Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments.

Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.

TRIM21 reduces H1N1-induced inflammation and apoptosis by regulating the TBK1-IRF3 signaling pathway in A549 cells.

Triple motif protein 21 (TRIM21) has an antiviral function that inhibits various viral infections. However, its role in the progress of influenza A virus (IAV) infection is unclear. In this study, we investigated the role and molecular mechanism of TRIM21 in IAV infection. RT-qPCR was used to determine the level of TRIM21 mRNA, and ELISA was used to measure the levels of IFN-α, IFN-ß, IL-6, and TNF-α. The levels of the TRIM21, NP, TBK1, IRF3, p-TBK1, and p-IRF3 proteins were estimated by Western blot. The results showed that, after IAV infection, TRIM21 was upregulated in clinical patient serum and A549 cells, and this was correlated with the IFN response. Overexpression of TRIM21 reduced IAV replication and transcription in in vitro cell experiments. TRIM21 also increased IFN-α and IFN-ß levels and decreased IL-6 and TNF-α levels in A549 cells. In addition, overexpression of TRIM21 inhibited IAV-induced apoptosis. Further experiments demonstrated that TBK1-IRF3 signaling was activated by TRIM21 and was involved in the inhibitory effect of TRIM21 on virus replication. In summary, our study suggests that TRIM21 inhibits viral replication by activating the TBK1-IRF3 signaling pathway, further inhibiting the infection process of IAV.

A novel data augmentation approach for influenza A subtype prediction based on HA proteins.

Influenza, a pervasive viral respiratory illness, remains a significant global health concern. The influenza A virus, capable of causing pandemics, necessitates timely identification of specific subtypes for effective prevention and control, as highlighted by the World Health Organization. The genetic diversity of influenza A virus, especially in the hemagglutinin protein, presents challenges for accurate subtype prediction. This study introduces PreIS as a novel pipeline utilizing advanced protein language models and supervised data augmentation to discern subtle differences in hemagglutinin protein sequences. PreIS demonstrates two key contributions: leveraging pre-trained protein language models for influenza subtype classification and utilizing supervised data augmentation to generate additional training data without extensive annotations. The effectiveness of the pipeline has been rigorously assessed through extensive experiments, demonstrating a superior performance with an impressive accuracy of 94.54% compared to the current state-of-the-art model, the MC-NN model, which achieves an accuracy of 89.6%. PreIS also exhibits proficiency in handling unknown subtypes, emphasizing the importance of early detection. Pioneering the classification of HxNy subtypes solely based on the hemagglutinin protein chain, this research sets a benchmark for future studies. These findings promise more precise and timely influenza subtype prediction, enhancing public health preparedness against influenza outbreaks and pandemics. The data and code underlying this article are available in https://github.com/CBRC-lab/PreIS.

Amino acids in the polymerase complex of shorebird-isolated H1N1 influenza virus impact replication and host-virus interactions in mammalian models.

A diverse population of avian influenza A viruses (AIVs) are maintained in wild birds and ducks yet the zoonotic potential of AIVs in these environmental reservoirs and the host-virus interactions involved in mammalian infection are not well understood. In studies of a group of subtype H1N1 AIVs isolated from migratory wild birds during surveillance in North America, we previously identified eight amino acids in the polymerase genes PB2 and PB1 that were important for the transmissibility of these AIVs in a ferret model of human influenza virus transmission. In this current study we found that PB2 containing amino acids associated with transmissibility at 67, 152, 199, 508, and 649 and PB1 at 298, 642, and 667 were associated with more rapid viral replication kinetics, greater infectivity, more active polymerase complexes and greater kinetics of viral genome replication and transcription. Pathogenicity in the mouse model was also impacted, evident as greater weight loss and lung pathology associated with greater inflammatory lung cytokine expression. Further, these AIVs all contained the avian-type amino acids of PB2-E627, D701, G590, Q591 and T271. Therefore, our study provides novel insights into the role of the AIV polymerase complex in the zoonotic transmission of AIVs in mammals.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Functionality of IAV packaging signals depends on site-specific charges within the viral nucleoprotein.

