Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity

viruses Article Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity Aitor Nogales 1,* , Laura Villamayor 2, Sergio Utrilla-Trigo 1, Javier Ortego 1, Luis Martinez-Sobrido 3 and Marta L. DeDiego 2,*   Citation: Nogales, A.; Villamayor, L.; Utrilla-Trigo, S.; Ortego, J.; Martinez-Sobrido, L.; DeDiego, M.L. Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity. Viruses 2021,13, 1760. https:// doi.org/10.3390/v13091760 Academic Editor: Feng Li Received: 14 July 2021 Accepted: 31 August 2021 Published: 3 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Center for Animal Health Research, CISA-INIA-CSIC, Valdeolmos, 28130 Madrid, Spain; [email protected] (S.U.-T.); [email protected] (J.O.) 2Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain; [email protected] 3Department of Disease Intervention and Prevention, Texas Biomedical Research Institute, San Antonio, TX 78227, USA; [email protected] *Correspondence: [email protected] (A.N.); [email protected] (M.L.D.) Abstract: Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996–1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence. Keywords: influenza A virus; NS1; PA-X; HPAIV; H5N1; shutoff 1. Introduction Influenza A viruses (IAV) belong to the Orthomyxoviridae family of viruses containing an eight-segmented, single-stranded, negative-sense RNA genome. IAV are divided into subtypes based on the two glycoproteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). To date, 18 different HA subtypes and 11 different NA subtypes (H1 through H18 and N1 through N11, respectively) have been described [1,2]. Although IAV can infect and replicate in multiple mammalian and avian species, the natural host reservoir species are wild aquatic birds [ 2 , 3 ]. Avian influenza virus (AIV) is a type of IAV that is isolated from and adapted to avian host species, and it is classified as low or highly pathogenic (LPAIV and HPAIV, respectively) based on its pathogenicity in domestic chickens. LPAIV strains are associated with mild clinical signs in broilers and reduction in egg production in layers, but promote secondary infections causing an increase in mortality [ 4 , 5 ]. On the other hand, HPAIV strains are responsible for systemic and fatal infections with high mortality rates in poultry [ 4 , 5 ]. HPAIV H5N1 occurs mainly in birds, is highly contagious, and is deadly. The first HPAIV H5N1 was detected in 1996 in geese in China [ 6 ] and continues to spread, posing a major challenge to both animal and human health. In 1997, the first human case of an H5N1 HPAIV was reported in a three-yearold boy in Hong Kong, China [ 7 ]. The same year, a total of 18 human infections with H5N1 HPAIV were reported in Hong Kong, leading to 6 deaths [ 7 ]. These human cases were preceded by outbreaks in poultry, and it is very likely that human infections were Viruses 2021,13, 1760. https://doi.org/10.3390/v13091760 https://www.mdpi.com/journal/viruses Viruses 2021,13, 1760 2 of 25 acquired directly from infected birds, without the involvement of an intermediate host. The outbreak in Hong Kong was efficiently controlled by the slaughter of all poultry in markets and farms in 1997 [ 8 ]. However, H5N1 HPAIV continues to circulate and evolve among poultry in Asia, and zoonotic spillover was again observed in early 2003 [ 9 ]. Since January 2003 until now, the cumulative number of laboratory-confirmed human cases of H5N1 HPAIV infections were 861, including 455 deaths, in 17 different countries (Available online: https://www.who.int/influenza/human_animal_interface/2020_01_ 20_tableH5N1.pdf?ua=1 (accessed on 5 July 2021)), most of these human infections being due to the close contact with infected birds. People infected with H5N1 HPAIV developed severe respiratory disease, with symptoms including fever, cough, shortness of breath, and pneumonia, which frequently progressed to acute respiratory distress syndrome (ARDS) and multiorgan failure [ 10 ]. Systemic spread and cytokine storm [ 11 ] have been described as possible disease-aggravating factors of HPAIV H5N1 infections, but the reasons for their high virulence in humans remains unclear. Although the effective transmission of H5N1 HPAIV from humans to humans has not yet been observed, humans are at risk of an H5N1 HPAIV pandemic. IAV non-structural 1 (NS1) protein is the main transcript of the NS segment and a key virulence factor, playing an important role in counteracting innate immune responses induced by the host through different mechanisms [ 12 , 13 ]. IAV NS1 inhibits activation of innate antiviral responses by sequestering double stranded (ds) RNA, a potential trigger of type I and III interferon (IFN) responses [ 14 ]. IAV NS1 also inhibits activation of IFN signaling through retinoic acid-inducible gene I (RIG-I), an intracellular sensor of virus infection, by binding to RIG-I and preventing its activation [ 15 – 17 ]. Moreover, the NS1 protein of some IAV strains interacts with components of the cellular pre-mRNA processing machinery, including the cleavage and polyadenylation specificity factor 30 (CPSF30), and poly(A)-binding protein II, inhibiting proper 3 0 end processing and blocking the nuclear export of cellular mRNAs, which lead to the inhibition of host gene expression, including IFN and pro-inflammatory responses [ 18 , 19 ]. Some publications have shown the relevance of amino acid residues in IAV NS1 functions and virus pathogenesis. For example, a five amino acid deletion at position 80–84 described in H5N1 IAV isolated from 2000 to 2015 [ 20 ] has been implicated in enhancing virulence in ducks, chickens, and mice [ 21 , 22 ], at least in part by enhancing resistance to tumor necrosis factor (TNF)-induced responses [ 23 ]. Likewise, IAV NS1 mutations L103F and I106M in H5N1 IAV circulating since 1998 are responsible for inhibition of host gene expression by enhancing the interaction of H5N1 IAV NS1 with CPSF30 [ 24 ]. Moreover, these mutations have been shown to enhance viral virulence in mice by increasing the systemic spread of the virus from the lungs, mainly to the brain [25] and by mediating interstitial pneumonia in mice [26]. IAV segment 3 encodes the polymerase acidic (PA) and PA-X proteins [ 27 ]. PA-X is translated as a +1 frameshift open reading frame (ORF) from the PA viral segment [ 27 ]. During translation, the ribosome shifts at a specific sequence in the PA mRNA, a U-rich region followed by a rare codon, which usually promotes ribosomal shifting because they are typically decoded more slowly [ 28 ]. This ribosomal frameshifting results in the expression of PA-X, which shares the same first N-terminal 191 amino acids with PA, including the endonuclease domain, and encodes a unique, short C-terminal sequence [ 27 ]. Most of the human and avian IAV strains contain a 61 amino acid long C-terminal extension (leading to a 252 amino acid PA-X) [ 29 ]. By contrast, some IAV, including the 2009 human pandemic H1N1 (pH1N1), canine, and certain swine IAV, possess a TGG (W) to TAG (stop) mutation at codon 42 in the X ORF, leading to a 232 amino acid long PA-X protein [ 30 ]. IAV PA-X selectively degrades RNAs transcribed by host RNA polymerase II (Pol II) [ 31 ], contributing to host-cell shutoff and inhibition of host antiviral responses [ 27 ]. Moreover, PA-X modulates host inflammation, immune response, apoptosis, cell differentiation, and tissue remodeling [ 27 ]. Interestingly, the role of PA-X in IAV pathogenesis seems to be strain specific [ 12 ]. Loss of PA-X expression increased viral replication, pathogenicity, and host inflammatory response for pH1N1 in mice [ 32 , 33 ] and for H5N1 IAV in mice, Viruses 2021,13, 1760 3 of 25 chickens, and ducks [ 34 ]. In addition, PA mRNA and protein synthesis were upregulated in PA-X-deficient pH1N1 and H5N1 virus-infected cells [ 33 ]. Using viral strains of pH1N1, H5N1 HPAIV, and a H9N2 LPAIV, it has been shown that viruses with full-length PA-X (252 amino acids) replicate more efficiently and were more pathogenic in mice than the corresponding viruses with truncated (232 amino acids) PA-X proteins [30]. Since IAV NS1 and PA-X proteins are virulence factors that modulate host innate immune responses, the interplay between both viral proteins is important for viral replication and pathogenicity. We have shown that regulation of innate immune responses by IAV NS1 and PA-X proteins determines virus fitness and pathogenesis in vitro and in vivo , respectively [ 12 , 35 – 37 ]. In addition, we have shown a functional co-evolution of NS1 and PA-X proteins in pH1N1 [ 36 ]. Here, we evaluated the fitness and pathogenicity of recombinant IAV encoding PA or NS1 genes from old (1996–1997) or recent (since 2016) H5N1 strains. Consistent with our previous findings [ 36 , 37 ], our studies suggest the importance of a co-evolution in the regulation of host gene expression by H5N1 NS1 and PA-X proteins, with effects on viral fitness and pathogenesis. 2. Materials and Methods 2.1. Cell Lines Madin-Darby canine kidney (MDCK, ATCC CCL-34), human lung epithelial carcinoma A549 (ATCC CCL-185), human embryonic kidney (HEK293T, ATCC CRL-11268), and chicken embryo fibroblast DF-1 (ATCC CRL-12203) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc, Manassas, VA, USA) containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37 ◦C in a 5% CO2incubator. 2.2. Analysis of NS1 and PA-X Sequences Human and avian H5N1 PA-X and NS1 sequences available at the Influenza Research Database (https://www.fludb.org/ (accessed on 23 June 2020)) were downloaded, introducing in the search the time periods shown in Tables 1–4, and either human or avian origin. The sequences were aligned using the “analyze sequence variation (SNP)” function available in this webpage. 