
==== Front
Emerg Microbes Infect
Emerg Microbes Infect
Emerging Microbes & Infections
2222-1751
Taylor & Francis

39221898
2400546
10.1080/22221751.2024.2400546
Version of Record
Emerging Seasonal and Pandemic Influenza Infection
Research Article
The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus
Emerging Microbes & Infections
P. WANG ET AL.
Wang Pingping abc*
Guo Jianing a*
Zhou Yefan abc
Zhu Min abc
Fang Senbiao de
Sun Fanyuan abc
Huang Chongqiang abc
Zhu Yaohui abc
Zhou Huabo f
Pan Boyu de
Qin Yifeng abc
Ouyang Kang abc
Wei Zuzhang abc
Huang Weijian abc
García-Sastre Adolfo ghi
Chen Ying abc
a Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, People’s Republic of China
b Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, People’s Republic of China
c Guangxi Key Laboratory of Animal Breeding, Disease Prevention and Control, Nanning, People’s Republic of China
d Department of Molecular Pharmacology, Tianjin Medical University Cancer Institute & Hospital, Tianjin, People’s Republic of China
e National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin Clinical Research Center for Cancer, Tianjin, People’s Republic of China
f Huabo Pet Hospital, Nanning, People’s Republic of China
g Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
h Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
i Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
CONTACT Ying Chen yingchen@gxu.edu.cn Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning 530004, People’s Republic of China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530004, People’s Republic of China; Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, Nanning 530004, People’s Republic of China;
Adolfo García-Sastre Adolfo.Garcia-Sastre@mssm.edu Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
* These authors are contributed equally to this article.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2024.2400546.

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ABSTRACT

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-β in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

KEYWORDS

Canine influenza virus
NS1 protein
four C-terminal residues
virulence
viral replication
National Natural Science Foundation of China 10.13039/501100001809 32160825 Guangxi Natural Science Foundation 10.13039/501100004607 2023GXNSFDA026042 The National Natural Science Foundation of China (32160825) and Guangxi Natural Science Foundation (2023GXNSFDA026042) supported this work.
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pmcIntroduction

Interspecies transmission of influenza viruses amongst and between hosts needs to overcome the species barriers and they need to interact successfully with host-specific factors in order to adapt new hosts. In the past two decades, the H3N8 equine influenza virus (EIV) and H3N2 avian influenza virus (AIV) accomplished the “host jump” from horses to dogs and birds to dogs, respectively [1,2]. Since the emergence of the H3N8 canine influenza virus (CIV) in dogs, it diverged from the EIVs circulating in horses and formed an independent lineage. Interestingly, there was no evidence that the adapted H3N8 CIV could naturally transfer back to horses [3]. The introduction of H3N2 AIVs into dogs resulted in the establishment of a new canine lineage in Asia, which not only showed a relatively broad host range, but also produced novel viruses by re-assortment with the 2009 pandemic H1N1 (pdm09/H1N1), H5N1 AIV and H1N1 swine influenza virus (SIV) [4–7]. Numerous studies have identified several key determinants of host adaptation, which were responsible for increasing the host range as well as their virulence. For example, the receptor binding specificity of hemagglutinin (HA), the changes of species-specific amino acids of the ribonucleoprotein complex (RNP) in their mammalian hosts and the immune antagonists induced by the non-structural protein 1 (NS1) [8–10].

The NS1 protein is encoded by the smallest fragment of the IAV genome and it is comprised of a N-terminal dsRNA-binding domain (RBD) (amino acids 1–73), a variable-length linker region (amino acids 74–84), a C-terminal effector domain (ED) and a variable tail (amino acids 85-end) [11,12]. There are two nuclear location sequences (NLSs) located at residues positioned at 35–41 and a C-terminal disordered tail (CTT), which are beneficial for transport through the nuclear membrane by interaction with importin-α [13]. The species-specific adaptation occurred at the C-terminal end of the ED, which is structurally disordered and varies in the length, indicating an interaction with the poly (A)-binding protein II (PAB II) [14]. Large-scale sequence analyses of the avian and mammalian species have also indicated that the NS1 protein is important for viral evolution and interspecies transmission. The last four C-terminal residues of the 230 aa long NS1 protein contain a consensus PDZ-domain ligand (PL) motif (e.g. ESEV or EPEV) at its terminus, and this has the potential to bind to PDZ domain-containing proteins [15]. There is a substantial amount of evidence to suggest that the four-amino-acid-motif in the C-terminal domain of the NS1 protein (e.g. ESEV, EPEV and RSKV) is a conserved species-specific motif, and that it has an important impact on the replication and pathogenicity of the viruses [16,17]. Additionally, the C-terminal ESEV extension of the NS1 protein contributes to increasing their transmission efficiency [18].

