with a truncated PA-X expression (Gao et al., 2015a), whereas triple-reassortant H1N2 swine influenza virus with truncated PA-X enhanced viral pathogenicity and replication in pigs (Xu et al., 2016).

The host shutoff activity of PA-X protein is expected to weaken the host antiviral response by inhibiting host protein synthesis including immune related gene expression. Previous studies reported that PA-X deficient virus markedly upregulated expression levels of apoptosis, inflammation and immune response related genes in mice or chicken lungs, using global gene expression profiling (Hu et al., 2015; Jagger et al., 2012). Recently, Hayashi et al. found that PA-X deficient virus induced a significantly greater amount of IFN-β mRNA as well as more anti-hemagglutinin neutralizing antibodies in mice when compared to the wild type virus (Hayashi, MacDonald, and Takimoto, 2015). Therefore, it suggests that PA-X is an important accessory protein of IAV as an immune modulator and virulence factor. However, the detailed underlying mechanisms of PA-X function and the effects of different expressions of PA-X protein on viral pathogenicity as well as host immune response remain unclear.

In this study, we used 2009 pH1N1 A/California/04/2009 (CA09) virus which expresses a truncated PA-X protein and its two mutated viruses generated by reverse genetics to investigate the impacts of three different expressions of PA-X protein (full-length, truncated and PA-X deficient forms) on viral replication, pathogenicity and host innate immune response.

Material and Methods

Cells

Human embryonic kidney (293T) cells were cultured in Opti-modified Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% antibiotic-antimycotic (Gibco). Madin-Darby canine kidney (MDCK) cells were grown in MEM containing 5% FBS, 1% antibiotic-antimycotic, 2mM L-glutamine (Gibco) and 1X MEM vitamin solution (Gibco). Human lung adenocarcinoma epithelial (A549) cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% FBS, 2mM L-glutamine, 1X MEM vitamin solution and 1% antibiotic-antimycotic. MEM infecting media containing 0.3% bovine serum albumins (BSA) (Sigma-Aldrich), 1% antibiotic-antimycotic and 1µg/ml TPCK-treated trypsin (Sigma-Aldrich) were used for virus infection of cells.

Plasmid constructions

Eight-plasmid reverse genetics for the 2009 pH1N1 A/California/04/2009 (CA09) virus has been established and described in our previous study (Qiao et al., 2012). Eight-gene segments of CA09 virus were cloned into a pHW2000 vector (Hoffmann et al., 2000). To generate the CA09_PA-X_Full virus that expresses a full-length PA-X, a single amino acid substitution from UAG (stop) to UGG (tryptophan) at position 42 in the C-terminal domain of PA-X protein was introduced based on pHW2000-PA_WT using a GeneArt® site-directed mutagenesis kit (Invitrogen) according to the manufacturer’s instructions (Fig. 1). The resulting plasmid was named pHW2000-PA_PA-X_Full. To construct the PA-X deficient virus that does not express truncated or full-length PA-X, three nucleotide mutations in frameshifting motif were introduced from UCC UUU CGU to UCC UUC AGA based on pHW2000-PA_WT by site-directed mutagenesis to prevent PA-X expression as described previously (Fig. 1) (Jagger et al., 2012). The resulting plasmid was named pHW2000-PA_PA-X_K/O. None of the mutations altered the PA open reading frame (ORF) as silent mutations which were confirmed by sequencing. To investigate expression of different PA-X forms, the coding regions of proposed different PA-X forms were cloned into pCAGGS vector and the resulting three plasmids were named pCAGGS_PA-X_WT, pCAGGS_PA-X_Full and pCAGGS_PA-X_K/O. To investigate effects of different expressions of PA-X protein on the polymerase activity, ORFs of wild type and two mutated PA genes as well as PB1, PB2 and NP genes of the CA09 virus were cloned into pCAGGS vector. The resulting plasmids were pCAGGS_PA_PA-X_WT, pCAGGS_PA_PA-X_Full, pCAGGS_PA_PA-X_K/O, pCAGGS_PB1, pCAGGS_PB2, and pCAGGS_NP that were confirmed by sequencing.

Fig. 1. Generation of PA-X deficient or full-length PA-X expression recombinant CA09 viruses.

Fig. 1

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(a) Schematic structure diagram of PA and PA-X proteins. PA-X protein contains the N-terminal 191aa identical to PA protein and a PA-X unique C-terminal domain (grey color box) of 41 or 61aa produced by +1 ribosomal frameshifting. 2009 pH1N1 virus expresses truncated PA-X protein with 41aa in the C-terminal domain due to a stop codon (UAG) at position 42. To generate the CA09_PA-X_Full virus, a single amino acid substitution from UAG (stop codon) to UGG (W) at position 42 in the C-terminal domain of PA-X protein was introduced. Three nucleotide mutations in frameshifting motif were introduced from UCC UUU CGU to UCC UUC AGA to inhibit PA-X expression. The bold letters indicate the nucleotide substitutions. (b) The expression of indicated PA-X proteins was determined by western blotting analysis from transfected 293T cells with pCAGGS_PA-X_WT, pCAGGS_PA-X_Full or pCAGGS_PA-X_K/O plasmids. Empty vector control was transfected with the empty pCAGGS plasmid.