2.3. Plasmids The NS1 and PA-X genes fused to an N-terminal HA epitope tag were cloned into the pCAGGS HA-NH 2 plasmid [ 38 , 39 ] using SphI and NheI restriction sites. The amino acid sequences were as follows: for PA-X OR , A/Hong Kong/483/1997, H5N1 strain, Pubmed protein ID: AAF74340.1; for PA-X CIR , A/mallard/Italy/3401/2005, H5N1 strain, Pubmed protein ID: AGJ72962.1; for NS1 OR, A/Hong Kong/156/97, H5N1 strain, Pubmed protein ID: O56264.1; and for NS1 CIR, A/duck/Bangladesh/27820/2015, H5N1 strain, Pubmed protein ID: AQY17641.1. To obtain the pDZ-NS CIR and pDZ-NS OR rescue plasmids, the coding regions of NS1 OR and NS1 CIR were cloned into a pDZ plasmid carrying non-overlapping NS1 and NEP genes (pDZ-NSs) using the porcine teschovirus 2A autoproteolytic cleavage site, as previously described [ 40 ]. Rescue plasmids encoding the PA OR and PA CIR proteins were obtained by cloning PA OR and PA CIR viral segments into the pHW2000 plasmid using BsmBI restriction sites. 2.4. Effect of NS1 and PA-X Proteins on Gene Expression HEK293T or DF-1 cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were transiently co-transfected, with 200 ng/well of pCAGGS plasmids encoding either NS1 and/or PA-X OR or CIR proteins fused to an N-terminal HA tag, or transfected with empty plasmid as internal control, together with 20 ng/well of a pCAGGS plasmid encoding Renilla luciferase (Rluc), using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). At 24 h post-transfection (h p.t), cells were lysed using passive lysis buffer (Promega, Madison, WI, USA), and Rluc activity was measured using a Biolux renilla lu- Viruses 2021,13, 1760 4 of 25 ciferase reagent (New England BioLabs, Ipswich, MA, USA) and a Lumicount luminometer (Apliskan, Thermo Scientific, Waltham, MA, USA). The means and standard deviations were calculated and a two-tailed Student ttest was used for statistical analysis using GraphPad Prism software (v8). 2.5. Effect of NS1 and PA-X Proteins on IFN Induction after SeV Infection To evaluate the effect of H5N1 IAV NS1 and PA-X proteins on induction of IFN responses, HEK293T cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were transiently co-transfected with 100 ng/well of pCAGGS plasmids encoding OR or CIR HAtagged NS1 and/or PA-X proteins, or empty plasmid as control, together with 50 ng/well of a plasmid expressing firefly luciferase (Fluc) under the control of the IFN-sensitive response element (ISRE) promoter (pISRE-Fluc) [ 41 ], together with 20 ng/well of a pCAGGS plasmid encoding Rluc, using calcium phosphate (Agilent Technologies, Santa Clara, CA, USA). At 24 h p.t, cells were washed with PBS and infected with a multiplicity of infection (MOI) of 3 with the Sendai virus (SeV), strain Cantell, as previously described [ 41 ]. At 24 h post-infection (h p.i), cells were lysed using passive lysis buffer (Promega). Luciferase expression in the cell lysates was determined using a dual-Glo luciferase kit (Promega) according to the manufacturer’s instructions. Measurements were recorded with a Lumicount luminometer (Apliskan, Thermo Scientific), and the mean values and standard deviations were calculated. Statistical analysis was performed using a two-tailed Student ttest and GraphPad Prism software (v8). 2.6. Analysis of NS1 and PA-X Expression by Western Blot For Western blotting, transfected cells were lysed in buffer containing 100 mM TrisHCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 20% β -mercaptoethanol and boiled for 5 min. Then, proteins in cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Subsequently, membranes were blocked for 1 h in PBS containing 5% dried skim milk and 0.1% Tween-20 and incubated with an anti-HA polyclonal antibody (Sigma, Stockholm, Sweden) or an anti-actin monoclonal antibody (Sigma) at 4 ◦ C overnight. Horseradish peroxidase (HRP) secondary antibodies (Merck life science, Darmstadt, Germany) specific for either mouse or rabbit immunoglobulins (Ig) were used to detect bound primary antibodies. Proteins in the membranes were detected with a SuperSignal West Femto maximum-sensitivity chemiluminescent substrate kit (Thermo Scientific) following the manufacturer’s instructions. 2.7. Generation of Recombinant Viruses Recombinant A/Puerto Rico/8/1934 (PR8) H1N1 viruses were generated as previously described [ 40 , 42 – 44 ]. Briefly, cocultures (1:1) of HEK293T and MDCK cells (6-well plate format, 1 × 10 6 cells/well, triplicates) were transiently co-transfected in suspension using polyethileneimine (PEI; Polysciences, Warrington, PA, USA) with 1 µ g of each of the six ambisense pHW2000 PR8 WT plasmids (pHW-PB2, -PB1, -HA, -NP, -NA, and -M), pDZ-NSs plasmids encoding NS1 OR or NS1 CIR , and the pHW2000 plasmids encoding PA OR or PA CIR [ 36 ]. At 12 h p.t, the medium was replaced with DMEM containing 0.3% bovine serum albumin (BSA), and 0.5 µg/mL of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). At 96 h p.t, tissue culture supernatants were collected and used to infect fresh monolayers of MDCK cells. At 3 days post-infection (dpi), recombinant viruses were plaque purified and virus stocks were prepared in confluent monolayers of MDCK cells. Viral stocks were titrated by immunofocus assay (focus-forming units [FFU]/mL) on MDCK cells [ 45 ]. MDCK cells (96-well plate format, 5 × 10 4 cells/well, triplicates) were infected with 10-fold serial dilutions of the tissue culture supernatants. After 8 h of viral infection at 37 ◦ C, cells were fixed and permeabilized with PBS containing 4% formaldehyde and 0.1% Triton X-100 for 20 min at room temperature. Then, cells were incubated in a blocking solution (PBS with 2.5% BSA) for 1 h at room temperature and subsequently incubated with the anti-NP monoclonal antibody (MAb) HB-65 (ATCC Viruses 2021,13, 1760 5 of 25 H16-L10-4R5) for 2 h at 37 ◦ C and washed with PBS. Cells were then incubated at 37 ◦ C for 1 h with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG secondary antibody (Life Technologies; Carlsbad, CA, USA). Viral titers (FFU/mL) were quantified by counting all the cells that were positive for NP expression in the triplicate wells, and the means and standard deviations were calculated as previously described [ 45 ]. Experiments involving infectious viruses were performed at the biosafety level 3 (BSL3) and animal (ABSL3) facilities in the Animal Health Research Center (INIA-CISA), Madrid (Spain). 2.8. Virus Growth Kinetics Confluent monolayers of canine MDCK, human A549 or avian DF-1 cells (4 × 10 5 cells/well, 12-well plates, triplicates) were infected (MOI of 0.001 for MDCK cells; MOI 0.01 for A549 and DF-1 cells), for 1 h at room temperature, and incubated in DMEM media supplemented with 0.3% BSA and 1 (MDCK) or 0.5 (A549 and DF-1) µ g/mL TPCK-treated trypsin, at 37 ◦ C. Tissue culture supernatants collected at 24, 48, and 72 h p.i were titrated on MDCK cells (96-well plates, 5 × 10 4 cells/well, triplicates) by immunofluorescence (FFU/mL), as described above. The means and standard deviations were calculated, and statistically significant differences were determined using a two-tailed Student ttest and GraphPad Prism software (v8). 2.9. Pathogenesis of Recombinant Viruses in Mice Seven-to-nine-week-old wild-type (WT) A129 mice were bred in the animal care facility of the Department of Animal reproduction at INIA and housed under pathogenfree conditions at the biosafety level 3 (BSL3) animal facilities in the Animal Health Research Center (INIA-CISA), Madrid (Spain). Animal experimental protocols were approved by the Ethical Review Committee at the INIA-CISA and Comunidad de Madrid (Permit number: PROEX 116/19), in strict accordance with EU guidelines 2010/63/UE about the protection of animals used for experimentation and the Spanish Animal Welfare Act 32/2007. For viral inoculations, mice were first anesthetized intraperitoneally (i.p.) with 100 mg/kg of body weight of ketamine (La Casa del Campo, Cádiz, Spain) and 5/mg/kg of xylacin (La Casa del Campo) and then infected intranasally (i.n.) with 30 µ L of the indicated recombinant viruses. Mice (N= 5/group) were examined each day for weight loss, clinical symptoms (such as malaise, respiratory distress, and lack of movement), and mortality. Percent body weight loss was determined relative to the starting weight. Mice losing more than 25% of their initial body weight were considered to have reached the experimental endpoint and were humanely euthanized. In another set of experiments, mice (N= 4/group) were infected as described above and, at 2 and 4 dpi, animals were euthanized and lungs were extracted. The right and left lobules were used for analyzing viral titers or for extracting the RNA, respectively. For viral titers, the right lobules were transferred to tubes containing 1 mL of DMEM and one 5 mm stainless steel bead (Qiagen, Hilden, Germany) and homogenized using the TissueLyser II (Qiagen) at 25 Hz for 5 min, followed by centrifugation at 5000 × gfor 10 min at 4 ◦ C. Viral titers were determined by immunofluorescence assay (FFU/mL) as outlined above. The left lobules were collected and submerged into RNAlater (Thermo Fisher Scientific), incubated overnight at 4 ◦ C and then stored at − 80 ◦ C until processing. For RNA extraction, tissue samples were transferred to tubes containing one 5 mm stainless steel bead (Qiagen) and 1 mL of RLT buffer (Qiagen) with β -mercaptoethanol. The samples were homogenized using the TissueLyser II (Qiagen) at 25 Hz for 5 min, followed by centrifugation at 5000 × gfor 10 min at 4 ◦ C. Then, total RNAs were purified using an RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Reverse transcriptase (RT) reactions were conducted with a high-capacity cDNA transcription kit (Applied Biosystems, Waltham, MA, USA) at 37 ◦ C for 2 h. Quantitative PCRs (qPCRs) were performed using TaqMan gene expression assays (Applied Biosystems) specific for the mRNA of chemokine (C-C) motif ligand 2 (CCL2, Mm00441242_m1), TNF (Mm00443258_m1), and IFN-induced protein with tetratricopeptide repeats 2 (IFIT2, Mm00492606_m1). The 2 −∆∆CT method was Viruses 2021,13, 1760 6 of 25 used for quantification, and values were presented as fold induction [ 46 ]. For virus titers and qPCR results, one-way analysis of variance (ANOVA) was performed using GraphPad Prism software (v8). 