The coordinated packaging of the segmented genome of the influenza A virus (IAV) into virions is an essential step of the viral life cycle. This process is controlled by the interaction of packaging signals present in all eight viral RNA (vRNA) segments and the viral nucleoprotein (NP), which binds vRNA via a positively charged binding groove. However, mechanistic models of how the packaging signals and NP work together to coordinate genome packaging are missing. Here, we studied genome packaging in influenza A/SC35M virus mutants that carry mutated packaging signals as well as specific amino acid substitutions at the highly conserved lysine (K) residues 184 and 229 in the RNA-binding groove of NP. Because these lysines are acetylated and thus neutrally charged in infected host cells, we replaced them with glutamine to mimic the acetylated, neutrally charged state or arginine to mimic the non-acetylated, positively charged state. Our analysis shows that the coordinated packaging of eight vRNAs is influenced by (i) the charge state of the replacing amino acid and (ii) its location within the RNA-binding groove. Accordingly, we propose that lysine acetylation induces different charge states within the RNA-binding groove of NP, thereby supporting the activity of specific packaging signals during coordinated genome packaging. IMPORTANCE: Influenza A viruses (IAVs) have a segmented viral RNA (vRNA) genome encapsidated by multiple copies of the viral nucleoprotein (NP) and organized into eight distinct viral ribonucleoprotein complexes. Although genome segmentation contributes significantly to viral evolution and adaptation, it requires a highly sophisticated genome-packaging mechanism. How eight distinct genome complexes are incorporated into the virion is poorly understood, but previous research suggests an essential role for both vRNA packaging signals and highly conserved NP amino acids. By demonstrating that the packaging process is controlled by charge-dependent interactions of highly conserved lysine residues in NP and vRNA packaging signals, our study provides new insights into the sophisticated packaging mechanism of IAVs.

Discovery of an ellipticine derivative as TLR3 inhibitor against influenza A virus and SARS-CoV-2.

Influenza and COVID-19 continue to pose global threats to public health. Classic antiviral drugs have certain limitations, coupled with frequent viral mutations leading to many drugs being ineffective, the development of new antiviral drugs is urgent. Meanwhile, the invasion of influenza virus can cause an immune response, and an excessive immune response can generate a large number of inflammatory storms, leading to tissue damage. Toll-like receptor 3 (TLR3) recognizes virus dsRNA to ignite the innate immune response, and inhibit TLR3 can block the excess immune response and protect the host tissues. Taking TLR3 as the target, SMU-CX1 was obtained as the specific TLR3 inhibitor by high-throughput screening of 15,700 compounds with IC50 value of 0.11 µM. Its anti-influenza A virus activity with IC50 ranged from 0.14 to 0.33 µM against multiple subtypes of influenza A virus and also showed promising anti-SARS-CoV-2 activity with IC50 at 0.43 µM. Primary antiviral mechanism study indicated that SMU-CX1 significantly inhibited PB2 and NP protein of viruses, it can also inhibit inflammatory factors in host cells including IFN-ß, IP-10 and CCL-5. In conclusion, this study demonstrates the potential of SMU-CX1 in inhibiting IAV and SARS-CoV-2 activity, thereby offering a novel approach for designing antiviral drugs against highly pathogenic viruses.

Atomic force microscopy of spherical intermediates on the pathway to fibril formation of influenza A virus nuclear export protein.

The nuclear export protein of the influenza A virus (NEP) is involved in many important processes of the virus life cycle. This makes it an attractive target for the treatment of a disease caused by a virus. Previously it has been shown, that recombinant variants of NEP are highly prone to aggregation in solution under various conditions with the formation of amyloid-like aggregates. In the present work, the amyloid nature of NEP aggregates was evidenced by Congo red binding assays. Atomic force microscopy has shown that NEP can form two types of spherical nanoparticles, which provide an alternative pathway for the formation of amyloid-like fibrils. Type I of these "fibrillogenic" spheres, formed under physiological conditions, represents the micelle-like particles with height 10-60 nm, which can generate worm-like flexible fibrils with the diameter 2.5-4.0 nm, length 20-500 nm and the Young's modulus ~73 MPa. Type II spherical aggregates with size of about 400-1000 nm, formed at elevated temperatures, includes fractions of drop-like and vesicle-like particles, generating more rigid amyloid-like fibrils with height of ~8 nm, and length of up to 2 µm. The hypothetical mechanism of fibril formation via nanospherical structures was suggested. RESEARCH HIGHLIGHTS: AFM has revealed two types of the influenza A virus nuclear export protein spherical aggregates. They provide an alternative pathway for the formation of amyloid-like fibrils. The mechanism of fibril formation via spherical structures is suggested.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Glycoblotting-Based Ovo-Sulphoglycomics Reveals Phosphorylated N-Glycans as a Possible Host Factor of AIV Prevalence in Waterfowls.