Rescue of wild type and mutated CA09 H1N1 recombinant viruses

Wild type (CA09_WT), PA-X knock-out (CA09_PA-X_K/O) and full-length PA-X expression (CA09_PA-X_Full) CA09 viruses were generated by reverse genetics using the eight-plasmid system. To rescue CA09_PA-X_K/O and CA09_PA-X_Full viruses, the pHW2000-PA_WT was replaced with pHW2000-PA_PA-X_K/O or pHW2000-PA_PA-X_Full, respectively. Briefly, MDCK and 293T cells were co-cultured in 6-well plates and transfected with eight constructed pHW2000 plasmids encoding viral genomic RNA segments using Lipofectamine 2000 (Invitrogen). After 48 hours post transfection, supernatants were collected and passaged three times on MDCK cells. The rescued viruses were confirmed by sequencing.

Replication kinetics

To evaluate the growth kinetics of recombinant viruses, MDCK and A549 cells were cultured in 12-well plates and infected with each virus at a multiplicity of infection (MOI) of 0.01 in triplicate. Supernatants were collected at 12, 24, 36 and 48 hours post-infection (hpi). Virus titers were determined by calculating the 50% tissue culture infective dose (TCID50)/ml in MDCK cells.

Plaque assay

The plaque assay was performed to compare the size of plaques formed by each recombinant virus on MDCK cells. A monolayer of confluent MDCK cells in 6-well plates was infected with 500 µl of each virus serially diluted 10-fold in infection media for 1 hour at 37°C. The infection media was removed and the infected cells were washed twice with phosphate-buffered saline (PBS). Then, 2 ml of agarose overlay that contains 2X MEM with 0.6% BSA and 2 µg/ml TPCK-treated trypsin (Sigma-Aldrich) and 2% SeaPlaque Agarose (Lonza) was added to the cells. The final concentration of MEM and Agarose in the agarose overlay was 1X MEM and 1% agarose with 0.3% BSA and 1 µg/ml TPCK-treated trypsin. The agarose overlay was removed at 48 hpi, and the cells were fixed and stained with 1% crystal violet in methanol for 10 minutes. The stained plates were washed with water and dried before taking pictures.

Western blot analysis

Confluent 293T cells were transfected with pCAGGS_PA-X_WT, pCAGGS_PA-X_K/O or pCAGGS_PA-X_Full using Lipofectamine 2000 (Invitrogen). Mock control was transfected with empty pCAGGS plasmid. At 18 hours post-transfection, total cell lysates were extracted using CelLytic M cell lysis reagent according to the manufacturer’s instructions (Sigma-Aldrich). The extracted cell lysates were loaded to 4–12% Bis-Tris polyacrylamide gel (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The blot was blocked using 5% skim milk and then incubated with a primary monoclonal antibody against PA-X (diluted 1:1000, kindly provided by Dr. Toru Takimoto, University of Rochester Medical Center, NY, USA) or β-actin (diluted 1:500, Santa Cruz) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse immunoglobulins (diluted 1:1000, Dako). Target proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate according to the manufacturer’s protocol (Thermo Scientific).

Viral RNP polymerase assay

The effects of different expressions of PA-X protein on polymerase activity were determined using ribonucleoprotein (RNP) minigenome assay as described previously (Bortz et al., 2011). Briefly, a reporter minigenome plasmid (pPolI-NS-Luc) that contains the firefly luciferase gene flanked by the noncoding regions of influenza A virus NS gene with a polymerase I (Pol I) promoter was used. Confluent 293T cells were co-transfected with pPolI-NS-Luc (100ng) and four pCAGGS vectors encoding PB2, PB1, NP and PA (PA_PA-X_WT, PA_PA-X_Full or PA_PA-X_K/O) (the amount of the PB2, PB1, PA and NP plasmids used were 50ng, 100ng, 100ng and 500ng respectively) along with pSV-Renilla (50 ng) carrying the Renilla luciferase gene as an internal control. As a negative control, cells were transfected with the same aforementioned plasmids mixture except the plasmid expressing PA. After 24 hours post-transfection, cells were collected using passive cell lysis buffer and luciferase activity was determined using a dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega). Luminescence was detected using FLUOstar Omega (BMG LABTECH). The relative luciferase amount was quantified by comparing to negative control cells which were transfected with pCAGGS blank plasmid instead of PA expression plasmid, after normalizing it to the Renilla luciferase internal control. Each co-transfection experiment was performed in triplicate.