3. Results 3.1. Identification and Selection of Amino Acid Changes in H5N1 IAV NS1 and PA-X Proteins We previously showed that the pH1N1 IAV that emerged in humans in 2009, has evolved to incorporate amino acid changes in the NS1 and PA-X proteins leading to increased and decreased shutoff activity, respectively [ 36 , 42 ]. To analyze whether the NS1 and PA-X proteins of other IAV subtypes also evolve over time, the sequences of NS1 and PA-X proteins of H5N1 IAV circulating in avian and human hosts during 1996–1997 (named as OR, from original), when the first avian and human cases were reported, were compared to the NS1 and PA-X proteins of H5N1 IAV circulating since 2016 (named as CIR, from circulating). No changes were observed in the PA frameshift sequence between OR and CIR H5N1 strains. However, eight amino acid changes (T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q) were observed in the most frequent sequences of PA-X (Figure 1A), whereas 18 amino acid changes (R55E, E60A, H63Q, P87S, E92D, L103F, I106M, T112A, R118K, D127N, N139D, A143T, D152E, E171D, T202A, S212P, E223A, and P228S) were identified in the most frequent sequences of NS1 (Figure 1B). In addition, a 5 amino acid deletion, between amino acids 80 and 84, leading to a 225 amino acid long NS1 protein, was observed in most of the viruses isolated from 2001 to 2015. However, since, in this work, we studied the consensus sequence from NS1 proteins from H5N1 viruses isolated in 1996–1997 and since 2016, both proteins used in this study were 230 amino acid in length (Figure 1B). We analyzed the prevalence of these mutations over time, showing the percentage of sequences encoding the different amino acids at each particular position in H5N1 PAX (Tables 1and 2) and NS1 (Tables 3and 4) proteins. For PA-X, amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, and R250Q, were selected rapidly in avian viruses ( Table 1 ), since more than 90% of the sequences isolated during 1998–2000 contain the amino acids encoded by the viruses preferentially circulating nowadays. However, although the amino acid changes T20A and G209E were selected quickly in avian viruses, a mixture of isolates containing T and A at position 20, and G and E residues at position 209 was present in the period of 2016–2020 (Table 1). The amino acid change L221R was selected later, with most of the H5N1 IAV isolates from 2001 to 2005 encoding the residue circulating nowadays (Table 1). Notably, H5N1 IAV containing the residues most common in avian isolates were also the ones most frequently infecting humans (Table 2), as could be expected taking into account that most of the infections in humans occur in subjects in close contact with infected avian species [ 47 ]. In H5N1 IAV isolated from humans, PA-X residues T20, A85, T118, G209, V212, L221, and Q250 were present in viruses infecting humans during 1996–1997. The residues I and V at position 127 were present at 42 and 58% of the H5N1 viruses infecting humans during 1996–1997. However, in recent years, the most frequent residues were T85, I118, V127, E209, A212, R221, and Q250, and a mixture of A and T at amino acid 20 (Table 2). These data suggest that most of the changes in the PA-X protein of H5N1 viruses are likely beneficial as they became fixed at the global level. However, whereas the amino acid changes G209E, V212A, L221R, and R250Q, encode silent mutations to the PA protein, the amino acid changes T20A, A85T, T118I, and I127V also affect the PA sequence, making difficult a priori to discern whether these changes were selected because they are beneficial for the PA-X and/or for the PA protein. Viruses 2021,13, 1760 7 of 25 Viruses 2021, 13, x FOR PEER REVIEW 7 of 26 level. However, whereas the amino acid changes G209E, V212A, L221R, and R250Q, encode silent mutations to the PA protein, the amino acid changes T20A, A85T, T118I, and I127V also affect the PA sequence, making difficult a priori to discern whether these changes were selected because they are beneficial for the PA-X and/or for the PA protein. Figure 1. Effect of H5N1 NS1 and PA-X amino acid changes on host gene expression. (A,B) Schematic representation of PA-X (A) and NS1 (B) amino acid residues most frequently present in H5N1 IAV circulating in avian and humans during 1996–1997 (OR, original), and from viruses circulating since 2016 (CIR, circulating). The PA-X +1 frameshift motif (UCC UUU CGU C) is indicated with a striped bar (A). (C–E) Human HEK293T (C,E) and avian DF-1 (D) cells were transiently co-transfected with a pCAGGS empty plasmid, or with pCAGGS plasmids expressing the indicated HA epitope-tagged NS1 and PA-X viral proteins, together with a Rluc expressing pCAGGS plasmid. NS1CIR protein encodes the amino acid changes R55E, E60A, H63Q, P87S, E92D, L103F, I106M, T112A, R118K, D127N, N139D, A143T, D152E, E171D, T202A, S212P, E223A, and P228S; whereas the PA-XCIR protein encodes the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q. At 24 h p.t, Rluc expression (C and D) was quantified using a luminometer. Error bars represent the standard deviations from triplicates. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001 (NS1OR versus NS1CIR, PAXOR versus PA-XCIR, NS1OR/PA-XOR versus NS1OR/PA-XCIR, NS1OR/PA-XOR versus and NS1CIR/PA-XOR and NS1OR/PA-XOR versus NS1CIR/PA-XCIR) using a Student’s t-test. R.u., relative units. NS1, PA-X, and actin protein expression levels in HEK293T transfected cells were analyzed by Western blot using cells extracts and antibodies specific for the HA tag, to Figure 1. Effect of H5N1 NS1 and PA-X amino acid changes on host gene expression. ( A , B ) Schematic representation of PA-X ( A ) and NS1 ( B ) amino acid residues most frequently present in H5N1 IAV circulating in avian and humans during 1996–1997 (OR, original), and from viruses circulating since 2016 (CIR, circulating). The PA-X +1 frameshift motif (UCC UUU CGU C) is indicated with a striped bar ( A ). ( C – E ) Human HEK293T ( C , E ) and avian DF-1 ( D ) cells were transiently co-transfected with a pCAGGS empty plasmid, or with pCAGGS plasmids expressing the indicated HA epitope-tagged NS1 and PA-X viral proteins, together with a Rluc expressing pCAGGS plasmid. NS1 CIR protein encodes the amino acid changes R55E, E60A, H63Q, P87S, E92D, L103F, I106M, T112A, R118K, D127N, N139D, A143T, D152E, E171D, T202A, S212P, E223A, and P228S; whereas the PA-X CIR protein encodes the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q. At 24 h p.t, Rluc expression ( C and D ) was quantified using a luminometer. Error bars represent the standard deviations from triplicates. *, p< 0.05; **, p< 0.005; ***, p< 0.0005; ****, p< 0.0001 (NS1 OR versus NS1 CIR , PA-X OR versus PA-X CIR , NS1 OR /PA-X OR versus NS1 OR /PA-X CIR , NS1 OR /PA-X OR versus and NS1 CIR /PA-X OR and NS1 OR /PA-X OR versus NS1 CIR /PA-X CIR ) using a Student’s t-test. R.u., relative units. NS1, PA-X, and actin protein expression levels in HEK293T transfected cells were analyzed by Western blot using cells extracts and antibodies specific for the HA tag, to detect NS1 and PA-X proteins (bottom and top bands in the upper blot, respectively); and actin (bottom blot) ( E ). Molecular mass markers (in kDa) are indicated on the left. The experiments were repeated three times, with similar results. Viruses 2021,13, 1760 8 of 25 For the NS1 protein, most of the amino acid changes were selected quickly in avian H5N1 viruses; however, interestingly, there was a high variability in the amino acids identified in these H5N1 IAV during the time frames analyzed (Table 3). There are some amino acid positions (e.g., 112, 127, 171, and 212) in which different residues were selected over time (Table 3). Furthermore, we noted that there was an amino acid change at residues 92 and 139 (E92D and N139D, respectively) in H5N1 viruses isolated from 1998 to 2015 in avian hosts. However, since 2016, an important variability was observed in these residues (Table 3). Sequences obtained from H5N1 viruses isolated in humans showed less variability (Table 4). Nevertheless, we observed that different residues were selected over time at positions 112, 127, 143, and 212 (Table 4). This analysis suggests a high complexity in the evolution of these virulence factors, which is most clear in H5N1 viruses isolated from avian species, the natural host. Importantly, since avian hosts include multiple species of wild birds, or poultry, the observed variability could be linked to differences between hosts and/or their innate immune systems. For that reason, evaluating the functional consequences of the observed changes is highly important. Table 1. Amino acid changes in PA-X proteins from H5N1 IAV isolated in avian. Position Amino Acid 1996–1997 1998–2000 2001–2005 2006–2010 2011–2015 2016–2020 20 T 40.7 0 0.93 1.59 70.77 31.86 A 59.3 100 98.75 97.97 29.09 68.13 Other 0 0 0.31 0.42 0.13 0 85 A 40.74 0 0.61 0.62 1.85 1.44 T 59.3 100 99.23 99.26 98.14 98.56 Other 0 0 0.15 0.1 0 0 118 T 42.86 0 0.77 0 0 23.7 I 57.14 100 99.22 99.68 93.84 70.19 Other 0 0 0 0.31 6.15 6.73 127 I 25 0 0.46 0.42 0.26 0 V 75 100 99.38 99.37 99.73 98.56 Other 0 0 0.62 0.21 0 1.44 209 G 46.42 0 0.92 0.42 1.04 24.88 E 53.57 100 98.92 99.48 98.29 75.11 Other 0 0 0.15 0.1 0.66 0 212 V 39.28 0 1.4 2.6 1.44 0 A 57.15 92 96.9 97.29 98.16 99.04 Other 3.57 8 1.7 0.1 0.39 0.95 221 L 50 68 1.69 0.21 