In our study, sequences analysis of the interspecies transmission of IAVs from horses (H3N8), birds (H3N2) and pigs (H1N1) to dogs showed that there was a conserved four C-terminal residue sequence of the NS1 protein in viruses obtained from different hosts. Whether these C-terminal residues were involved in the virulence of the viruses or if they were responsible for the replication in their new hosts was not clear. Here, we generated a series of NS1 mutant viruses by using reverse genetic techniques. These contained the four C-terminal residues from different origins (avian H3N2, equine H3N8 and swine H1N1 as well as the corresponding subtypes of canine origin) and used the naturally NS-truncated H1N1 CIV as the backbone. The mutant viruses were investigated for their virulence in mice as well as their capacity to antagonize the effects of IFN in human cell lines in order to understand the viral determinants of the NS1 protein that mediated host adaptation in dogs. A better understanding of influenza viruses will aid towards the consistent threat that these viruses pose to human health worldwide.

Materials and methods

Cells, viruses and reagents

Madin-Darby canine kidney (MDCK), human alveolar basal epithelial (A549), human embryonic kidney (293 T), porcine kidney (PK-15), Vero and chicken fibroblast (DF-1) cells were cultured and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin–streptomycin. Cell cultures incubated in a 37 ℃, 5% CO2 incubator. The eight viral segments of swine-origin H1N1 CIV, A/canine/Guangxi/LZ57/2015 (H1N1) (rΔNS1 wt), with a natural deletion of 135 aa at the C-terminus of the NS1 protein was rescued previously by reverse genetics [19]. VSV-GFP and SeV were kindly provided by the Guangxi Institute of Veterinary Medicine. The primary antibodies were anti-Flag-Tag antibody (Affinity, USA, Cat No. T0003) and GAPDH Monoclonal antibody (Proteintech, USA, Cat No. 60004-1-Ig). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (YIFEIXUE, Nanjing, China, Cat No. YFSA01) were used as the secondary antibodies for western blotting.

Construction of plasmids

Seven mutated NS segments were amplified by overlapping extension-PCR (SOE-PCR) by using primers of differently mutated ΔNS1 gene sequences. These mutated NS segments were digested with Sap I, and cloned into the pDZ vector. All of the constructs of the NS gene were then verified by sequencing and rescued in combination with the seven viral gene segments, following the previously described method [19]. Moreover, the Flag tags at the C terminus were fused with ΔNS1 mutant sequences and these were cloned into a eukaryotic expression plasmid, pCAGGS, between the EcoR I and Kpn I sites. All primer sequences used are available upon request.

Viral growth kinetics

Confluent MDCK, A549, PK-15 and DF-1 cells were infected with rΔNS1 mutant viruses with a multiplicity of infection (MOI) of 0.01, 0.1, 0.01 and 0.1, respectively. The cells were then overlaid with serum-free DMEM containing 1 μg/mL TPCK trypsin and incubated at 37℃, 5% CO2 for 1 h. The cell supernatants were harvested every 12 h until 72 hours post-infection (h.p.i.). Each time-point was performed in triplicate. Cell debris was removed by centrifugation at 12,000 rpm for 5 min and the samples were titrated using plaque assays on MDCK cells and analyzed by the Reed-Muench method, as previously described [19].

Mouse experiments

All procedures were carried out in accordance with the guidelines of the Animal Experimental Ethical Inspection of Guangxi University. The Guangxi University Animal Care & Welfare Committee (approval number GXU-2021-084) approved all of the animal experiments described in this study.

6-week-old female specific pathogen-free BALB/c mice (Charles River, Beijing) were lightly anesthetized and inoculated intranasally with 106 PFU/50 μL of wt and the seven rΔNS1 mutant viruses in a volume of 50 μL. Animals from the control group were inoculated intranasally with 50 μL PBS. On day 5 post infection (d.p.i.), three mice in each group were euthanized and tissue samples were collected during necropsy. Tissue samples were either frozen for virological analysis and titration by using plaque assays or fixed in 4% paraformaldehyde for histological analysis. Clinical signs, body weights and the survival rate were monitored daily until 14 d.p.i. When mice had lost 25% of their pre-inoculation weight, they were humanely euthanized according to the study protocol.

To determine the mean dose lethal to 50% of infected mice (MLD50) of the mutated viruses, mice were inoculated intranasally with 10-fold serial dilutions of each virus (from 102 to 106 PFU per mouse). As mentioned above, the health status and daily weight changes were monitored until 14 d.p.i. The MLD50 was calculated according to the Reed-Muench method.

Analysis of IFN-α/β mRNA by qPCR

To measure the mRNA levels of type I IFN in the infected A549 cells, cells were infected with the wt and mutant NS1 viruses with a MOI of 1. At 6, 12 and 24 h.p.i., the cells were washed with 4℃ PBS and they were processed by using Buffer RL. Subsequently, the total RNA was extracted by using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme) according to the manufacturer’s protocol. Reverse transcription was performed by using 5 × HiScript II qRT SuperMix (Vazyme) (+gDNA wiper). Quantitative PCR (qPCR) was performed in a final volume of 20 μL by using 1 μL of DNA (diluted 10 times), each primer at 0.2 μM and 10 μL and 2 × ChamQ Universal SYBR qPCR Master Mix. The qPCR was performed with the LightCycler 96 instrument (Roche applied Biosystems). The following program was used: pre-incubation (30 s at 95°C), a 2-step amplification (10 s at 95°C and 30 s at 60°C) followed by 40 cycles and a melting step (15 s at 95°C, 60 s at 60°C and 15 s at 95°C). The qPCR relative quantitative calculation method (2-△△t) was used for data analysis.