Sulfated N-glycans play a crucial role in the interaction between influenza A virus (IAV) and its host. These glycans have been found to enhance viral replication, highlighting their significance in IAV propagation. This study investigated the expression of acidic N-glycans, specifically sulfated and phosphorylated glycans, in the egg whites of 72 avian species belonging to the Order Anseriformes (waterfowls). We used the glycoblotting-based sulphoglycomics approach to elucidate the diversity of acidic N-glycans and infer their potential role in protecting embryos from infections. Family-specific variations in sulfated and phosphorylated N-glycan profiles were identified in waterfowl egg whites. Different waterfowl species exhibited distinct expressions of sulfated trans-Gal(+) and trans-Gal(-) N-glycan structures. Additionally, species-specific expression of phosphorylated N-glycans was observed. Furthermore, it was found that waterfowl species with high avian influenza virus (AIV) prevalence displayed a higher abundance of phosphorylated hybrid and high-mannose N-glycans on their egg whites. These findings shed light on the importance of phosphorylated and sulfated N-glycans in understanding the role of acidic glycans in IAV propagation.

Impaired influenza A virus replication by the host restriction factor SAMHD1 which inhibited by PA-mediated dephosphorylation of the host transcription factor IRF3.

BACKGROUND: Influenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV. METHODS: To investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication. RESULTS: In this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1's ability to restrict H5N1 is related to phosphorylation at 592-tyrosine. CONCLUSIONS: In conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.

Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck.

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

The avian influenza A virus receptor SA-α2,3-Gal is expressed in the porcine nasal mucosa sustaining the pig as a mixing vessel for new influenza viruses.

Influenza A viruses (IAVs) originate from wild birds but have on several occasions jumped host barriers and are now also circulating in humans and mammals. The IAV host receptors (glycans with galactose linked to a sialic acid (SA) in an α2,3 or α2,6 linkage) are crucial host factors restricting inter-species transmission. In general, avian-origin IAVs show a preference for SA-α2,3 (avian receptor), whereas IAVs isolated from humans and pigs prefer SA-α2,6 (human receptor). N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two major SAs. Neu5Ac is expressed in all species, whereas Neu5Gc is only expressed in a limited number of domestic species such as pigs and horses, but not in humans. Despite that previous studies have shown that the IAV host receptor distribution appears to be similar in pigs and humans, none of these studies have investigated the expression of Neu5Gc-α2,6 in situ in porcine tissues. Thus, the aim of this study was to elucidate the distribution of IAV host receptors expressed in the porcine respiratory tract and relate the expression to the viral tropism of diverse host-adapted IAVs. The IAV receptor (SA-α2,3 and SA-α2,6) distribution and the presence of specifically Neu5Gc-α2,6 in the porcine nasal, tracheal, and lung tissues was investigated by lectin histochemistry. Furthermore, IAV immunohistochemistry was performed on tissues from pigs experimentally infected with IAVs, either adapted to pigs or humans, to investigate the significance of the IAV host receptors and the tropism of the diverse host-adapted IAVs. We document for the first time the expression of the avian receptor on the surface of the porcine nasal mucosa and an equal expression of Neu5Ac-α2,6 and Neu5Gc-α2,6 on the surface of the tracheal epithelium and alveoli. In all IAV-infected pigs, we found a low amount of IAV-positive cells in the trachea despite a high expression of the human receptor. Cumulatively, these findings suggest that optimal IAV replication involves a complex interplay between the viruses and their host receptors and that there might be other less clearly defined host factors that determine the site of replication.

A quantitative model for virus uncoating predicts influenza A infectivity.

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.

Cholesterol and M2 Rendezvous in Budding and Scission of Influenza A Virus.

The cholesterol of the host cell plasma membrane and viral M2 protein plays a crucial role in multiple stages of infection and replication of the influenza A virus. Cholesterol is required for the formation of heterogeneous membrane microdomains (or rafts) in the budozone of the host cell that serves as assembly sites for the viral components. The raft microstructures act as scaffolds for several proteins. Cholesterol may further contribute to the mechanical forces necessary for membrane scission in the last stage of budding and help to maintain the stability of the virus envelope. The M2 protein has been shown to cause membrane scission in model systems by promoting the formation of curved lipid bilayer structures that, in turn, can lead to membrane vesicles budding off or scission intermediates. Membrane remodeling by M2 is intimately linked with cholesterol as it affects local lipid composition, fluidity, and stability of the membrane. Thus, both cholesterol and M2 protein contribute to the efficient and proper release of newly formed influenza viruses from the virus-infected cells.

Discovery and Characterization of IFITM S-Palmitoylation.

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane-sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.

ZBP1 Drives IAV-Induced NLRP3 Inflammasome Activation and Lytic Cell Death, PANoptosis, Independent of the Necroptosis Executioner MLKL.