0.26 0.95 R 50 32 97.69 99.58 99.6 98.08 Other 0 0 0.62 0.21 0.13 0.95 250 R 46.43 4 6.33 0.52 1.7 1.91 Q 46.43 96 92.73 98.84 97.37 95.21 Other 7.14 0 0.93 0.63 0.92 2.87 Frequencies of identified mutations in H5N1 IAV PA-X proteins isolated from avian at the indicated time periods. Publicly available sequences in the Influenza Research Database (https://www.fludb.org/brc/home. spg?decorator=influenza (accessed on 23 June 2020)) were downloaded and the frequencies represented according to the year of isolation. The original amino acid present in 1996–1997 H5N1 IAV isolates is depicted first, whereas the amino acid most frequently found nowadays is depicted second. The numbers of sequences available at the Influenza Research Database are 27, 25, 644, 939, 739, and 204 sequences, for periods 1996–1997, 1998–2000, 2001–2005, 2006—2010, 2011–2015, and 2016–2020, respectively. Viruses 2021,13, 1760 9 of 25 Table 2. Amino acid changes in PA-X proteins from H5N1 IAV isolated in humans. Position Amino Acid 1996–1997 1998–2000 2001–2005 2006–2010 2011–2015 2016–2020 20 T 100 ND 0 1.35 50 ND A 0 ND 100 98.65 50 ND 85 A 100 ND 0 2.7 0 ND T 0 ND 98.71 97.3 100 ND Other 0 ND 1.28 0 0 ND 118 T 100 ND 0 0 0 ND I 0 ND 100 100 100 ND 127 I 42.1 ND 0 0 0 ND V 57.89 ND 100 100 100 ND Other 0 ND 0 1.34 0 ND 209 G 100 ND 1.26 2 0 ND E 0 ND 98.73 98 100 ND 212 V 100 ND 1.26 0.67 0 ND A 0 ND 98.73 99.32 100 ND 221 L 100 ND 0 0 0 ND R 0 ND 98.73 100 100 ND Other 0 ND 1.26 0 0 ND 250 R 0 ND 1.26 0 3.57 ND Q 100 ND 98.73 100 96.43 ND Frequencies of identified mutations in H5N1 IAV PA-X proteins isolated from humans at the indicated time periods. Publicly available sequences in the Influenza Research Database (https://www.fludb.org/brc/home. spg?decorator=influenza (accessed on 23 June 2020)) were downloaded and the frequencies represented according to the year of isolation. The original amino acid present in 1996–1997 H5N1 IAV isolates is depicted first, whereas the amino acid most frequently found nowadays is depicted second. The numbers of sequences available at the Influenza Research Database are 19, 0, 77, 148, 28, and 0 sequences for periods 1996–1997, 1998–2000, 2001–2005, 2006–2010, 2011–2015, and 2016–2020, respectively. Table 3. Amino acid changes in NS1 proteins from H5N1 IAV isolated in avian. Position Amino Acid 1996–1997 1998–2000 2001–2005 2006–2010 2011–2015 2016–2020 55 R 75 65.38 1.5 0.84 0.32 3.04 E 25 34.62 96.98 94.1 87.93 96.34 Other 0 0 1.5 5.67 11.74 1.83 60 E 70 65.38 1.5 2.22 0.54 3 A 30 34.62 9.39 97.21 80.54 87.95 Other 0 0 0.1 0.55 18.91 9 63 H 60 3.84 1.07 0.18 0 2.41 Q 25 30.77 98.06 99.81 98.91 94.57 Other 15 65.38 0.86 0 1.08 3.01 87 P 75 65.38 3.11 0.83 2.17 4.89 S 25 34.61 93.23 98.98 97.5 96.34 Other 0 0 3.65 0.18 0.32 0 92 E 55 0 0.43 0.092 44.45 37.35 D 45 100 99.46 99.91 55.21 62.65 Other 0 0 0.1 0 0.32 0 103 L 60 3.8 1.18 0.092 0 2.4 F 25 26.92 97.42 97.4 99.78 94.57 Other 15 69.23 1.39 2.5 0.21 3 106 I 60 11.53 1.29 0.28 0 0 M 40 84.61 98.71 99.72 100 100 Other 0 3.8 0 0 0 0 Viruses 2021,13, 1760 16 of 25 Viruses 2021, 13, x FOR PEER REVIEW 16 of 26 Figure 4. Effect of H5N1 NS1 and PA-X amino acid changes on IFN responses. (A,B) Human HEK293T cells were transiently co-transfected with a pCAGGS empty plasmid or with pCAGGS plasmids expressing the indicated HA epitopetagged PA-X variants, together with a plasmid expressing Fluc under an ISRE dependent promoter, and a pCAGGS plasmid constitutively expressing Rluc. PA-XCIR protein encodes the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q. PAOR-XCIR encodes the amino acid changes G209E, V212A, L221R, and R250Q. PACIR-XOR encodes the amino acid changes T20A, A85T, T118I, and I127V. At 24 h.p.t, cells were infected with SeV (Cantell strain, MOI 3) or left mock-infected (M), and 20 h later expression of Fluc (A) and Rluc (B) was analyzed by luminescence. Data show the mean and standard deviations from cells in triplicate. ns, non-significant; *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001 using a Student’s t-test. Experiments were repeated 3 times in triplicate, with similar results. R.u., relative units. 3.5. Effect of H5N1 NS1 and PA-X Mutations on Viral Growth To analyze whether amino acid changes in H5N1 NS1 and PA-X modulating host shutoff and IFN responses affect viral replication, we generated four recombinant viruses: PAOR/NS1OR, PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR. Since IAV NS1 open reading frame (ORF) partially overlaps with the nuclear export protein (NEP), we used an NS segment encoding non-overlapping NS1 and NEP ORFs [36,42–44]. All the recombinant viruses encoded the PR8 segments with the exception of NS1, which were from H5N1 IAV circulating in 1996–1997 (NS1OR) or since 2016 (NS1CIR); and the H5N1 PA viral segment, which was from H5N1 viruses circulating during 1996–1997 (PAOR), or containing the PAX mutations of H5N1 viruses circulating since 2016 (PACIR). Consequently, for the PA segment, the PA-XCIR protein encoded the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q, and the PACIR encoded the amino acid substitutions T20A, A85T, T118I, and I127V, which affect both the PA and PA-X proteins. To assess whether the mutations incorporated in the H5N1 NS1 and PA-X proteins affect virus growth, canine MDCK, human A549, or avian DF-1 cells were infected at low MOI (0.001 for MDCK and 0.01 for A549 and DF-1 cells), and virus titers in tissue culture supernatants were measured at 24, 48, and 72 h p.i. All viruses grew similarly in the three cell lines and no significant differences were observed (Figure 5), although viruses containing PAOR (PAOR/NS1OR and PAOR/NS1CIR) grew to slightly higher titers compared with viruses encoding the PACIR at 24 and 48 h p.i. (Figure 5). Figure 4. Effect of H5N1 NS1 and PA-X amino acid changes on IFN responses. ( A , B ) Human HEK293T cells were transiently co-transfected with a pCAGGS empty plasmid or with pCAGGS plasmids expressing the indicated HA epitope-tagged PA-X variants, together with a plasmid expressing Fluc under an ISRE dependent promoter, and a pCAGGS plasmid constitutively expressing Rluc. PA-X CIR protein encodes the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q. PA OR -X CIR encodes the amino acid changes G209E, V212A, L221R, and R250Q. PA CIR -X OR encodes the amino acid changes T20A, A85T, T118I, and I127V. At 24 h.p.t, cells were infected with SeV (Cantell strain, MOI 3) or left mock-infected (M), and 20 h later expression of Fluc ( A ) and Rluc ( B ) was analyzed by luminescence. Data show the mean and standard deviations from cells in triplicate. ns, non-significant; *, p< 0.05; **, p< 0.005; ***, p< 0.0005; ****, p< 0.0001 using a Student’s t-test. Experiments were repeated 3 times in triplicate, with similar results. R.u., relative units. 3.5. Effect of H5N1 NS1 and PA-X Mutations on Viral Growth To analyze whether amino acid changes in H5N1 NS1 and PA-X modulating host shutoff and IFN responses affect viral replication, we generated four recombinant viruses: PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR . Since IAV NS1 open reading frame (ORF) partially overlaps with the nuclear export protein (NEP), we used an NS segment encoding non-overlapping NS1 and NEP ORFs [ 36 , 42 – 44 ]. All the recombinant viruses encoded the PR8 segments with the exception of NS1, which were from H5N1 IAV circulating in 1996–1997 (NS1 OR ) or since 2016 (NS1 CIR ); and the H5N1 PA viral segment, which was from H5N1 viruses circulating during 1996–1997 (PA OR ), or containing the PA-X mutations of H5N1 viruses circulating since 2016 (PA CIR ). Consequently, for the PA segment, the PA-X CIR protein encoded the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q, and the PA CIR encoded the amino acid substitutions T20A, A85T, T118I, and I127V, which affect both the PA and PA-X proteins. To assess whether the mutations incorporated in the H5N1 NS1 and PA-X proteins affect virus growth, canine MDCK, human A549, or avian DF-1 cells were infected at low MOI (0.001 for MDCK and 0.01 for A549 and DF-1 cells), and virus titers in tissue culture supernatants were measured at 24, 48, and 72 h p.i. All viruses grew similarly in the three cell lines and no significant differences were observed (Figure 5), although viruses containing PA OR (PA OR /NS1 OR and PA OR /NS1 CIR ) grew to slightly higher titers compared with viruses encoding the PACIR at 24 and 48 h p.i. (Figure 5). Viruses 2021,13, 1760 17 of 25 Viruses 2021, 13, x FOR PEER REVIEW 17 of 26 Figure 5. Growth kinetics of recombinant viruses containing amino acid changes in H5N1 IAV NS1 and PA proteins: MDCK (A), A549 (B), or DF-1 (C) cells were infected (MOI of 0.001 for MDCK or 0.01 for A549 and DF-1) in triplicates with the recombinant viruses encoding the indicated H5N1 NS1 and PA proteins (PAOR/NS1OR, PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR). Virus titers in cell culture supernatants were determined at the indicated h p.i. by immunofocus assay (FFU/mL). No statistically significant differences among PAOR/NS1OR vs. PAOR/NS1CIR, PACIR/NS1OR, or PACIR/NS1CIR, using 2-way ANOVA (p < 0.001), were found. 