The RNP component, PA and NP proteins, were also detected in co-transfected 293T cells with different combinations of RNP complex plasmids as well as in MDCK cells infected with each indicated virus at an MOI of 1 at 12 hpi by using Western blotting. The first antibodies including the monoclonal antibody against PA (diluted 1:1000, kindly provided by Dr. Toru Takimoto), a polyclonal rabbit anti-NP antibody (diluted 1: 1000, GenScript) and a monoclonal antibody against β-actin (diluted 1:500, Santa Cruz) were used. The detected protein bands were quantified using densitometry with AlphaEase FC Software (Alpha Innotech). The PA and NP protein expressions were normalized to β-actin expression.

GFP expression assay

Confluent 293T cells were co-transfected with 500ng of pEGFP-N1 (Clontech) and 500ng of each PA plasmid that expresses different PA-X forms (pCAGGS_PA-WT, pCAGGS_PA_PA-X_K/O and pCAGGS_PA_PA-X_Full). After 24 hours post-transfection, cells were lysed with passive lysis buffer and the fluorescent intensity was measured using FLUOstar Omega (BMG LABTECH). The fluorescent intensity was standardized to the pCAGGS empty vector transfected control cells. The expressed GFP proteins and β-actin were detected from total cell lysates by western blotting to determine the relative expression of GFP protein using anti-GFP antibody (1:200 dilution, Santa Cruz) or anti-β-actin antibody (1:500 dilution, Santa Cruz). The detected protein bands were quantified using densitometry with AlphaEase FC Software (Alpha Innotech). The GFP protein expressions were normalized to β-actin expression.

Pathogenicity study in mice

A total of 56 six-week-old female BALB/c mice were randomly divided into four groups (14 mice/group). Mice from each group were inoculated intranasally (IN) with 3.75 × 105 TCID50 of each virus in a volume of 50 µl under slight anesthesia with isoflurane. For the control group, mice were mock-infected with 50 µl of virus-free MEM. Mice were monitored daily for clinical signs and weighed daily until 14 days post infection (dpi). Mice that lost more than 25% of their original body weight were humanly euthanized. Three mice from each group were euthanized at 3 and 7 dpi. Lungs were collected from each mouse to assess virus replication, cytokine gene expressions and histopathological analysis. For virus titration, 10% lung homogenates were made using fresh MEM with 1% antibiotic-antimycotic, and virus titers in the lung homogenates were determined on MDCK cells.

qRT-PCR

Total RNAs from lung homogenates were extracted using the RNeasy Plus Mini kit according to the manufacturer’s protocol (Qiagen). The cDNA was synthesized from 1 µg of total RNA of each sample using the SuperScript III Reverse Transcriptase (Invitrogen) with Oligo(dT)20 by reverse transcription reaction. The gene specific primers and probes were obtained from the TaqMan Gene Expression Assays (Applied Biosystems). The quantitative real-time PCR assays (qRT-PCR) were performed with equal volumes of cDNA from each sample and gene specific primers and probes using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems). Amplification was conducted using the CXF96 Touch real-time PCR system (Bio-Rad) with the following cycle program: 1 cycle at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. The expression fold change of each group was calculated relative to control group after normalization to 18S ribosomal RNA internal control using 2−ΔΔCt method (Livak and Schmittgen, 2001). Each experiment was performed in triplicate.

Histopathology and immunohistochemistry

Lung samples from mice were fixed in 10% neutral buffered formalin and processed by the histopathology and immunohistochemistry laboratory of the Kansas State Veterinary Diagnostic Laboratory (KSVDL). A board-certified veterinary pathologist evaluated histopathological lesions of routinely processed hematoxylin and eosin (H&E) stained tissues. Each slide was examined by a veterinary pathologist in a blinded fashion. For the microscopic lung lesions in mice, lungs were graded on the following seven criteria: Subjective percentage of lung involved in the histological section examined (4 point scale of 1–4); airway epithelial necrosis, neutrophilic airway inflammation, peribronchiolar lymphocyte cuffing, interstitial pneumonia, airway epithelial hyperplasia (all on a 3 point scale of 1–3); and lastly the presence and absence of hyaline membranes (2 point scale, 0=absent and 1=present). Immunohistochemistry (IHC) staining for mouse lungs was conducted to detect influenza virus antigen in tissues using an anti-influenza A H1N1 rabbit polyclonal antibody. Antigen retrieval was performed using EDTA at pH 9.0 for 10 minutes at 100 °C. The antibody was diluted 1:4000 and detected with the Leica Bond Polymer Refine Detection Kit on the Leica Bond-Max.

Statistical analysis

For statistical analysis among groups, analysis of variance (ANOVA) test was used in GraphPad Prism version 5.0 (GraphPad Software). A p value of 0.05 or less was considered statistically significant.