IFN-antagonizing activity of the viruses

A549 cells were grown in 12-well plates and infected with wt and mutant NS1 viruses at a MOI of 2. After infection for 24 h, the cell supernatants were UV-treated for 45 min on ice and collected. The UV-inactivated supernatants were verified for inactivation by using plaque assays on MDCK cells. Then Vero cells were incubated with the UV-inactivated supernatants for 24 h.p.i., followed by inoculation of VSV-GFP (MOI = 0.1). The levels of VSV-GFP expression were determined using a fluorescence microscope (EVOS, Thermo Fisher Scientific, USA). Sendai virus (SeV), which can act as an inducer of interferon, was used as a positive control (40 HAU/mL).

Quantification of IFN-β production by ELISA

To measure the secretion of IFN-β, A549 cells were infected with wt and mutant NS1 viruses at a MOI of 2. The cell supernatants which were infected with mutant viruses and stimulated by poly (I: C) were harvested at 24 h.p.i. Production of IFN-β in culture supernatants was measured by using a human IFN-β ELISA Kit (Beyotime, China), according to the manufacturer’s instructions. Three sets of samples infected with each mutant virus were collected and ELISA was used to measure each sample in duplicate.

Molecular dynamic (MD) simulations

In order to check the effects of different mutations on proteins, the homology modeling results were employed for MD simulations carried out by using Gromacs software (version 2021.5) with the AMBER ff02 force field as topology parameters for proteins. Hydrogen atoms were added to the initial protein model using the leap module, setting ionizable residues as their default protonation states at a neutral pH value. The proteins were solubilized in a cubic periodic box of the explicit TIP3P water model that extended to a minimum 10 Å distance from the box surface to any atom of the solute. Sodium ions were randomly replaced with water molecules in the box which acted to neutralize the entire system. The particle mesh Ewald (PME) method for simulation of periodic boundaries was used to estimate the long-range electrostatic interactions and this was set to a cutoff of 10 Å. All bond lengths were constrained using the SHAKE algorithm and the integration time step was set to 2 fs using the Verlet leapfrog algorithm [20].

To eliminate possible bumps between the solute and the solvent, the entire system was minimized into two steps. Firstly, the protein was restrained with a harmonic potential of the form k (Δx)2 with a force constant k = 100 kca mol−1Å−2. The water molecules and counterions were optimized using the steepest descent method of 2500 steps, followed by the conjugate gradient method for 2500 steps. Secondly, the entire system was optimized by using the first step method without any constraints. These two minimization steps were followed by an annealing simulation with a weak restraint (k = 100 kca mol−1Å−2) for the complex. Then the entire system was heated gradually in an NVT ensemble from 0 to 298 K over 50 ps. After the heating phase, a 100 ns MD simulation was performed under a pressure of 1 atm for each protein. The constant temperature was selected to be 298 K with the NPT ensemble and this was maintained using a Langevin thermostat with a collision frequency of 2 ps−1. Constant pressure was maintained by employing the isotropic position scaling algorithm with a relaxation time of 2 ps. In this study, 20 snapshots (every 50 ps) were extracted from the last 10 ns trajectory for each complex. The binding free energy calculations were carried out by using the MM-PBSA method (https://ambermd.org/tutorials/advanced/tutorial3/py_script /section4.php).

Western blotting and qPCR analysis

293 T cells were transfected with ΔNS1 plasmids containing Flag-Tags. After transfection at 16, 24, and 32 hours, the cells were washed with cold PBS once and lysed using RIPA lysis buffer containing protease inhibitors at 4℃ for 15 min. The cell lysates were collected and then they were centrifuged at 12,000 rpm for 10 min. These were subjected to western blotting analysis by incubating with primary antibodies to Flag-Tag (Affinity, USA) and a monoclonal antibody to mouse GAPDH (Proteintech, USA), followed by a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (YIFEIXUE, China). Finally, the eECL Western Blot Kit (CWBIO, China) was used for color imaging. In addition, the cells were lysed at the specified time-points, and the total cell RNA was extracted by using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme) according to the manufacturer’s protocol. Subsequently, 5×HiScript II qRT SuperMix (Vazyme, Nanjing, China, Cat No. R223-01) (+gDNA wiper) was used for reverse transcription. QPCR was then performed using a 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China, Cat No. Q711-02) in order to quantify the mRNA levels of ΔNS1.

Statistical analysis

All experimental data were statistically analyzed using GraphPad Prism 6.0 (GraphPad software, San Diego, CA, USA). Analysis of variance (ANOVA) was used to determine whether the differences between groups were statistically significant. The P values of * (P < 0.05), ** (P < 0.01), *** (P < 0.001), **** (P < 0.0001) were considered statistically significant differences.