3.6. Effect of H5N1 NS1 and PA-X Mutations on Viral Pathogenesis in Vivo To analyze the effect of H5N1 NS1 and PA-X amino acid changes in viral pathogenesis and on the induction of innate immune responses, A129 mice were infected with 1000 and 10,000 FFU of the different viruses per mice. First, we evaluated weight loss and mortality during 14 days (Figure 6). Mice infected with 1000 FFU of the viruses encoding H5N1 NS1CIR and either PAOR or PACIR (PAOR/NS1CIR and PACIR/NS1CIR) did not significantly lose weight (Figure 6A), and none of them succumbed to viral infection (Figure 6B). However, mice infected with the viruses encoding H5N1 NS1OR and either PAOR or PACIR (PAOR/NS1OR and PACIR/NS1OR) started losing weight at day 6 p.i., and all of them succumbed to viral infection by days 10 or 11, respectively (Figure 6A,B, respectively). All the mice infected with 10,000 FFU, irrespective of the recombinant virus used in the infections, lost weight (Figure 6A) and all of them died from viral infection (Figure 6B). These data suggested that viruses encoding H5N1 NS1OR (PAOR/NS1OR and PACIR/NS1OR) were more virulent than viruses encoding H5N1 NS1CIR (PAOR/NS1CIR and PACIR/NS1CIR) and that the presence of H5N1 PAOR or PACIR did not significantly affect viral pathogenesis, at least in the A129 mouse model of infection, and with experimental conditions used in these studies. Figure 5. Growth kinetics of recombinant viruses containing amino acid changes in H5N1 IAV NS1 and PA proteins: MDCK ( A ), A549 ( B ), or DF-1 ( C ) cells were infected (MOI of 0.001 for MDCK or 0.01 for A549 and DF-1) in triplicates with the recombinant viruses encoding the indicated H5N1 NS1 and PA proteins (PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ). Virus titers in cell culture supernatants were determined at the indicated h p.i. by immunofocus assay (FFU/mL). No statistically significant differences among PA OR /NS1 OR vs. PA OR /NS1 CIR , PA CIR /NS1 OR , or PA CIR /NS1 CIR , using 2-way ANOVA (p< 0.001), were found. 3.6. Effect of H5N1 NS1 and PA-X Mutations on Viral Pathogenesis In Vivo To analyze the effect of H5N1 NS1 and PA-X amino acid changes in viral pathogenesis and on the induction of innate immune responses, A129 mice were infected with 1000 and 10,000 FFU of the different viruses per mice. First, we evaluated weight loss and mortality during 14 days (Figure 6). Mice infected with 1000 FFU of the viruses encoding H5N1 NS1 CIR and either PA OR or PA CIR (PA OR /NS1 CIR and PA CIR /NS1 CIR ) did not significantly lose weight (Figure 6A), and none of them succumbed to viral infection (Figure 6B). However, mice infected with the viruses encoding H5N1 NS1 OR and either PA OR or PA CIR (PA OR /NS1 OR and PA CIR /NS1 OR ) started losing weight at day 6 p.i., and all of them succumbed to viral infection by days 10 or 11, respectively (Figure 6A,B, respectively). All the mice infected with 10,000 FFU, irrespective of the recombinant virus used in the infections, lost weight (Figure 6A) and all of them died from viral infection (Figure 6B). These data suggested that viruses encoding H5N1 NS1 OR (PA OR /NS1 OR and PA CIR /NS1 OR ) were more virulent than viruses encoding H5N1 NS1 CIR (PA OR /NS1 CIR and PA CIR /NS1 CIR ) and that the presence of H5N1 PA OR or PA CIR did not significantly affect viral pathogenesis, at least in the A129 mouse model of infection, and with experimental conditions used in these studies. Viruses 2021,13, 1760 18 of 25 Viruses 2021, 13, x FOR PEER REVIEW 18 of 26 Figure 6. Virulence of recombinant viruses containing amino acid changes in H5N1 NS1 and PA proteins: Groups of 7-to8-week-old A129 female mice (N = 5/group) were infected with 1000 (103; A) or 10,000 (104; B) FFU/mouse of the recombinant viruses encoding the indicated H5N1 NS1 and PA proteins (PAOR/NS1OR, PAOR/NS1CIR, PACIR/NS1OR, PACIR/NS1CIR). Weight loss (left panels) and survival (right panels) were evaluated daily for 2 weeks. To study whether the higher virulence of viruses encoding H5N1 NS1OR (PAOR/NS1OR and PACIR/NS1OR) correlated with higher virus titers in the lungs of infected animals, another set of A129 mice was similarly infected with 1000 and 10,000 FFU/mice, and viral titers were analyzed at days 2 and 4 after infection (Figure 7). Viral titers in the lungs of animals inoculated with 1000 or 10,000 FFU were similar for the four viruses at day 2 p.i. Interestingly, viral titers at day 4 p.i were slightly higher (between 4to 8-fold) for viruses encoding H5N1 NS1OR (PAOR/NS1OR and PACIR/NS1OR) than viruses encoding H5N1 NS1CIR (PAOR/NS1CIR and PACIR/NS1CIR) for both viral doses (Figure 7A,B). These data correlate with the higher virulence observed for H5N1 NS1OR-encoding viruses (PAOR/NS1OR and PACIR/NS1OR) (Figure 6). Figure 6. Virulence of recombinant viruses containing amino acid changes in H5N1 NS1 and PA proteins: Groups of 7-to-8-week-old A129 female mice (N= 5/group) were infected with 1000 (10 3 ; A ) or 10,000 (10 4 ; B ) FFU/mouse of the recombinant viruses encoding the indicated H5N1 NS1 and PA proteins (PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , PACIR/NS1CIR). Weight loss (left panels) and survival (right panels) were evaluated daily for 2 weeks. To study whether the higher virulence of viruses encoding H5N1 NS1 OR (PA OR /NS1 OR and PA CIR /NS1 OR ) correlated with higher virus titers in the lungs of infected animals, another set of A129 mice was similarly infected with 1000 and 10,000 FFU/mice, and viral titers were analyzed at days 2 and 4 after infection (Figure 7). Viral titers in the lungs of animals inoculated with 1000 or 10,000 FFU were similar for the four viruses at day 2 p.i . Interestingly, viral titers at day 4 p.i were slightly higher (between 4to 8-fold) for viruses encoding H5N1 NS1 OR (PA OR /NS1 OR and PA CIR /NS1 OR ) than viruses encoding H5N1 NS1 CIR (PA OR /NS1 CIR and PA CIR /NS1 CIR ) for both viral doses (Figure 7A,B). These data correlate with the higher virulence observed for H5N1 NS1 OR -encoding viruses (PAOR/NS1OR and PACIR/NS1OR) (Figure 6). Viruses 2021,13, 1760 19 of 25 Viruses 2021, 13, x FOR PEER REVIEW 19 of 26 Figure 7. Viral titers of recombinant viruses containing amino acid changes in H5N1 NS1 and PA proteins: Groups of 7to-8-week-old A129 male mice (N = 8/group) were infected with 1000 (103; A) or 10,000 (104; B) FFU/mouse of the indicated viruses (PAOR/NS1OR, PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR). Mice were sacrificed at 2 (N = 4) and 4 (N = 4) d p.i, and right lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/mL. *, p < 0.05 (PAOR/NS1OR vs. PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR) using 2-way ANOVA (N = 4 per time point). Nonsignificant when p > 0.05. It has been shown that increased levels of pro-inflammatory cytokine induction correlate with higher virulence of H5N1 IAV [11]. To analyze whether the higher virulence of H5N1 NS1OR-encoding viruses (PAOR/NS1OR and PACIR/NS1OR) correlated with higher induction of pro-inflammatory cytokines (TNF, CCL2) or interferon-stimulated genes (ISGs, IFIT2), and whether the induction of these genes correlated with the shutoff activity of NS1 and PA-X, mRNA levels in the lungs of mice infected with 1000 and 10,000 FFU/mice were measured by RT-qPCR at 2 and 4 d p.i (Figure 8A,B). Expression of TNF, CCL2, and IFIT2 was induced in the lungs of infected compared to mock-infected control lungs, with the exception of mice infected with 1000 FFU at 4 d p.i. Interestingly, at 2 d p.i, expression of these cytokines was higher in the lungs of A129 mice infected with the highest dose of viruses encoding H5N1 NS1OR, than those mice infected with H5N1 NS1CIR-encoding viruses (Figure 8A,B). Moreover, expression of TNF and IFIT2 at 4 d p.i in mice infected with 10,000 FFU and expression of CCL2 and IFIT2 at 2 d p.i in mice infected with 1000 FFU, were higher in A129 mice infected with H5N1 PAOR and NS1OR (PAOR/NS1OR) and lower in A129 mice infected with H5N1 PACIR and NS1CIR (PACIR/NS1CIR) viruses. These data suggest that H5N1 NS1CIR, and, to a lower extent, PA-XCIR, contribute to decrease inflammatory responses after virus infection, likely due to the increased inhibition of host gene expression mediated by H5N1 NS1CIR and PA-XCIR compared with NS1OR and PA-XOR, respectively. Figure 7. Viral titers of recombinant viruses containing amino acid changes in H5N1 NS1 and PA proteins: Groups of 7-to-8-week-old A129 male mice (N= 8/group) were infected with 1000 (10 3 ; A ) or 10,000 (10 4 ; B ) FFU/mouse of the indicated viruses (PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ). Mice were sacrificed at 2 (N= 4) and 4 (N= 4) d p.i, and right lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/mL. *, p< 0.05 (PA OR /NS1 OR vs. PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ) using 2-way ANOVA (N= 4 per time point). Non-significant when p> 0.05. It has been shown that increased levels of pro-inflammatory cytokine induction correlate with higher virulence of H5N1 IAV [ 11 ]. To analyze whether the higher virulence of H5N1 NS1 OR -encoding viruses (PA OR /NS1 OR and PA CIR /NS1 OR ) correlated with higher induction of pro-inflammatory cytokines (TNF, CCL2) or interferon-stimulated genes (ISGs, IFIT2), and whether the induction of these genes correlated with the shutoff activity of NS1 and PA-X, mRNA levels in the lungs of mice infected with 1000 and 10,000 FFU/mice were measured by RT-qPCR at 2 and 4 d p.i (Figure 8A,B). Expression of TNF, CCL2, and IFIT2 was induced in the lungs of infected compared to mock-infected control lungs, with the exception of mice infected with 1000 FFU at 4 d p.i. Interestingly, at 2 d p.i, expression of these cytokines was higher in the lungs of A129 mice infected with the highest dose of viruses encoding H5N1 NS1 OR , than those mice infected with H5N1 NS1 CIR -encoding viruses ( Figure 8A,B ). Moreover, expression of TNF and IFIT2 at 4 d p.i in mice infected with 10,000 FFU and expression of CCL2 and IFIT2 at 2 d p.i in mice infected with 1000 FFU , were higher in A129 mice infected with H5N1 PA OR and NS1 OR (PA OR /NS1 OR ) and lower in A129 mice infected with H5N1 PA CIR and NS1 CIR (PA CIR /NS1 CIR ) viruses. These data suggest that H5N1 NS1 CIR , and, to a lower extent, PA-X CIR , contribute to decrease inflammatory responses after virus infection, likely due to the increased inhibition of host gene expression mediated by H5N1 NS1 CIR and PA-X CIR compared with NS1 OR and PA-XOR, respectively. Viruses 2021,13, 1760 20 of 25 Viruses 2021, 13, x FOR PEER REVIEW 20 of 26 Figure 8. Induction of innate immune responses in vivo by recombinant viruses encoding H5N1 NS1 and/or PA variants: Groups of 7-to-8-week-old A129 male mice (N = 8/group) were infected with 1000 (103; A) or 10,000 (104; B) FFU/mouse of the indicated viruses (PAOR/NS1OR, PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR) or left mock-infected (N = 4). Mice were sacrificed at 2 (N= 4) and 4 (N= 4) d p.i. Left lungs were collected and total RNA was extracted to quantify the levels of TNF (left panels), CCL2 (middle panels), and IFIT2 (right panels) by RT-qPCR. Levels of expression in infected mice were compared to those in mock-infected mice. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001 (PAOR/NS1OR vs. PAOR/NS1CIR, PACIR/NS1OR, and PACIR/NS1CIR) using 2 way ANOVA (n = 4 per time point). Non-significant when p > 0.05. 4. Discussion IAV evolve in their hosts to optimize fitness and transmission. In addition, to successfully replicate in their natural hosts and to evade antiviral responses induced during viral infection, IAV encode specific proteins, such as the NS1 and PA-X, to counteract host antiviral responses, allowing the virus to replicate in IFN competent systems [12]. In this work, we assessed the contribution of NS1 and PA-X proteins from different H5N1 IAV, as well as the role of specific amino acid mutations in the PA-X protein of H5N1 IAV on their ability to inhibit host gene expression, viral replication, and pathogenesis. Influenza NS1 and PA-X subvert antiviral responses through the inhibition of host gene expression, including IFNs and ISGs, many of which display antiviral activity [12]. For IAV NS1 protein, one of the mechanisms to inhibit host gene expression involves its binding to CPSF30 [49,57]. This binding inhibits cellular pre-mRNA processing, causing a global inhibition of host gene expression [49,57]. However, there are other mechanisms by which IAV NS1 protein inhibits antiviral responses [12,13]. In fact, the property of inhibiting host gene expression is not conserved among all IAV strains [12]. Previously, we described that, whereas the NS1 protein from pH1N1 IAV circulating in 2009 did not inhibit host gene expression, 6 amino acid changes were selected in the viruses circulating Figure 8. Induction of innate immune responses in vivo by recombinant viruses encoding H5N1 NS1 and/or PA variants: Groups of 7-to-8-week-old A129 male mice (N= 8/group) were infected with 1000 (10 3 ; A ) or 10,000 (10 4 ; B ) FFU/mouse of the indicated viruses (PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ) or left mock-infected (N= 4) . Mice were sacrificed at 2 (N= 4) and 4 (N= 4) d p.i. Left lungs were collected and total RNA was extracted to quantify the levels of TNF (left panels), CCL2 (middle panels), and IFIT2 (right panels) by RT-qPCR. Levels of expression in infected mice were compared to those in mock-infected mice. *, p< 0.05; **, p< 0.005; ***, p< 0.0005; ****, p< 0.0001 ( PAOR/NS1OR vs. PAOR/NS1CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ) using 2 way ANOVA (n= 4 per time point). Nonsignificant when p> 0.05. 4. Discussion IAV evolve in their hosts to optimize fitness and transmission. In addition, to successfully replicate in their natural hosts and to evade antiviral responses induced during viral infection, IAV encode specific proteins, such as the NS1 and PA-X, to counteract host antiviral responses, allowing the virus to replicate in IFN competent systems [ 12 ]. In this work, we assessed the contribution of NS1 and PA-X proteins from different H5N1 IAV, as well as the role of specific amino acid mutations in the PA-X protein of H5N1 IAV on their ability to inhibit host gene expression, viral replication, and pathogenesis. Influenza NS1 and PA-X subvert antiviral responses through the inhibition of host gene expression, including IFNs and ISGs, many of which display antiviral activity [ 12 ]. For IAV NS1 protein, one of the mechanisms to inhibit host gene expression involves its binding to CPSF30 [ 49 , 57 ]. This binding inhibits cellular pre-mRNA processing, causing a global inhibition of host gene expression [ 49 , 57 ]. However, there are other mechanisms by which IAV NS1 protein inhibits antiviral responses [ 12 , 13 ]. In fact, the property of inhibiting host gene expression is not conserved among all IAV strains [ 12 ]. Previously, Viruses 2021,13, 1760 21 of 25 we described that, whereas the NS1 protein from pH1N1 IAV circulating in 2009 did not inhibit host gene expression, 6 amino acid changes were selected in the viruses circulating afterward, leading to an NS1 protein able to induce cellular shutoff [ 36 , 42 ]. Similarly, in this work, we describe that whereas the NS1 protein from H5N1 IAV strains circulating during 1996–1997 do not efficiently inhibit host gene expression, likely including IFN responses, H5N1 viruses circulating afterward have selected amino acid changes in NS1 leading to an increase ability to induce cellular shutoff (Figures 1and 2). In accordance with our data, a previous study showed that amino acid changes L103F and I106M, which are also different between H5N1 NS1 OR and NS1 CIR , increase the ability of H5N1 NS1 to inhibit host gene expression and IFN responses [ 24 , 25 ] because these amino acid changes allow binding to CPSF30 [ 24 ]. Similarly, for influenza H7N9 strains, it has been shown that the NS1 amino acid change I106M can restore CPSF30 binding together with the ability to block host gene expression [ 55 ]. In the case of influenza H9N2 strains, amino acid substitutions L103F, I106M, P114S, G125D, and N139D in A/quail/Hong Kong/G1/97 H9N2 NS1 resulted in binding to CPSF30 and, in consequence, inhibition of host gene expression [ 58 ]. For avian H9N2 viruses, whereas the NS1 protein from recent isolates does not inhibit host gene expression, the NS1 proteins from earlier H9N2 strains does, suggesting that in the case of avian host infected with H9N2 strains, encoding a NS1 protein lacking the ability to inhibit host gene expression is beneficial for the virus [58]. We also showed that H5N1 IAV circulating nowadays encode a PA-X protein with eight amino acid changes (T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q) different from the PA-X proteins from H5N1 viruses circulating during 1996–1997, and that H5N1 PA-X CIR displays increased ability to inhibit host gene expression as compared with PA-X OR (Figures 1and 2). However, less is known about the amino acid residues in H5N1 PA-X contributing to its ability to inhibit host gene expression. We show that, among the eight amino acid differences between H5N1 PA-X OR and PA-X CIR , two of them, T118I and V127I, are responsible for the increased ability of H5N1 PA-X to inhibit host gene expression, whereas amino acid changes T20A and A85T have little or no effect on PA-X ´ s host shutoff activity (Figures 3and 4). Notably, substitution I118T is located within the endonuclease active site in the PA-X N-terminal domain [ 59 ]. Similar to our results, using an H9N2 strain, it has been shown that amino acid change T118I increases the ability of PA-X to inhibit host gene expression, whereas the amino acid change T20A has no effect on PA-X-mediated inhibition of host gene expression [ 59 ]. Interestingly, four of these amino acid changes (T20A, A85T, T118I, and I127V), especially two of them that mostly affect PA-X ´ s activity (T118I, I127V), also affect the protein sequence of PA; therefore, it is difficult to know whether these identified amino acid changes were selected due to a beneficial effect on PA-X, PA, or both. Furthermore, the selection of these mutations could be influenced by amino acid changes that occurred somewhere else in the viral genome, including NS1 [ 36 ]. Notably, by analyzing the evolution of pH1N1 in humans, we found that there are multiple amino acid changes in NS1 and PA-X proteins from circulating seasonal viruses compared to the viruses isolated at the origin of the 2009 pandemic . These amino acid substitutions are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes [ 36 ]. Notably, a recombinant pH1N1 virus containing PA-X and NS1 genes from recently circulating pH1N1 viruses has better viral fitness and is more pathogenic compared with the original pH1N1 [ 36 ]. Recombinant viruses encoding the PA-X amino acid change R195K, showed decreased shutoff activity and showed increased replication and transmission in ferrets but not in chickens [ 60 ], suggesting that this amino acid change might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans [ 60 ]. In the case of canine IAV, genetic analysis indicated that within the PA-X, X-ORFs of equine-derived H3N8 and avian-derived H3N2 viruses encoded 61 amino acids but were truncated after introduction into dogs [ 61 ]. Truncation of PA-X suppressed expression from co-transfected plasmids in cells and enhanced viral pathogenicity and transmission in dogs, suggesting that truncation of PA-X might be Viruses 2021,13, 1760 22 of 25 important for the adaptation of influenza viruses to dogs [ 61 ]. Importantly, these results demonstrate an evolution in the abilities of NS1 and PA-X proteins to induce host shutoff and show how this is beneficial for IAV. In addition, several in vitro studies have shown that multiple amino acids in PA-X can affect PA-X ´ s shutoff activity, and that differences between IAV strains or subtypes can occur [36,38,39,56,62,63]. To analyze the combined effect of H5N1 NS1 and PA-X amino acid changes on virus replication, pathogenesis, and the induction of innate immune responses, we generated four recombinant viruses (PA OR /NS1 OR , PA OR /NS1 CIR , PA CIR /NS1 OR , and PA CIR /NS1 CIR ). All the recombinant viruses encoded the PR8 