Results

Generation of wild type pH1N1 and its mutated viruses expressing different PA-X forms

To investigate the effects of three different expressions of PA-X protein on viral pathogenicity and host innate immune response, we rescued a recombinant wild type A/California/04/2009 (CA09_WT) virus that expresses truncated PA-X protein with 232aa and its two mutant viruses (CA09_PA-X_K/O and CA09_PA-X_Full) using reverse genetics. CA09_PA-X_Full virus expressing a full-length PA-X with 252aa was generated by substituting stop codon (UAG) with tryptophan (UGG) at the 42aa position in the C-terminal domain of PA-X (Fig. 1a). CA09_PA-X_K/O virus, which does not express the PA-X, was generated through mutations in the frameshift motif from UUU CGU to UUC AGA according to the previous study (Jagger et al., 2012) (Fig. 1a). Due to the very low frameshifting efficiency (~1.3%) of PA-X gene (Jagger et al., 2012), it is very difficult to detect PA-X expression in virus infected cells. To confirm that the generated viruses were able to express the expected PA-X, we transfected 293T cells with different PA-X ORF encoding plasmids (pCAGGS_PA-X_WT, pCAGGS_PA-X_Full or pCAGGS_PA-X_K/O) to detect expressed PA-X proteins. Both truncated PA-X (232aa) and full-length PA-X (252aa) were detected by western blotting analysis in each plasmid transfected cells, while no PA-X was detected in transfected cells with pCAGGS_PA-X_K/O plasmid that the PA-X is proposed to be knocked out (Fig. 1b).

PA-X expression increases virus replication and polymerase activity in vitro

To further characterize wild type and its mutated viruses in vitro, we first performed a plaque assay in MDCK cells. Both CA09_PA-X_K/O and CA09_PA-X_Full mutated viruses as well as the CA09_WT formed similar small size of plaques in MDCK cells (Fig. 2a). Next, we examined virus replication kinetics of three viruses in MDCK and A549 cells. CA09_WT and its two mutated viruses replicated efficiently in both MDCK and A549 cells. In contrast, CA09_PA-X_Full virus grew to a higher titer than the other two viruses (CA09_PA-X_WT and CA09_PA-X_K/O) in both cell lines and a significant higher titer was observed at 24 and 36 hpi in A549 cells (Fig. 2b). The viral yields of PA-X deficient virus (CA09_PA-X_K/O) was significantly lower than both viruses expressing either a full-length or truncated PA-X (CA09_PA-X_Full and CA09_PA-X_WT) in MDCK cells (Fig. 2b). The data indicates that PA-X expression enhances virus replication, particularly when the CA09 virus expresses the full-length PA-X.

Fig. 2. Impact of different expressions of PA-X protein on plaque morphology, growth kinetics and polymerase activity.

Fig. 2

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(a) Plaque sizes formed by recombinant viruses in MDCK cells at 3 days post-infection (dpi). (b) Growth kinetics of indicated recombinant viruses in MDCK or A549 cells infected at an MOI of 0.01. Each data point on the curve indicates the means of the results in triplicate, and the error bars indicate standard errors of the mean (SEM). The asterisks (*) indicate a statistically significant difference with the other groups (p<0.05).

To determine whether PA-X expression enhances virus replication through effects on the viral RNA polymerase activity, a minigenome assay was performed by measuring the reporter gene expression levels which indirectly indicates the viral polymerase activity. Transfection of combination with the plasmid pCAGGS-PA_PA-X_K/O showed significantly lower polymerase activity when compared to those with either pCAGGS-PA_PA-X_WT or pCAGGS-PA_PA-X_Full, with 2.0 and 3.7 fold differences, respectively (Fig. 3a). In addition, the polymerase complex with the pCAGGS-PA_PA-X_Full combination displayed the strongest polymerase activity (Fig. 3a).

Fig. 3. Effects of different expressions of PA-X protein on the polymerase activity and expression of PA protein.

Fig. 3

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(a) Comparison of polymerase activities of vRNPs with indicated different expressions of PA-X protein in 293T cells. The dotted line represents the limit of detection of relative luminescence unit which is 5. The grey color boxes indicated transfected genes. Relative luminescence unit of each combination of polymerase complexes indicates the average of data of three independent experiments. Expression of PA and NP proteins in cell lysates (b) from 293T cells transfected with different combinations of RNP complex plasmids, and (c) from MDCK cells infected with each indicated virus at an MOI of 1 for 12 hrs. Relative PA and NP proteins compared to the PA-X_WT cells were quantified by densitometry after normalization to β-actin expression. The value of PA-X_WT was set to 100%. The results of each group are the average of data from two independent experiments. The asterisks (*) indicate a statistically significant difference between groups (*: p<0.05. **: p<0.001 and ***: p<0.0001).