Results

Generation of influenza virus mutants containing four altered C-terminal residues of the ΔNS1 protein

Although several sequence changes have been observed in the NS1 protein, there appeared to be a strong selection pressure on its four C-terminal residues in their respective hosts and these have evolved in a species-specific manner [15]. Sequence alignment of the representative avian H3N2, equine H3N8 and swine H1N1 as well as their corresponding subtypes of CIVs have indicated that there were species-specific motifs at the C terminus of the NS1 protein (Table S1). The dominant motifs of NS1 protein from different hosts were selected in order to investigate their biological characterization both in vitro and in vivo. The SKQK (equine H3N8) and LPPK (canine H3N2) motifs were found to be predominant in the majority of NS1 sequences from equine H3N8 and canine H3N2 viruses, respectively. However, both of these were C-terminal truncated NS1 proteins, which were shorter with 219 aa (H3N8) and 217 aa (H3N2) in length, respectively. In this study, we selected the putative PDZ-binding EPEV (equine H3N8) and non-PL ESEI (canine H3N2) motifs in the 230 aa-NS1 protein and these were mutated. Six rΔNS1 mutant viruses with the conserved C-terminal motifs of the NS1 protein of swine, equine, avian and canine origins, and one last four residues-truncated virus were generated by reverse genetics using the A/canine/Guangxi/ LZ57/2015 genetic as the backbone (Figure 1). Figure 1. A schematic diagram of the viral A/canine/Guangxi/LZ57 (H1N1)-NS1 protein containing the length of 84 amino acids with the 135-amino acids truncation of NS1 protein at the C-terminus. The RNA-binding domain (1–73 aa), linker (74–84 aa) and the C-terminal effector domain (85-end) are indicated. The full length of NS1 protein usually contains 219 amino acids. The last four C-terminal amino acids of NS1 were either deleted (rΔNS1-trunc) or replaced with the motifs of canine SEQK and swine PEQK from H1N1, canine KPEI and equine EPEV from H3N8, and canine ESEI and avian ESEV from H3N2 influenza viruses, respectively. nt, nucleotide; aa, amino acid.

Growth properties of wt and mutant NS1 viruses in canine-, human-, chicken- and swine-derived cells

To explore the replication capacity of the different mutant strains, we compared their growth properties in MDCK (canine), A549 (human), PK-15 (swine) and DF-1 (chicken) cells infected at a low MOI. There were no significant differences at 48 h.p.i. in MDCK cells, but rΔNS1-KPEI (H3N8 canine) and rΔNS1-ESEI (canine H3N2) viruses exhibited stronger replicative capacities in both A549 and PK-15 cells. Interestingly, in swine-derived cells (PK-15) infected at a MOI of 0.01, the rΔNS1-SEQK viruses replicated more efficiently than in the other cell lines, whereas rΔNS1-trunc, rΔNS1-PEQK (swine H1N1) and rΔNS1-EPEV (equine H3N8) viruses showed restricted replication in swine cells. When chicken-derived cells were infected, the rΔNS1-KPEI (canine H3N8) and rΔNS1-ESEI (canine H3N2) viruses produced higher viral yields when compared to those with the SEQK and EPEV motifs (Figure 2A). The plaque phenotypes of these mutant viruses seemed to be similar to wt-like plaques (Figure 2B and C), indicating that mutations of the four C-terminal residues did not affect their cell-to-cell spreading. Taken together, these results revealed that the presence of the last four C-terminal residues could change the replicative ability of the viruses in different host cells. There were significant differences in A549 and PK-15 cells. The KPEI and ESEI motifs showed better replicative advantage when compared to other C-terminal motifs. Figure 2. Growth kinetics of the parental and mutated viruses in different cell lines. (A) MDCK, A549, PK-15 and DF-1 cells were infected with wt and mutant NS1 viruses at MOIs of 0.01, 0.1, 0.01 and 0.1, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The supernatants were harvested every 12 h and titrated by using plaque assays in MDCK cells. The results represent the average of three samples infected in parallel. (B) Plaque phenotypes of wt and the mutant NS1 viruses in MDCK cells. After infection with 10-fold dilutions, the infected MDCK cells were fixed in 10% paraformaldehyde and then they were stained with 0.8% (w/v) crystal violet solution. (C) The areas of individual plaque formation were measured using ImageJ software. The diameters of selected plaques were calculated and these were standardized when compared to wt viruses.

The contribution of the four C-terminal residues of ΔNS1 protein towards the enhancement of viral virulence in mice