genes with the exception of NS1 and PA/PA-X ORFs, which were from H5N1 IAV circulating in 1996–1997 (NS1 OR and PA OR , respectively) or since 2016 (NS1 CIR and PA CIR , respectively). Consequently, for the PA segment, the PA-X CIR protein encoded the amino acid changes T20A, A85T, T118I, I127V, G209E, V212A, L221R, and R250Q, and the PA CIR encoded the amino acid substitutions T20A, A85T, T118I, and I127V, which affect both to the PA and PA-X proteins. Remarkably, viruses encoding the NS1 OR were more virulent in A129 mice, measured by body weight losses and survival rates (Figure 6), and replicated slightly more efficiently in the lungs of infected A129 mice than the viruses encoding H5N1 NS1 CIR (Figure 7). To analyze possible reasons for these changes in virulence, we analyzed the response of pro-inflammatory cytokines and ISGs after viral infection. Inflammatory response has two distinct roles in the pathogenesis of IAV. Whereas activation is protective against viral infection, an uncontrolled response can cause severe damage. Interestingly, viruses encoding H5N1 NS1 OR (PA OR /NS1 OR and PA OR /NS1 CIR ) induced increased levels of TNF, CCL2, and IFIT2 expression (Figure 8A,B) compared with viruses encoding H5N1 NS1 CIR (PA OR /NS1 CIR and PA CIR /NS1 CIR ). This is likely due to the reduced ability of H5N1 NS1 OR to mediate cellular shutoff compared with H5N1 NS1 CIR (Figure 2). These highest levels of pro-inflammatory cytokine induction observed for viruses encoding H5N1 NS1 OR are likely responsible for their increased virulence (Figures 6and 7). In fact, it has been shown that increased levels of pro-inflammatory cytokines and viral loads correlate with higher virulence with H5N1 IAV [ 11 ]. Similarly, mice and macaques infected with seasonal H1N1 virus induced lower levels of pro-inflammatory cytokines and chemokines than animals infected with the 2009 pH1N1 virus, resulting in decreased pathogenicity [ 64 ]. In pigs, two swine-origin pH1N1 viruses, derived from a human patient and from swine, were more virulent than a swine-origin 1918-like classical IAV [ 65 ]. Interestingly, these IAV induced higher expression of pro-inflammatory genes compared with the swine-origin 1918-like classical IAV, suggesting that both pH1N1 isolates are more virulent, at least in part, due to differences in the host transcriptional response during acute infection [65]. Altogether, our data suggest that circulating H5N1 IAV has adapted to induce less inflammatory responses and be less virulent than original circulating H5N1 IAV. However, additional experiments are needed to further analyze these differences. We are aware of the limitations from our study because it will be important to test this hypothesis in different animal models, including avian hosts. Moreover, recombinant viruses containing the original and modified PA/PA-X and/or NS1 proteins in the backbone of an H5N1 IAV should be used. Finally, because NS1 and PA/PA-X are multifunctional proteins, the specific role of the amino acid changes observed between original and circulating strains needs further analysis. In any case, our results indicate a functional co-evolution of PA-X and NS1 in H5N1 IAV, which is important to inhibit host gene expression, as we have shown previously [ 36 , 37 ]. Remarkably, our results also demonstrate the importance of conducting H5N1 IAV surveillance and to monitor these and other mutations contributing to H5N1 IAV pathogenesis to predict and hopefully prevent potential future H5N1 IAV pandemics. Author Contributions: Conceptualization, A.N., L.M.-S. and M.L.D.; formal analysis, A.N., L.V., J.O., L.M.-S. and M.L.D.; investigation, A.N., L.V., S.U.-T., L.M.-S. and M.L.D.; resources, A.N. and M.L.D.; data curation, A.N., L.V. and M.L.D.; writing—original draft preparation, A.N. and M.L.D.; writing—review and editing, A.N., L.V., S.U.-T., J.O., L.M.-S. and M.L.D.; funding acquisition, A.N. and M.L.D. All authors have read and agreed to the published version of the manuscript. Viruses 2021,13, 1760 23 of 25 Funding: This work was supported with funds from Comunidad de Madrid (Spain), reference 2017-T1/BMD-5155 and the Spanish Ministry of Science, Innovation and Universities (RTI-2018094213-A-I00) to M.L.D and a “Ramon y Cajal” Incorporation grant (RYC-2017) from Spanish Ministry of Science, Innovation and Universities to A.N. Institutional Review Board Statement: Animal experimental protocols were approved by the Ethical Review Committee at the INIA-CISA and Comunidad de Madrid (Permit number: PROEX 116/19), in strict accordance with EU guidelines 2010/63/UE about the protection of animals used for experimentation and the Spanish Animal Welfare Act 32/2007. Informed Consent Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgments: We would like to thank members at our institutes for their efforts in keeping them fully operational during the COVID-19 pandemic. Conflicts of Interest: The authors declare no conflict of interest. References 1. Cheung, T.K.W.; Poon, L.L.M. Biology of Influenza a Virus. Ann. N. Y. Acad. Sci. 2007,1102, 1–25. [CrossRef] [PubMed] 2. Tong, S.; Zhu, X.; Li, Y.; Shi, M.; Zhang, J.; Bourgeois, M.; Yang, H.; Chen, X.; Recuenco, S.; Gomez, J.; et al. New World Bats Harbor Diverse Influenza A Viruses. PLoS Pathog. 2013,9, e1003657. [CrossRef] 3. Wille, M.; Holmes, E.C. The Ecology and Evolution of Influenza Viruses. Cold Spring Harb. Perspect. Med. 2020 ,10, a038489. [CrossRef] 4. Mo, I.P.; Brugh, M.; Fletcher, O.J.; Rowland, G.N.; Swayne, D.E. Comparative Pathology of Chickens Experimentally Inoculated with Avian Influenza Viruses of Low and High Pathogenicity. Avian Dis. 1997,41, 125–136. [CrossRef] 5. Alexander, D.J. A Review of Avian Influenza in Different Bird Species. Vet. Microbiol. 2000,74, 3–13. [CrossRef] 6. Xu, X.; Subbarao, N.; Cox, N.J.; Guo, Y. Genetic Characterization of the Pathogenic Influenza A/Goose/Guangdong/1/96 (H5N1) Virus: Similarity of Its Hemagglutinin Gene to Those of H5N1 Viruses from the 1997 Outbreaks in Hong Kong. Virology 1999 ,261, 15–19. [CrossRef] [PubMed] 7. Chan, P.K.S. Outbreak of Avian Influenza A(H5N1) Virus Infection in Hong Kong in 1997. Clin. Infect. Dis. 2002 ,34 (Suppl. 2), S58–S64. [CrossRef] 8. Uyeki, T.M.; Peiris, M. Novel Avian Influenza A Virus Infections of Humans. Infect. Dis. Clin. North Am. 2019 ,33, 907–932. [CrossRef] [PubMed] 9. Hui, D.S.-C. Review of Clinical Symptoms and Spectrum in Humans with Influenza A/H5N1 Infection. Respirology 2008 ,13 (Suppl. 1), S10–S13. [CrossRef] 10. Gu, J.; Xie, Z.; Gao, Z.; Liu, J.; Korteweg, C.; Ye, J.; Lau, L.T.; Lu, J.; Gao, Z.; Zhang, B.; et al. H5N1 Infection of the Respiratory Tract and beyond: A Molecular Pathology Study. Lancet 2007,370, 1137–1145. [CrossRef] 11. De Jong, M.D.; Simmons, C.P.; Thanh, T.T.; Hien, V.M.; Smith, G.J.D.; Chau, T.N.B.; Hoang, D.M.; Chau, N.V.V.; Khanh, T.H.; Dong, V.C.; et al. Fatal Outcome of Human Influenza A (H5N1) Is Associated with High Viral Load and Hypercytokinemia. Nat. Med. 2006,12, 1203–1207. [CrossRef] 12. Nogales, A.; Martinez-Sobrido, L.; Topham, D.J.; DeDiego, M.L. Modulation of Innate Immune Responses by the Influenza A NS1 and PA-X Proteins. Viruses 2018,10, 708. [CrossRef] 13. Hale, B.G.; Randall, R.E.; Ortín, J.; Jackson, D. The Multifunctional NS1 Protein of Influenza A Viruses. J. Gen. Virol. 2008 ,89, 2359–2376. [CrossRef] [PubMed] 14. Wang, W.; Riedel, K.; Lynch, P.; Chien, C.Y.; Montelione, G.T.; Krug, R.M. RNA Binding by the Novel Helical Domain of the Influenza Virus NS1 Protein Requires Its Dimer Structure and a Small Number of Specific Basic Amino Acids. RNA 1999 ,5, 195–205. [CrossRef] 15. Guo, Z.; Chen, L.; Zeng, H.; Gomez, J.A.; Plowden, J.; Fujita, T.; Katz, J.M.; Donis, R.O.; Sambhara, S. NS1 Protein of Influenza A Virus Inhibits the Function of Intracytoplasmic Pathogen Sensor, RIG-I. Am. J. Respir. Cell Mol. Biol. 2007 ,36, 263–269. [CrossRef] 16. Mibayashi, M.; Martínez-Sobrido, L.; Loo, Y.-M.; Cárdenas, W.B.; Gale, M.; García-Sastre, A. Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus. J. Virol. 2007,81, 514–524. [CrossRef] 17. Opitz, B.; Rejaibi, A.; Dauber, B.; Eckhard, J.; Vinzing, M.; Schmeck, B.; Hippenstiel, S.; Suttorp, N.; Wolff, T. IFNbeta Induction by Influenza A Virus Is Mediated by RIG-I Which Is Regulated by the Viral NS1 Protein. Cell Microbiol. 2007 ,9, 930–938. [CrossRef] 18. Chen, Z.; Li, Y.; Krug, R.M. Influenza A Virus NS1 Protein Targets Poly(A)-Binding Protein II of the Cellular 3 0 -End Processing Machinery. EMBO J. 1999,18, 2273–2283. [CrossRef] 19. Noah, D.L.; Twu, K.Y.; Krug, R.M. Cellular Antiviral Responses against Influenza A Virus Are Countered at the Posttranscriptional Level by the Viral NS1A Protein via Its Binding to a Cellular Protein Required for the 3 0 End Processing of Cellular Pre-MRNAS. Virology 2003,307, 386–395. [CrossRef] Viruses 2021,13, 1760 24 of 25 20. Li, K.S.; Guan, Y.; Wang, J.; Smith, G.J.D.; Xu, K.M.; Duan, L.; Rahardjo, A.P.; Puthavathana, P.; Buranathai, C.; Nguyen, T.D.; et al. Genesis of a Highly Pathogenic and Potentially Pandemic H5N1 Influenza Virus in Eastern Asia. Nature 2004 ,430, 209–213. [CrossRef] 21. Long, J.-X.; Peng, D.-X.; Liu, Y.-L.; Wu, Y.-T.; Liu, X.-F. Virulence of H5N1 Avian Influenza Virus Enhanced by a 15-Nucleotide Deletion in the Viral Nonstructural Gene. Virus Genes 2008,36, 471–478. [CrossRef] 22. Li, Y.; Chen, S.; Zhang, X.; Fu, Q.; Zhang, Z.; Shi, S.; Zhu, Y.; Gu, M.; Peng, D.; Liu, X. A 20-Amino-Acid Deletion in the Neuraminidase Stalk and a Five-Amino-Acid Deletion in the NS1 Protein Both Contribute to the Pathogenicity of H5N1 Avian Influenza Viruses in Mallard Ducks. PLoS ONE 2014,9, e95539. [CrossRef] 23. Li, W.; Wang, G.; Zhang, H.; Xin, G.; Zhang, D.; Zeng, J.; Chen, X.; Xu, Y.; Cui, Y.; Li, K. Effects of NS1 Variants of H5N1 Influenza Virus on Interferon Induction, TNFalpha Response and P53 Activity. Cell Mol. Immunol. 