To further investigate how different expressions of PA-X protein affect viral RNA polymerase activity, production of the RNP component, PA and NP proteins, were analyzed by using Western blotting of cell lysates from 293T cells transfected with RNP component plasmids. A significantly higher expression of PA protein was detected in the transfection of combination with either full-length PA-X or truncated PA-X plasmid when compared to the combination with PA-X deficient construction (Fig. 3b). However, the combination with the full-length PA-X expression showed the highest of expression of PA protein while a similar level of NP protein expression was observed among three groups (Fig. 3b). Moreover, a significantly higher expression of PA protein was found in the CA09_PA-X_Full virus infected cells when compared to those infected with the either CA09_WT or CA09_PA_PA-X_K/O virus (Fig. 3c). Similarly, no significant difference was observed in NP expression level among three groups (Fig. 3c). In summary, PA-X expression enhances production of PA protein, thereby increasing viral RNA polymerase activity and resulting in enhanced viral replication.

PA-X expression inhibits co-transfected gene expression

Recent studies revealed that PA-X protein is involved in the shutoff of host protein synthesis (Desmet et al., 2013; Jagger et al., 2012). To compare the contribution of different expressions of PA-X protein to shutoff activity, 293T cells were co-transfected with eGFP expression plasmid and individual PA expression plasmids (pCAGGS_PA-WT, pCAGGS_PA_PA-X_Full, or pCAGGS_PA_PA-X_K/O) for 24h. Relative GFP expression levels were determined by measuring relative fluorescence intensity and western blotting analysis. The reductions of GFP fluorescence intensity were observed from the cells co-transfected with PA expression plasmids (pCAGGS_PA-WT, pCAGGS_PA_PA-X_Full, or pCAGGS_PA_PA-X_K/O); approximately 46%, 57% or 28% of decreased fluorescence signals were found when compared to the control, respectively (Fig. 4a and b). The GFP protein expression levels were also reduced by 21%, 52% and 7%, respectively, in the presence of pCAGGS_PA-WT, pCAGGS_PA_PA-X_Full and pCAGGS_PA_PA-X_K/O, when compared to transfection with an empty pCAGGS vector (Fig. 4c). In the presence of both full-length and truncated PA-X, the GFP expression levels were significantly suppressed in contrast to the PA-X deficient condition (Fig. 4b and c). Furthermore, the GFP expression was markedly reduced in cells co-transfected with pCAGGS_PA_PA-X_Full compared to co-transfection with either pCAGGS_PA-WT or pCAGGS_PA_PA-X_K/O (Fig. 4b and c). These results indicate that full-length PA-X in CA09 virus has the most significant effect on shutoff activity of co-expressed protein synthesis, while loss of PA-X results in decreasing the shutoff ability.

Fig. 4. Effects of different expressions of PA-X protein on co-transfected GFP expression.

Fig. 4

Fig. 4

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293T cells were co-transfected for 24h with GFP expression plasmid and indicated PA expression plasmids encoding different PA-X forms. (a) Fluorescence images of GFP expression at 24h post transfection with indicated PA expression plasmids. The empty vector control was co-tranfected with GFP expression vectors with empty pCAGGS plasmid. (b) Relative GFP fluorescence intensity of each group compared to the control cells. (c) Relative expression levels of GFP proteins compared to the empty vector control cells using western blotting and densitometry. The GFP protein expressions were normalized to β-actin expression. The value of the empty vector control was set to 100%. The results of each group are the average of data from three independent experiments. The asterisks (*) indicate a statistically significant difference between groups (*: p<0.05. **: p<0.001 and ***: p<0.0001).

PA-X expression increases virus replication and pathogenicity in mice

To investigate the effect of different expressions of PA-X protein on virus pathogenicity in vivo, BALB/c mice were intranasally inoculated with 3.75 × 105 TCID50 of each virus and monitored daily. Control animals did not show any clinical signs during the study. All infected mice in each group displayed clinical signs such as depression, less activities and obvious weight loss starting at 3 dpi when compared to the control animals. However, both PA-X expression viruses (CA09_PA-X_WT and CA09_PA-X_Full) induced more severe body weight loss (80.5% and 78.4% by 7 dpi and 8 dpi, respectively) than CA09_PA-X_K/O virus (body weight loss to 85.5% by 7 dpi) (Fig. 5a). All mice infected with the CA09_PA-X_K/O virus survived, whereas the CA09_PA-X_Full virus caused 25% mortality and the CA09_PA-X_WT virus resulted in 12.5% mortality in infected animals (Fig. 5b). Virus titers in mouse lungs infected with either CA09_PA-X_WT or CA09_PA-X_Full were higher than those infected with the CA09_PA-X_K/O at 3 dpi (Fig. 6a), and a significant difference in virus titers was observed between the CA09_PA-X_Full and CA09_PA-X_K/O infected groups at 3 dpi. In contrast, a similar virus titer in mouse lungs was found in three infection groups at late time point (7 dpi) (Fig. 6a).

Fig. 5. Impact of different expressions of PA-X protein on pathogenicity of CA09 virus in BALB/c mice.