We infected six-week-old BALB/c mice (groups of six) intranasally with 106 PFU of wt and mutant NS1 viruses. The body weight of each mouse was recorded daily up to 14 d.p.i. (Figure 3A). The mice infected with rΔNS1-KPEI (canine H3N8), rΔNS1-ESEV (avian H3N2) and rΔNS1-ESEI (canine H3N2) viruses displayed a faster body weight loss than those infected with other rΔNS1 mutant viruses after viral infection. Furthermore, we evaluated the survival rates of the infected mice (Figure 3A). It was found that those mice infected rΔNS1-KPEI, rΔNS1-ESEV and rΔNS1-ESEI viruses succumbed to viral infection by day 7, whereas 33% of the mice infected with the rΔNS1 wt virus survived up until day 14 p.i. The rΔNS1-trunc virus that lacked the NS1 protein final C-terminal residues, as well as the rΔNS1-SEQK, rΔNS1-PEQK and rΔNS1-EPEV viruses, caused only a mild weight loss. Subsequently, we determined the viral loads in lung tissues and turbinate samples of the infected mice at 5 d.p.i. (Figure 3A), and the results showed an altered replication ability in the mice. The rΔNS1 mutant viruses containing ESEV, KPEI, EPEV and ESEI exhibited better replication in mouse lungs, which was similar to the wt viruses. In contrast, the mutant viruses with SEQK, PEQK and truncation had decreased the viral yields in the lungs. Additionally, only the EPEV and ESEV viruses showed advantageous replication capacities in the turbinate samples. Consistent with these observations in body weights and survival rates, histopathological analysis of the lung tissues further identified that the three mutant viruses (KPEI, ESEI and ESEV) caused more severe bronchitis and alveolitis with significant thickening of bronchus and loss of epithelial cells. The alveolar spaces were filled with infiltration of large numbers of inflammatory cells, including neutrophils, macrophages and cellular debris, when compared to the rΔNS1 wt viruses. In contrast, the viruses containing the truncated, swine H1N1 (PEQK), canine H1N1 (SEQK) and equine H3N8 (EPEV) viruses caused only mild alveolitis in the lungs (Figure 3B). Figure 3. The effect of changes in four amino acid residues at the C-terminus end of the NS1 protein on the pathogenicity of the viruses in mice. Six-week-old female BALB/c mice were intra-nasally inoculated with either a 106 PFU/50 μL dose of each virus or PBS. (A) Infected mice were monitored daily for body weight changes and survival. Three of the infected mice were euthanized on the 5 d.p.i. The mouse lungs and nasal turbinate samples were collected for viral titration. The virus titers were determined by using the plaque assay in MDCK cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (B) Histopathological changes of mouse lungs infected with wt and mutant NS1 viruses. At 5 d.p.i., three mice in each group were sacrificed and their lungs were fixed with 4% paraformaldehyde. The tissues were then embedded in paraffin and then tissue sections were cut and stained with hematoxylin and eosin. Scale bars, 100 μm. *, Bronchus. (C) The virulence of wt and mutant NS1 viruses in mice. Groups of four mice were inoculated intranasally with 10-fold serial dilutions of each virus preparation (from 102 to 106 PFU per mouse). When a body weight of 25% was observed, the mice were assumed to be near death and they were euthanized. The survival of mice was monitored until 14 d.p.i. after inoculations. The amino acids in the NS1 protein were at positions 81–84.

In order to confirm whether the enhanced virulence was due to the changes in the four C-terminal residues of ΔNS1, the MLD50 of the rΔNS1 mutant viruses were tested by intranasal inoculations. As shown in Figure 3C, when the four C-terminal residues were altered with KPEI (canine H3N8) and ESEV (avian H3N2), these changes significantly enhanced the virulence of the viruses in mice, which was reflected by up to a 100-fold higher MLD50 values when compared with the rΔNS1 wt viruses (both were 3.75). However, the rΔNS1-ESEI (canine H3N2) virus had a similar MLD50 value to that of rΔNS1 wt (5.25 and 5.75, respectively). Our results further emphasized that the canonical ESEV PL domain (avian H3N2) conferred the highest pathogenicity in mice, but it did not increase their replication in human and chicken cell lines. Interestingly, the KPEI (canine H3N8) and ESEI (canine H3N2), not only exhibited enhanced virulence, but also showed more efficient replication in cultured human and swine cell lines among the C-terminal mutant viruses. This suggested that the final C-terminal motifs of the ΔNS1 protein, including the PL and unrecognized domains, may play a key role in modulating the virulence of these viruses.

Correlation of increased virulence of wt and mutant NS1 viruses with IFN production