2010,7, 235–242. [CrossRef] 24. Twu, K.Y.; Kuo, R.-L.; Marklund, J.; Krug, R.M. The H5N1 Influenza Virus NS Genes Selected after 1998 Enhance Virus Replication in Mammalian Cells. J. Virol. 2007,81, 8112–8121. [CrossRef] [PubMed] 25. Spesock, A.; Malur, M.; Hossain, M.J.; Chen, L.-M.; Njaa, B.L.; Davis, C.T.; Lipatov, A.S.; York, I.A.; Krug, R.M.; Donis, R.O. The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins. J. Virol. 2011,85, 7048–7058. [CrossRef] 26. Dankar, S.K.; Miranda, E.; Forbes, N.E.; Pelchat, M.; Tavassoli, A.; Selman, M.; Ping, J.; Jia, J.; Brown, E.G. Influenza A/Hong Kong/156/1997(H5N1) Virus NS1 Gene Mutations F103L and M106I Both Increase IFN Antagonism, Virulence and Cytoplasmic Localization but Differ in Binding to RIG-I and CPSF30. Virol. J. 2013,10, 243. [CrossRef] [PubMed] 27. Jagger, B.W.; Wise, H.M.; Kash, J.C.; Walters, K.-A.; Wills, N.M.; Xiao, Y.-L.; Dunfee, R.L.; Schwartzman, L.M.; Ozinsky, A.; Bell, G.L.; et al. An Overlapping Protein-Coding Region in Influenza A Virus Segment 3 Modulates the Host Response. Science 2012 , 337, 199–204. [CrossRef] 28. Firth, A.E.; Jagger, B.W.; Wise, H.M.; Nelson, C.C.; Parsawar, K.; Wills, N.M.; Napthine, S.; Taubenberger, J.K.; Digard, P.; Atkins, J.F. Ribosomal Frameshifting Used in Influenza A Virus Expression Occurs within the Sequence UCC_UUU_CGU and Is in the +1 Direction. Open Biol. 2012,2, 120109. [CrossRef] 29. Shi, M.; Jagger, B.W.; Wise, H.M.; Digard, P.; Holmes, E.C.; Taubenberger, J.K. Evolutionary Conservation of the PA-X Open Reading Frame in Segment 3 of Influenza A Virus. J. Virol. 2012,86, 12411–12413. [CrossRef] 30. Gao, H.; Sun, H.; Hu, J.; Qi, L.; Wang, J.; Xiong, X.; Wang, Y.; He, Q.; Lin, Y.; Kong, W.; et al. Twenty Amino Acids at the C-Terminus of PA-X Are Associated with Increased Influenza A Virus Replication and Pathogenicity. J. Gen. Virol. 2015 ,96, 2036–2049. [CrossRef] 31. Khaperskyy, D.A.; Schmaling, S.; Larkins-Ford, J.; McCormick, C.; Gaglia, M.M. Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein. PLoS Pathog. 2016,12, e1005427. [CrossRef] 32. Hu, J.; Mo, Y.; Gao, Z.; Wang, X.; Gu, M.; Liang, Y.; Cheng, X.; Hu, S.; Liu, W.; Liu, H.; et al. PA-X-Associated Early Alleviation of the Acute Lung Injury Contributes to the Attenuation of a Highly Pathogenic H5N1 Avian Influenza Virus in Mice. Med. Microbiol. Immunol. 2016,205, 381–395. [CrossRef] 33. Gao, H.; Sun, Y.; Hu, J.; Qi, L.; Wang, J.; Xiong, X.; Wang, Y.; He, Q.; Lin, Y.; Kong, W.; et al. The Contribution of PA-X to the Virulence of Pandemic 2009 H1N1 and Highly Pathogenic H5N1 Avian Influenza Viruses. Sci. Rep. 2015,5, 8262. [CrossRef] 34. Hu, J.; Mo, Y.; Wang, X.; Gu, M.; Hu, Z.; Zhong, L.; Wu, Q.; Hao, X.; Hu, S.; Liu, W.; et al. PA-X Decreases the Pathogenicity of Highly Pathogenic H5N1 Influenza A Virus in Avian Species by Inhibiting Virus Replication and Host Response. J. Virol. 2015 ,89, 4126–4142. [CrossRef] [PubMed] 35. Hilimire, T.A.; Nogales, A.; Chiem, K.; Ortego, J.; Martinez-Sobrido, L. Increasing the Safety Profile of the Master Donor Live Attenuated Influenza Vaccine. Pathogens 2020,9, 86. [CrossRef] 36. Nogales, A.; Martinez-Sobrido, L.; Chiem, K.; Topham, D.J.; DeDiego, M.L. Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA in Humans. J. Virol. 2018,92, e01206-18. [CrossRef] 37. Nogales, A.; Rodriguez, L.; DeDiego, M.L.; Topham, D.J.; Martínez-Sobrido, L. Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus. J. Virol. 2017,91, e00720-17. [CrossRef] 38. Chiem, K.; Martinez-Sobrido, L.; Nogales, A.; DeDiego, M.L. Amino Acid Residues Involved in Inhibition of Host Gene Expression by Influenza A/Brevig Mission/1/1918 PA-X. Microorganisms 2021,9, 1109. [CrossRef] [PubMed] 39. Chiem, K.; López-García, D.; Ortego, J.; Martinez-Sobrido, L.; DeDiego, M.L.; Nogales, A. Identification of Amino Acid Residues Required for Inhibition of Host Gene Expression by Influenza A/Viet Nam/1203/2004 H5N1 PA-X. J. Virol. 2021 , JVI0040821. [CrossRef] [PubMed] 40. Nogales, A.; DeDiego, M.L.; Topham, D.J.; Martínez-Sobrido, L. Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines. J. Virol. 2016,90, 6291–6302. [CrossRef] 41. Kochs, G.; García-Sastre, A.; Martínez-Sobrido, L. Multiple Anti-Interferon Actions of the Influenza A Virus NS1 Protein. J. Virol. 2007,81, 7011–7021. [CrossRef] [PubMed] 42. Clark, A.M.; Nogales, A.; Martinez-Sobrido, L.; Topham, D.J.; DeDiego, M.L. Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses. J. Virol. 2017,91, e00721-17. [CrossRef] 43. Nogales, A.; Martinez-Sobrido, L.; Topham, D.J.; DeDiego, M.L. NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses. J. Virol. 2017 ,91. [CrossRef] [PubMed] Viruses 2021,13, 1760 25 of 25 44. DeDiego, M.L.; Nogales, A.; Lambert-Emo, K.; Martinez-Sobrido, L.; Topham, D.J. NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses. J. Virol. 2016,90, 9693–9711. [CrossRef] [PubMed] 45. Nogales, A.; Baker, S.F.; Martínez-Sobrido, L. Replication-Competent Influenza A Viruses Expressing a Red Fluorescent Protein. Virology 2015,476, 206–216. [CrossRef] [PubMed] 46. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta C(T)) Method. Methods 2001,25, 402–408. [CrossRef] [PubMed] 47. Chen, X.; Wang, W.; Wang, Y.; Lai, S.; Yang, J.; Cowling, B.J.; Horby, P.W.; Uyeki, T.M.; Yu, H. Serological Evidence of Human Infections with Highly Pathogenic Avian Influenza A(H5N1) Virus: A Systematic Review and Meta-Analysis. BMC Med. 2020 ,18, 377. [CrossRef] [PubMed] 48. Das, K.; Ma, L.-C.; Xiao, R.; Radvansky, B.; Aramini, J.; Zhao, L.; Marklund, J.; Kuo, R.-L.; Twu, K.Y.; Arnold, E.; et al. Structural Basis for Suppression of a Host Antiviral Response by Influenza A Virus. Proc. Natl. Acad. Sci. USA 2008 ,105, 13093–13098. [CrossRef] 49. Nemeroff, M.E.; Barabino, S.M.; Li, Y.; Keller, W.; Krug, R.M. Influenza Virus NS1 Protein Interacts with the Cellular 30 KDa Subunit of CPSF and Inhibits 30end Formation of Cellular Pre-MRNAs. Mol. Cell. 1998,1, 991–1000. [CrossRef] 50. Gaucherand, L.; Porter, B.K.; Levene, R.E.; Price, E.L.; Schmaling, S.K.; Rycroft, C.H.; Kevorkian, Y.; McCormick, C.; Khaperskyy, D.A.; Gaglia, M.M. The Influenza A Virus Endoribonuclease PA-X Usurps Host MRNA Processing Machinery to Limit Host Gene Expression. Cell. Rep. 2019,27, 776–792.e7. [CrossRef] [PubMed] 51. Levene, R.E.; Shrestha, S.D.; Gaglia, M.M. The Influenza A Virus Host Shutoff Factor PA-X Is Rapidly Turned over in a StrainSpecific Manner. J. Virol. 2021,95, e02312-20. [CrossRef] 52. Oishi, K.; Yamayoshi, S.; Kozuka-Hata, H.; Oyama, M.; Kawaoka, Y. N-Terminal Acetylation by NatB Is Required for the Shutoff Activity of Influenza A Virus PA-X. Cell Rep. 2018,24, 851–860. [CrossRef] [PubMed] 53. Nogales, A.; Chauché, C.; DeDiego, M.L.; Topham, D.J.; Parrish, C.R.; Murcia, P.R.; Martínez-Sobrido, L. The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression. J. Virol. 2017 , 91, e00877-17. [CrossRef] [PubMed] 54. Hale, B.G.; Steel, J.; Medina, R.A.; Manicassamy, B.; Ye, J.; Hickman, D.; Hai, R.; Schmolke, M.; Lowen, A.C.; Perez, D.R.; et al. Inefficient Control of Host Gene Expression by the 2009 Pandemic H1N1 Influenza A Virus NS1 Protein. J. Virol. 2010 ,84, 6909–6922. [CrossRef] 55. Ayllon, J.; Domingues, P.; Rajsbaum, R.; Miorin, L.; Schmolke, M.; Hale, B.G.; García-Sastre, A. A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence. J. Virol. 2014 ,88, 12146–12151. [CrossRef] 56. Oishi, K.; Yamayoshi, S.; Kawaoka, Y. Identification of Novel Amino Acid Residues of Influenza Virus PA-X That Are Important for PA-X Shutoff Activity by Using Yeast. Virology 2018,516, 71–75. [CrossRef] [PubMed] 57. Twu, K.Y.; Noah, D.L.; Rao, P.; Kuo, R.-L.; Krug, R.M. The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target. J. Virol. 2006,80, 3957–3965. [CrossRef] [PubMed] 58. Rodriguez, L.; Nogales, A.; Iqbal, M.; Perez, D.R.; Martinez-Sobrido, L. Identification of Amino Acid Residues Responsible for Inhibition of Host Gene Expression by Influenza A H9N2 NS1 Targeting of CPSF30. Front. Microbiol. 2018,9, 2546. [CrossRef] 59. Clements, A.L.; Sealy, J.E.; Peacock, T.P.; Sadeyen, J.-R.; Hussain, S.; Lycett, S.J.; Shelton, H.; Digard, P.; Iqbal, M. Contribution of Segment 3 to the Acquisition of Virulence in Contemporary H9N2 Avian Influenza Viruses. J. Virol. 2020 ,94, e01173-20. [CrossRef] [PubMed] 60. Sun, Y.; Hu, Z.; Zhang, X.; Chen, M.; Wang, Z.; Xu, G.; Bi, Y.; Tong, Q.; Wang, M.; Sun, H.; et al. An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts. J. Virol. 2020 ,94, e01817-19. [CrossRef] 61. Liu, L.; Song, S.; Shen, Y.; Ma, C.; Wang, T.; Tong, Q.; Sun, H.; Pu, J.; Iqbal, M.; Liu, J.; et al. Truncation of PA-X Contributes to Virulence and Transmission of H3N8 and H3N2 Canine Influenza Viruses in Dogs. J. Virol. 2020 ,94, e00949-20. [CrossRef] [PubMed] 62. Oishi, K.; Yamayoshi, S.; Kawaoka, Y. Identification of Amino Acid Residues in Influenza A Virus PA-X That Contribute to Enhanced Shutoff Activity. Front. Microbiol. 2019,10, 432. [CrossRef] 63. Hayashi, T.; Chaimayo, C.; McGuinness, J.; Takimoto, T. Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity. J. Virol. 2016,90, 7131–7141. [CrossRef] [PubMed] 64. Itoh, Y.; Shinya, K.; Kiso, M.; Watanabe, T.; Sakoda, Y.; Hatta, M.; Muramoto, Y.; Tamura, D.; Sakai-Tagawa, Y.; Noda, T.; et al. In Vitro and in Vivo Characterization of New Swine-Origin H1N1 Influenza Viruses. Nature 2009 ,460, 1021–1025. [CrossRef] [PubMed] 65. Ma, W.; Belisle, S.E.; Mosier, D.; Li, X.; Stigger-Rosser, E.; Liu, Q.; Qiao, C.; Elder, J.; Webby, R.; Katze, M.G.; et al. 2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation of Genes Related to Inflammatory and Immune Responses, Cell Death, and Lipid Metabolism in Pigs. J. Virol. 2011,85, 11626–11637. [CrossRef]