Fig. 5

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(a) Average body weights of surviving mice in each group up to 14 dpi are represented as percentages of the original weight on day 0. a,b and c on the value point of each day address significant differences (p<0.05) between infected groups (a: CA09_WT and CA09_PA-X_K/O, b: CA09_PA-X_K/O and CA09_PA-X_Full, c: CA09_WT and CA09_PA-X_Full). The error bars indicate standard errors of the mean (SEM). (b) Survival rate of mice infected with indicated viruses.

Fig. 6. Virus replication and histopathological scores in lungs of infected mice with either wild type CA09 or its mutated viruses.

Fig. 6

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(a) Virus titers in lungs (10% lung homogenates) of infected mice were determined at 3 and 7 dpi by calculating the 50% tissue culture infective dose (TCID50)/ml in MDCK cells. (b) Microscopic lung scores are presented as average scores ± SEM of three mice in each group at 3 and 7 dpi. The asterisks (*) indicate a statistically significant difference between groups (*: p<0.05). The error bars represent standard errors of the mean (SEM).

Histopathological analysis showed that no lung lesions were found in control mice, while animals from three infection groups exhibited lung lesions at both 3 and 7 dpi (Fig. 6b). Lesions consisted of varying degrees and percentage of lung affected with neutrophilic bronchiolitis, segmental loss of bronchiole epithelium, cuffing of bronchioles by lymphocytes and interstitial pneumonia (expansion of alveolar septa by histiocytes) (Fig. 7). Histologically, lung lesions varied in severity depending on dpi and the infected virus. At 3 dpi, both CA09_PA-X_WT and CA09_PA-X_Full viruses induced less histopathological lung lesions than the CA09_PA-X_K/O virus (Fig. 6b and Fig. 7). Furthermore, a significant difference in lung lesions was found between CA09_PA-X_K/O and CA09_PA-X_Full virus infected groups (Fig. 6b). More severe lung lesions were observed in both CA09_PA-X_WT and CA09_PA-X_Full infected mice at 7 dpi than at 3 dpi, whereas a similar lung lesion score was seen in CA09_PA-X_K/O infected mice at both days. Influenza antigens were detected in bronchiole epithelium and pneumocytes in infected mice using the H1N1 polyclonal antibody by immunohistochemistry analysis (Fig. 7). Control mice had no staining in the lung by immunohistochemistry analysis (Fig. 7).

Fig. 7. H&E and IHC staining of mouse lung sections at 3 dpi.

Fig. 7

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H&E stained sections of mice lungs infected with indicated viruses showed typical influenza pneumonia. Negative control: There are no lesions in the bronchiole. There is no antigen deposition in the H1N1 immunohistochemistry. CA09_WT and CA09_PA-X_Full: A small number of neutrophils are present in the bronchiole lumen (asterisk) and there is mild cuffing of the bronchiole by lymphocytes (arrow). IHC cytoplasmic staining with H1N1 antibody is present in the cytoplasm of bronchioles and pneumocytes (brown staining). CA09_PA-X_K/O: The bronchiole is filled with neutrophils (asterisk) and there is segmental loss of bronchiole epithelium and moderate cuffing of the bronchiole by lymphocytes (arrow). Adjacent alveolar lumina are expanded by lymphocytes and histocytes. IHC cytoplasmic staining with H1N1 antibody is present in the cytoplasm of bronchioles and pneumocytes (brown staining). Bars, 100 µm.

PA-X expression suppresses the early inflammatory response in lungs of infected mice

To investigate the impact of different expressions of PA-X protein on the host antiviral immune response, expression of representative cytokine genes related with the inflammatory response in lungs of infected mice was determined. Overall, CA09_PA-X_K/O virus induced stronger cytokine responses than both CA09_WT and CA09_PA-X_Full virus in mice at 3dpi (Fig. 8). In particular, CA09_PA-X_Full virus significantly reduced the gene expression levels of TNF-α, IL-1β, IL-6 and IL-12α when compared to CA09_PA-X_K/O virus at 3 dpi (Fig. 8). However, all three viruses induced comparable levels of gene expressions of IL-1β, IL-6, IL-12α and IFN-β at the late time point (7 dpi). At 7 dpi, IFN-α, IFN-β and IFN-γ were highly expressed, while lower levels of gene expressions of TNF-α, IL-1β, IL-6 and IL-12α were observed in infected mice with each virus compared to the early infection time point (3 dpi). Taken together, the PA-X deficient virus elicited stronger cytokine response in lungs of infected mice at the early time point (3dpi) than both PA-X expression viruses. Moreover, all tested cytokines and IFNs were more diminished in animals infected with CA09_PA-X_Full virus in contrast to those infected with CA09_WT expressing a truncated PA-X at the early time point (3 dpi).

Fig. 8. The mRNA expression levels of pro-inflammatory cytokines and IFNs in lungs of mice infected with either wild type CA09 or its mutated viruses.