The multifunctional NS1 protein was largely responsible for countering the IFN antiviral response by inhibiting of the activation of IRF3 as well as IFN transcription of the processed IFN pre-mRNA molecules [11]. We then determined whether the differences in replication and virulence of these rΔNS1 mutant viruses was directly correlated with the ability of them to inhibit the IFN production in the host cells. We measured the mRNA levels of type I IFN by collecting cell samples after viral infection of A549 cells and found there was no significant difference in the levels of IFN-α mRNA between the rΔNS1 mutant viruses at 12 and 24 h.p.i. However, with the rΔNS1-KPEI and ESEI viruses-infected cells, higher levels of IFN-α mRNA were detected at the early time-points (P < 0.0001) (Figure 4A). In addition, high levels of IFN-β mRNA were also detected in the rΔNS1-KPEI- (canine H3N8) and rΔNS1-EPEV-infected viruses at 6 h.p.i. (P < 0.0001). The rΔNS1-EPEV viruses produced the highest levels of IFN-β mRNA at 12 h.p.i. (P < 0.0001). In particular, the rΔNS1-SEQK (canine H1N1 viruses) could induce the higher level of IFN-β at 6,12, 24 h.p.i. (P < 0.0001) (Figure 4B). Subsequently, the supernatants collected from A549-infected cells were inactivated by pretreatment with UV light, and inoculated into cultured IFN-production-deficient Vero cells, followed by infection with VSV-GFP (MOI = 0.1). The levels of secreted IFN-α/β were determined in the supernatants from the infected A549 cells by using a bioassay based on blocking the GFP expression in VSV-GFP-infected cells. It was found that when the cells were infected with Sendai virus (SeV), a well-described IFN-β inducer, this blocked the GFP expression [21]. Similarly, VSV-GFP replication was completely abolished in cells pretreated with the supernatants obtained from rΔNS1-SEQK-infected cells until the dilution of ratio reached 1:32 (Figure 4C). However, the cells infected with the viruses expressed high levels of GFP, suggesting that high levels of IFN were not produced in these rΔNS1 mutant viruses-infected cells. Figure 4. The interferon-antagonizing activity of the mutant NS1 viruses in A549 cells. A549 cells were infected with wt and mutant NS1 viruses, after which they were incubated at 37 °C. (A,B) The cells were collected and lysed at 6, 12 and 24 h.p.i. and IFN-α (A) and IFN-β (B) were then measured qPCR. (C) The cell supernatants were treated with UV light and collected at 24 h.p.i. Then Vero cells were treated with the UV-inactivated supernatants and subsequently inoculated with VSV-GFP. At 24 h.p.i. with VSV-GFP, the expression levels of GFP were assessed. SeV, a beta IFN inducer, was used as a positive control in VSV-GFP experiments. (D) IFN-β levels in the cell supernatants were measured using a human IFN-β ELISA Kit at 24 h.p.i. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (E) A549 cells were infected with wt and mutant NS1 viruses at a MOI of 2, and the cells were also treated with 1μg/mL poly (I:C) at 12 h.p.i. The total RNA was extracted from lysed cells and qPCR was employed to measure the relative expression levels of mRNA. The data are the means and standard deviations from three independent experiments (*, P < 0.05; **, P < 0.01; ****, P < 0.0001).

To further determine the expression and secretion levels of IFN-β in the treated cells, the levels of IFN-β in the supernatants at 24 h.p.i were measured by ELISAs. Interestingly, we detected 118.10 and 572.72 pg/mL of IFN-β in the supernatants of the rΔNS1-trunc and rΔNS1-SEQK viruses-infected cells, respectively (Figure 4D). Further, we examined the expression levels of IFN mRNA by treating cells with poly (I:C), which had been shown to increase its stability. It also was found that the replacement of the SEQK motifs caused the upregulation of high-level expression of IFN-β (Figure 4E). Overall, these results indicated that the C-terminal motifs increased the virulence of the viruses in mice in a NS1-independent manner, although there was an exception, in that the higher levels of IFN-β induced by the rΔNS1-SEQK viruses resulted in its lower virulence.

Mutations in the four C-terminal residues decreased stability of the ΔNS1 protein without decreasing mRNA levels

Previous studies have indicated that there was a correlation between the NS1 protein expression and the strong ability of the virus to antagonize the IFN-α/β response in the host cells [22,23]. We employed MD to imitate the protein structure in order to explore the effects of the C-terminal motifs on the ΔNS1 protein. The equilibration of MD trajectories was monitored from the convergence of the root-mean square deviation. The results showed that most of systems reached equilibration except for KPEI and ESEI, which fluctuated by 14 and 12 Å, respectively, suggesting that the changes in the C-terminal motifs resulted in formation of unstable structures for the resulting ΔNS1 proteins (Figure 5A). To further analyze the effects of the interaction of the C-terminal motifs on the A and B chains of ΔNS1 protein, the C-terminal tail orientations for the KPEI and ESEI systems indicated that the binding networks were almost lost, resulting in obvious denaturation of the protein structures. In contrast, the presence of the TTSH (wt), SEQK, PEQK, EPEV and ESEV tended to stabilize the C-terminal structure (Figure 5B), which further reflected the major contribution of the C-terminal residues to the stability of the truncated NS1 protein (ΔNS1). Figure 5. Analysis of stability of wt and mutant ΔNS1 proteins. (A) Root-mean-square deviation plots of backbone atoms during an 80 ns evolution time dependent in 8 parallel trajectories from the corresponding initial structures. (B) Conformation variation analysis of the eight NS1 mutant proteins. Intensive binding interaction network around the C-terminal tail region was formed based on the dimeric 84 aa-length NS1 protein structure (left). Marked conformational changes were observed when the image was magnified. This was due to the mutation of the C-terminal residues with SEQK, EPEV, ESEV and PEQK as well as its removal, as shown on the right. The underlined amino acids represent the interactions between them. (C) 293 T cells were transfected with NS1 plasmids containing either Flag-Tags or empty plasmids. At 16, 24 and 32 hours after transfection, the cell lysates were collected and western blotting was performed to determine the levels of the NS1 proteins. (D) RNA was extracted from some of the cells and the mRNA levels of NS1 was determined by qPCR.