Influenza A virus (IAV) continues to pose a significant global health threat, causing severe respiratory infections that result in substantial annual morbidity and mortality. Recent research highlights the pivotal role of innate immunity, cell death, and inflammation in exacerbating the severity of respiratory viral diseases. One key molecule in this process is ZBP1, a well-recognized innate immune sensor for IAV infection. Upon activation, ZBP1 triggers the formation of a PANoptosome complex containing ASC, caspase-8, and RIPK3, among other molecules, leading to inflammatory cell death, PANoptosis, and NLRP3 inflammasome activation for the maturation of IL-1ß and IL-18. However, the role for other molecules in this process requires further evaluation. In this study, we investigated the role of MLKL in regulating IAV-induced cell death and NLRP3 inflammasome activation. Our data indicate IAV induced inflammatory cell death through the ZBP1-PANoptosome, where caspases and RIPKs serve as core components. However, IAV-induced lytic cell death was only partially dependent on RIPK3 at later timepoints and was fully independent of MLKL throughout all timepoints tested. Additionally, NLRP3 inflammasome activation was unaffected in MLKL-deficient cells, establishing that MLKL and MLKL-dependent necroptosis do not act upstream of NLRP3 inflammasome activation, IL-1ß maturation, and lytic cell death during IAV infection.

Systemic perturbations in amino acids/amino acid derivatives and tryptophan pathway metabolites associated with murine influenza A virus infection.

BACKGROUND: Influenza A virus (IAV) is the only influenza virus causing flu pandemics (i.e., global epidemics of flu disease). Influenza (the flu) is a highly contagious disease that can be deadly, especially in high-risk groups. Worldwide, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness and in about 290,000 to 650,000 respiratory deaths. We intend to reveal the effect of IAV infection on the host's metabolism, immune response, and neurotoxicity by using a mouse IAV infection model. METHODS: 51 metabolites of murine blood plasma (33 amino acids/amino acid derivatives (AADs) and 18 metabolites of the tryptophan pathway) were analyzed by using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry with Electrospray Ionization at the acute (7 days post-infection (dpi)), resolution (14 dpi), and recovery (21 dpi) stages of the virus infection in comparison with controls. RESULTS: Among the 33 biogenic amino acids/AADs, the levels of five amino acids/AADs (1-methylhistidine, 5-oxoproline, α-aminobutyric acid, glutamine, and taurine) increased by 7 dpi, whereas the levels of ten amino acids/AADs (4-hydroxyproline, alanine, arginine, asparagine, cysteine, citrulline, glycine, methionine, proline, and tyrosine) decreased. By 14 dpi, the levels of one AAD (3-methylhistidine) increased, whereas the levels of five amino acids/AADs (α-aminobutyric acid, aminoadipic acid, methionine, threonine, valine) decreased. Among the 18 metabolites from the tryptophan pathway, the levels of kynurenine, quinolinic acid, hydroxykynurenine increased by 7 dpi, whereas the levels of indole-3-acetic acid and nicotinamide riboside decreased. CONCLUSIONS: Our data may facilitate understanding the molecular mechanisms of host responses to IAV infection and provide a basis for discovering potential new mechanistic, diagnostic, and prognostic biomarkers and therapeutic targets for IAV infection.

Deep mutational scanning reveals the functional constraints and evolutionary potential of the influenza A virus PB1 protein.

IMPORTANCE: The influenza virus polymerase is important for adaptation to new hosts and, as a determinant of mutation rate, for the process of adaptation itself. We performed a deep mutational scan of the polymerase basic 1 (PB1) protein to gain insights into the structural and functional constraints on the influenza RNA-dependent RNA polymerase. We find that PB1 is highly constrained at specific sites that are only moderately predicted by the global structure or larger domain. We identified a number of beneficial mutations, many of which have been shown to be functionally important or observed in influenza virus' natural evolution. Overall, our atlas of PB1 mutations and their fitness impacts serves as an important resource for future studies of influenza replication and evolution.

Palmitoylation of the hemagglutinin of influenza B virus by ER-localized DHHC enzymes 1, 2, 4, and 6 is required for efficient virus replication.

IMPORTANCE: Influenza viruses are a public health concern since they cause seasonal outbreaks and occasionally pandemics. Our study investigates the importance of a protein modification called "palmitoylation" in the replication of influenza B virus. Palmitoylation involves attaching fatty acids to the viral protein hemagglutinin and has previously been studied for influenza A virus. We found that this modification is important for the influenza B virus to replicate, as mutating the sites where palmitate is attached prevented the virus from generating viable particles. Our experiments also showed that this modification occurs in the endoplasmic reticulum. We identified the specific enzymes responsible for this modification, which are different from those involved in palmitoylation of HA of influenza A virus. Overall, our research illuminates the similarities and differences in fatty acid attachment to HA of influenza A and B viruses and identifies the responsible enzymes, which might be promising targets for anti-viral therapy.