Fig. 8

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The mRNA expressions of the indicated mouse cytokines and IFNs were quantified from lungs of infected mice by using qRT-PCR analysis. The expression fold change of each group was calculated relative to the control group after normalization to 18S ribosomal RNA internal control using 2−ΔΔCt method. Data represent the average values ± SEM of three mice in each group on the days indicated. The asterisks (*) indicate a statistically significant difference between groups (*: p<0.05. **: p<0.01 and ***: p<0.001).

Discussion

Since the PA-X protein was identified, multiple functions including effects on the viral virulence and modulation of the host immune response (Gao et al., 2015b; Gao et al., 2015c; Hayashi, MacDonald, and Takimoto, 2015; Hu et al., 2015; Jagger et al., 2012) and suppression of host protein synthesis by strong host shutoff activity (Desmet et al., 2013; Hayashi, MacDonald, and Takimoto, 2015; Jagger et al., 2012) have been described. Furthermore, several studies compared the roles of full-length and truncated PA-X proteins in viral pathogenicity and endonuclease activity (Bavagnoli et al., 2015; Gao et al., 2015a; Xu et al., 2016). In contrast to previous studies, we investigated the contribution of three different expressions of PA-X protein (full-length, truncated and PA-X deficient forms) in 2009 pH1N1 virus which expresses truncated PA-X protein to viral replication, pathogenicity and host immune response. Our results showed that the expression of either full-length or truncated PA-X enhanced viral replication and pathogenicity of CA09 virus in cells as well as in mice and polymerase activity in vitro in contrast to the PA-X deficient expression (Fig. 2, 3 and 5). Furthermore, the full-length PA-X expression resulted in more efficient replication in cells, higher virulence in mice and higher polymerase activity when compared to the truncated PA-X expression (Fig. 2, 3 and 5). Our results are consistent with former findings that full-length PA-X in pH1N1, H5N1 and H9N2 viruses enhanced virus replication in cells and pathogenicity in mice compared to corresponding viruses with truncated PA-X (Gao et al., 2015a). In addition, wild type 2009 pH1N1 virus with truncated PA-X expression showed increased viral growth in cells and virulence in mice compared to PA-X deficient virus (Hayashi, MacDonald, and Takimoto, 2015). These results indicate that the enhanced viral replication seen with PA-X expression is most likely due to promotion of the polymerase activity by increased expression of PA protein. In addition, the longer C-terminal domain (61aa) of PA-X has a stronger effect on the polymerase activity with higher expression of PA protein compared to truncated PA-X. In contrast, a previous study revealed that PA-X expression had no significant effect on viral growth of 1918 H1N1 virus in MDCK cells and reduced viral pathogenicity in mice (Jagger et al., 2012). Additionally, other previous studies showed that PA-X deficient pH1N1 and H5N1 viruses increase viral replication in A549 cells or Vero cells along with higher polymerase activity and enhanced viral virulence in mice compared to corresponding wild type viruses (Gao et al., 2015b; Hu et al., 2015). These results indicate that the effect of PA-X expression on viral replication and pathogenicity is virus strain-dependent. Interestingly, we showed that PA-X deficiency in pH1N1 virus resulted in decreased viral replication and polymerase activity in vitro, whereas Gao et al. showed that loss of PA-X expression in pH1N1 virus enhanced viral replication with a higher polymerase activity than the wild type virus (Gao et al., 2015b). One reason for the discrepant results on the PA-X deficiency in pH1N1 virus between two studies could be due to the different mutation approaches used in the frameshifting site in order to inhibit the PA-X expression. Whether different mutation approaches used (UCCUUUCGU to UCCUUCAGA used in our study and UCCUUUCGC to AGCUUCAGA in the previous study) induce different expression levels of PA-X protein leading to this discrepancy remains unknown and will be investigated in future studies.