In order to further verify the effect of the NS1 C-terminal amino acids on the stability of NS1 protein, we constructed eight H1N1 ΔNS1 mutant plasmids with Flag tags, and these were transfected into 293 T cells. At 16, 24 and 32 hours after transfection, cells were lysed and the cell extracts were analyzed by western blotting. Unexpectedly, there were apparent stability differences with the changes of the four C-terminal residues in the ΔNS1 protein. The ΔNS1 wt protein was expressed at the early stages and reached its maximum expression levels at 32 hours after transfection. Similarly, the C-terminal residues with SEQK, EPEV and ESEV motifs also had higher protein expression levels at 24 hours after transfection. However, the protein expression with the C-terminal KPEI and ESEI motifs was significantly less than that those of other six mutants until 32 hours after transfection (Figure 5C). In order to determine whether the reduction observed in these mutant ΔNS1 protein levels was a consequence of their decrease in their mRNA levels, the mRNA was extracted and analyzed by qPCR (Figure 5D). The results showed no significant difference in mRNA levels between the different strains, suggesting that the difference in C-terminal amino acids may affect the post-transcriptional process.

Discussion

The NS1 protein is one of the most polymorphic and mutation-tolerant proteins found in IAVs, which diverged early during their evolution and have subsequently evolved in a host lineage-dependent manner [24]. In addition, it is thought that these proteins have been directly involved in the viral adaptation to new host species [10]. Although there are species-associated polymorphisms in the NS1 protein, conservation of its species-specific C-terminal motif indicates that the last four C-terminal residues possess a strong selective advantage in its natural hosts.

In this study, a novel triple recombinant swine-origin H1N1 IAV (A/canine/Guangxi/LZ57, LZ57) initially introduced from pigs to dogs was the main subject of our studies. In particular, the NS segment with naturally deleted 243–378 bases, which results in a frameshift mutation containing only the 84 aa-length NS1 protein at the N-terminus (Figure 1). Previous studies indicated that the replication characteristics of IAVs with a truncated NS1 were severely compromised in IFN-responsive competent cells and they were attenuated in mice [25–30]. In line with previous studies, the N terminal 84 aa-NS1 protein resulted in the lower virulence in mice, but it did not affect the replication efficiency in cultured cells [19]. This suggested that the C-terminal NS1 effector domain sequences were not dispensable with respect to viral replication in cultured cells [31]. We cannot rule out an effect on the other sections of the NS1 protein, but these large truncations appeared to affect several functions of its remaining modules by altering its structure. The analysis of large sequences of H1N1, H3N8 and H3N2 IAVs derived from pigs, horses, birds and dogs have indicated that there was a conserved four C-terminal residue section of NS1. This was observed to be essential for interspecies transmission, including the recognition of the canonical PDZ domain ligands (e.g. ESEV) as well as the unrecognized dominant motifs (Table S1). Therefore, here we evaluated whether the differences in the replication and virulence between the H1N1 (swine PEQK and canine SEQK), H3N8 (equine EPEV and canine KPEI) and H3N2 (avian ESEV and canine ESEI) IAVs derived from different hosts, could be attributed to amino acid changes in the ED-truncated NS1 protein.

Here, we were able to show for the first time that the C-terminal ESEI and KPEI domains of NS1 protein circulating in canines increased the replication of the viruses in human cells, and conferred disease-related functions and enhanced virulence in mice. However, the C-terminal ESEV motif did not display efficient replication in human cells, even when it showed increased virulence in mice. Large-scale sequence analysis revealed that the avian PL motifs were found in highly pathogenic human infections in 1997 (EPEV) and 2003 (ESEV). In addition, in the 1918 pandemic, the virus motif (KSEV) interacted with the PDZ protein and this was identified as the determinant of host adaptation and virulence [15,16]. Consistent with previous observations regarding ESEV viruses, our results indicated that the avian H3N2 C-terminal ESEV motif was still regarded a pathogenic factor. The reason for the increased virulence of the ESEV virus was correlated to its interactions with the PDZ domain and several intracellular proteins that could potentially disrupt essential biochemical pathways. For example, the NS1-ESEV PBM (PDZ-binding motif) could bind to the cellular scaffolding proteins, Dlg-1, Scribble, MAGI-1, MAGI-2 and MAGI-3, leading to enhanced viral replication by direct protein–protein interactions. Specifically, an interaction between NS1 and Scribble was shown to inactivate its pro-apoptotic activity in host cells [32,33].

The results of this study re-emphasized the important role of the avian ESEV motif in increasing virulence in mice on the genetic background of the ED-truncated NS1 protein, but not its replication capability in human and chicken cells, confirming that the ESEV PL domain may be indispensable for viral replication [34]. However, it was found that the C-terminal ESEV motif contributed little to the virulence of the highly pathogenic H5N1 virus in mice and chickens [35], suggesting a contribution of the ESEV PL motif to pathogenicity in the backbone of the ED-truncated LZ57 virus. Moreover, our results were contrary to those of Jackson in that the C-terminal EPEV motif in our studies did not show any virulence in mice, although higher titers were observed in the lungs and turbinate samples of virus-infected mice [16]. Golebiewski et al. (2011) showed that the non-canonical EPEV PBM may be either unable to bind PDZ proteins or target different PDZ proteins by using binding assays in vitro [33]. Consistently, Wang et al. (2012) also indicated the EPEV virus was only able to replicate in the lungs infected by the classical swine H1N1 influenza virus [36].