The virus-induced host shutoff activity is expected to help the virus avoid the host antiviral responses by inhibiting antiviral factor expression (Narayanan et al., 2008; Vreede and Fodor, 2010). Influenza virus infection can cause shutoff of host protein synthesis to inhibit host antiviral defense and allow viral replication (Nemeroff et al., 1998; Noah, Twu, and Krug, 2003; Plotch et al., 1981). Recent studies revealed that PA-X protein in influenza virus infection has a major role in host shutoff which modulates host immune response by preventing antiviral related gene expressions (Hayashi, MacDonald, and Takimoto, 2015; Hu et al., 2015; Jagger et al., 2012). When we confirmed expression of expected PA-X proteins in transfected 293T cells with different PA-X ORF encoding plasmids, we could barely detect PA-X proteins because of PA-X self-suppression to PA-X expression plasmids (Fig. 1b). In addition, our in vitro study showed that both full-length and truncated PA-X can shutoff co-transfected eGFP expression (Fig. 4). Furthermore, we found that PA-X expressions in CA09 virus resulted in reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12α), type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) at 3 dpi (Fig. 8). This result may account for the reduced inflammatory reactions and histopathological damages in lungs of mice infected with CA09_PA-X_WT and CA09_PA-X_Full viruses compared to CA09_PA-X_K/O infected mice, especially at early time point of infection (Fig. 6b and 7). In addition, it is possible that inhibition of host antiviral response through host shutoff activity of PA-X promoted viral replication in lungs of infected mice, which may contribute to enhance viral virulence of both PA-X expression viruses in mice in contrast to the PA-X deficient virus (Fig. 5 and 6a). However, previous studies showed that virulence of both 1918 H1N1 and H5N1 HPAIV viruses was attenuated due to PA-X expressions when compared to their respective PA-X deficient viruses, even though PA-X expressions in those viruses suppressed host genes associated with immune response in vivo (Hu et al., 2015; Jagger et al., 2012). One possible explanation of this discrepancy between this study and previous studies is that both influenza viruses, unlike 2009 pH1N1 virus, induce a massive cytokine response, known as a cytokine storm which results in serious immunopathology and acute lung injury (Peiris, Hui, and Yen, 2010; Perrone et al., 2008). This excessive immune response inhibits viral replication but causes serious pathological damage in infected lungs, which contribute to increased viral pathogenicity in these virulent influenza viruses. In contrast, absence of PA-X expression in 2009 pH1N1 virus elicits a more appropriate host immune response which is critical for virus clearance and reduces viral virulence. Furthermore, this indicates that the effects of PA-X protein on viral pathogenicity and host immune response are in a virus strain-dependent manner.

Although PA-X protein is highly conserved among different influenza viruses, the length of its C-terminal domain varies among influenza viruses (Jagger et al., 2012; Rash et al., 2014; Shi et al., 2012). Most human and avian influenza viruses express a full-length PA-X protein with 61aa in the C-terminal domain, while truncated PA-X protein possessing 41aa in the C-terminal region is common in canine, swine and 2009 pH1N1 influenza viruses (Jagger et al., 2012; Shi et al., 2012). We have shown that the full-length PA-X protein displays stronger host shutoff activity than the truncated PA-X protein in vitro and in vivo (Fig. 4 and 8), which is consistent with previous findings (Gao et al., 2015a). However, two recent studies described that the first 15aa in the C-terminal region of PA-X are sufficient for maximum shutoff activity, while the addition of 20 aa of C-terminal end is not critical to shutoff activity of PA-X (Hayashi et al., 2016; Oishi, Yamayoshi, and Kawaoka, 2015). The reason for the different observations in shutoff activities of truncated PA-X between these studies could be due to differences of PA-X expression systems used, resulting in different protein expression levels, thereby showing a difference of the host shutoff activity. Because the whole PA expression plasmid was used in our study and the former studies, which could generate full-length or truncated PA-X by ribosomal frameshifting (Gao et al., 2015a), while PA-X expression plasmids were used in latter studies (Gao et al., 2015a; Hayashi et al., 2016; Oishi, Yamayoshi, and Kawaoka, 2015). Comprehensive phylogenetic and evolutionary analysis revealed that truncated PA-X appears in particular hosts such as dogs and swine which indicates species specificity of PA-X protein (Shi et al., 2012). Additionally, truncated PA-X protein is considered to be associated with the adaptation to these new hosts (Shi et al., 2012; Xu et al., 2016). However, the role of the additional 20aa to the C-terminal end of PA-X protein remains unclear and further investigations are needed.

In summary, our data indicate that expression of either full-length or truncated PA-X protein in 2009 pH1N1 virus helps viral replication in vitro, likely through promoting the polymerase activity, and enhances viral virulence in vivo by inhibiting host innate immune response through host shutoff activity. Moreover, full-length PA-X protein displays a stronger effect on viral replication and pathogenicity compared to truncated PA-X. Thus, our study provides a better understanding on the contribution of different expressions of PA-X protein forms to viral pathogenicity and the host immune response. Additional studies are needed to understand the role of PA-X protein in influenza virus evolution and host adaptation.

Highlights.

  • Impact of different expressions of PA-X protein including full-length, truncated or deficient PA-X on 2009 pandemic H1N1 virus replication, pathogenicity and host immune response was determined.

  • Expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X.

  • The full-length PA-X expression exhibited a greater effect on virus replication, pathogenicity and host immune response than the truncated PA-X form.

Acknowledgments

We would like to thank Hui He, Haixia Liu, Nan Cao and Chester McDowell for helping with the animal studies. We would also like to thank Dr. Toru Takimoto in University of Rochester Medical Center for providing PA and PA-X monoclonal antibodies. The authors thank staffs from the Comparative Medicine Group at Kansas State University for supporting the animal studies, and Jennifer Phinney from the Kansas State Veterinary Diagnostic Laboratory for technical assistance with H&E and IHC staining. This work was partially supported by Kansas State University Start-Up Fund (SRO# 001), and by an NIAID funded Center of Excellence for Influenza Research and Surveillance, under contract number HHSN266200700006C.

Footnotes

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Conflicts of interest

The authors declare no conflicts of interest.

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