Previous studies have found that the avian ESEV virus replicated more efficiently in mouse cells and had increased virulence in mice, whereas the human RSKV virus preferred to replicate in human and duck cells with increased replication in ducks, indicating modulation of the C-terminal domain of NS1 protein in a species-specific manner [17]. Our results showed that the canine ESEI and KPEI viruses conferred stronger replication advantages in human rather than in other cells, whereas the canine SEQK motif, rather than the swine PEQK, could only replicate more efficiently in swine cell lines. The PEQK and SEQK motifs were circulating in the 2009 pandemic and in the classical swine H1N1 virus, respectively, and they both lack any potential C-terminal PDZ domain ligand motifs. This was similar to earlier reports which indicated the PEQK motif had minimal effects on replication, pathogenicity and transmission [36,37]. Although the PEQK and SEQK motifs were avirulent in mice, it was initially reported that the presence of the SEQK motif was able to modulate the production of IFN-β among these rΔNS1 mutant viruses. With respect to the highly pathogenic H5N1 virus, Soubies et al. (2010) pointed out its higher pathogenicity correlated with a higher level of type I IFN induction in the tissues and organs of infected mice and ducks [17]. However, this was contrary for the low pathogenic H1N1 virus in that the SEQK mutant virus was avirulent in mice, but induced high levels of type I IFN induction in A549 cells. Previous observations indicated that there was no correlation between the increased pathogenicity caused by NS1 mutant viruses and inhibition of IFN synthesis during infections [16]. A single mutation in the C-terminal region of the ED was found to have largely conferred the IRF3 and IFN-β transcription phenotypes of the NS1 protein [38]. Whether the substitution of proline (P) at position 81 with serine (S) in this C-terminal motif of H1N1 virus did not block the activation of IRF3 and IFN-β transcription or there is an alternative mechanism involving an interaction with targeted proteins or signals still needs to be further studied.

In addition, it has been found that the NS1 protein of the H5N1 subtype influenza virus has a deletion of amino acids at position 80–84, and it has also been found that the deletion in the junction region could participate in the regulation of viral virulence by affecting the binding affinity of dsRNA or its interaction with host proteins [39,40]. The dimerization and multimerization of the NS1 protein were shown to be essential for its RNA binding activities to functionally replace its C-terminal domain [23]. In our study, the missing amino acids at positions 81–84 were replaced on based on natural truncation, and it was found that these changes could affect the stability and dimerization of the NS1 protein. Therefore, it was likely that this was caused by the difference in the C-terminal amino acids, and further research is needed as to whether this could be related to the deletion of the amino acids at position 80–84. There are several possible explanations for the fact that the KPEI and ESEI motifs modulated the replication and virulence when the ED domain was absent. Firstly, the ESEI and KPEI motifs may play a role in the post-transcriptional and translational regulatory functions in order to compensate for the truncated ED domain. Secondly, the last C-terminal residues had the potential to bind the PDZ domain-containing proteins and the ability to target different host proteins by interactions with the nucleoli and this could have been related to the pathogenicity of the viruses in mice. Finally, there were unsteady-state expression levels of the KPEI and ESEI, which inhibited the induction of IFN-β mRNA and enhanced the viruses’ virulence in mice. There may also be a correlation between the ability of rΔNS1 mutant viruses to inhibit the induction of IFN-β mRNA and their virulence in mice. Further investigations into these hypotheses will still need to be carried out.

Taken together, these results suggested that the C-terminal domains of influenza viruses were largely responsible for their virulence and viral fitness in a host-specific manner in the context of the N-terminal 84 aa-length NS1 protein. Interestingly, the non-PL domain of the ESEI and KPEI motifs could also be pathogenic factors, which conferred replication and fitness advantage, as well as enhance virulence, whereas the typical avian PL motifs (ESEV) only contributed to the high pathogenicity of viruses in mice. The findings in this study revealed a novel mechanism of interspecies adaptation without the presence of the PL domain, and this will provide new insights for the development of antiviral therapy against influenza viruses in the future.

Supplementary Material

Supplementary Figure_Intracellular localization in infected MDCK cells.tif

Table S1_n.doc

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors contribution

Pingping Wang and Jianing Guo: Methodology, validation and formal analysis. Pingping Wang wrote the original paper. Yefan Zhou and Min Zhu: Methodology and investigation. Senbiao Fang and Boyu Pan: Methodology of NS1 protein using MD. Fanyuan Sun, Chongqiang Huang and Yaohui Zhu and Huabo Zhou: Data curation. Yifeng Qin, Kang Ouyang, Zuzhang Wei and Weijian Huang: Conceptualization and supervision. Adolfo García-Sastre: Writing – review & editing. Ying Chen: Conceptualization, resources, writing-review and editing, supervision, project administration and funding acquisition. We are also grateful to Dr Dev Sooranna, Imperial College London, for editing the manuscript. All the authors read and approved the final manuscript.
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