Revealing the mechanism underlying the effects of γ-aminobutyric acid-dioscorin interactions on dioscorin structure and emulsifying properties by molecular dynamic simulations.

Many studies have shown that γ-aminobutyric acid (GABA) exhibits various beneficial biological activities, including gut-modulating, neuro-stimulating, and cardio-protecting activities. Naturally, GABA exists in small amounts in yam, which is primarily synthesized by the decarboxylation of L-glutamic acid in the presence of glutamate decarboxylase. Dioscorin, the major tuber storage protein of yam, has been shown to have good solubility and emulsifying activity. However, how GABA interacts with dioscorin and affects their properties has yet to be clarified. In this research, the physicochemical and emulsifying properties of GABA-fortified dioscorin, which was dried by spray drying and freeze drying, were studied. As results, the freeze-dried (FD) dioscorin produced more stable emulsions, while the spray-dried (SD) dioscorin adsorbed more rapidly to oil/water (O/W) interface. The fluorescence spectroscopy, ultraviolet spectroscopy and circular dichroism spectroscopy showed that GABA changed the structure of dioscorin, by exposing its hydrophobic groups. The addition of GABA significantly promoted the adsorption of dioscorin to the O/W interface and prevented droplets coalescence. The results of molecular dynamics simulation (MD) showed that GABA destroyed the H-bond network between dioscorin and water, increased surface hydrophobicity and finally improved the emulsifying properties of dioscorin.

Effects of spray drying and freeze drying on the structure and emulsifying properties of yam soluble protein: A study by experiment and molecular dynamics simulation.

This study focused on the effects of freeze drying (FD) and sprays drying (SD) on the structure and emulsifying properties of yam soluble protein (YSP). The results showed that the surface hydrophobicity (Ho) value, free sulfhydryl group (SH) content, turns content, denaturation temperature and enthalpy value of spray-dried YSP (SD-YSP) were higher than freeze-dried YSP (FD-YSP), but the apparent hydrodynamic diameter (Dh) value of SD-YSP was smaller. The smaller Dh, higher Ho and free SH led to higher percentage of adsorbed proteins and stronger binding between protein and oil droplet in emulsions. Thus, the emulsifying properties of SD-YSP were better, and the SD-YSP-stabilized emulsion had better dynamical rheological properties. Molecular dynamics (MD) simulations suggested that some intramolecular disulfide bonds and hydrogen bonds of dioscorin were broken, and some helices transformed into turns during the SD process. These structural changes resulted in better thermal stability and emulsification properties of SD-YSP.

A Novel lncRNA SAAL Suppresses IAV Replication by Promoting Innate Responses.

Influenza A virus (IAV) infection has traditionally been a serious problem in animal husbandry and human public health security. Recently, many studies identified that long noncoding RNAs play an important role in the antiviral immune response after the infection of the influenza virus. However, there are still lots of IAV-related lncRNAs that have not been well-characterized. Using RNA sequencing analysis, we identified a lncRNA, named Serpina3i Activation Associated lncRNA (SAAL), which can be significantly upregulated in mice after IAV infection. In this study, we found that overexpression of SAAL inhibited the replication of A/WSN/33(WSN). SAAL upregulated Serpina3i with or without WSN infection. Overexpression of Serpina3i reduced influenza virus infection. Meanwhile, knockdown of Serpina3i enhanced the replication of WSN. Furthermore, knockdown of Serpina3i abolished the SAAL-mediated decrease in WSN infection. Overexpression of SAAL or Serpina3i positively regulated the transcription of interferon ß (IFN-ß) and several critical ISGs after WSN infection. In conclusion, we found that the novel lncRNA SAAL is a critical anti-influenza regulator by upregulating the mRNA level of Serpina3i.

TREX (transcription/export)-NP complex exerts a dual effect on regulating polymerase activity and replication of influenza A virus.

Influenza A viruses effectively hijack the intracellular "resources" to complete transcription and replication, which involve extensive interactions between the viral and host proteins. Herein, we screened the host factors, which belong to DExD/H-box protein family members, RNA-binding proteins or mitochondrial anchoring proteins, to investigate their effects on polymerase activity. We observed DDX39B and DDX39A, DEAD-box RNA-Helicases, exert a dual effect on regulating polymerase activity and replication of influenza A viruses. We further revealed that DDX39B and DDX39A interact with viral NP and NS1 proteins. Interestingly, the viral NP proteins could reverse the inhibitory effect of excess DDX39B or DDX39A on polymerase activity. Mechanistically, the TREX complex subunits, THOC1, THOC4 and CIP29, were recruited to DDX39B-DDX39A-NP complex in an ATP-dependent manner, via the interaction with DDX39B or DDX39A, followed by excess TREX-NP complexes interfere with the normal oligomerization state of NP depending on the ratio between the viral and host proteins. On the other hand, the TREX complex, an evolutionarily conserved protein complex, is responsible for the integration of several mRNA processing steps to export viral mRNA. Knockdown of TREX complex subunits significantly down-regulated viral titers and protein levels, accompanied by retention of viral mRNA in the nucleus. Taken together, screening the host factors that regulate the replication of influenza virus advances our understanding of viral pathogenesis and our findings point out a previously unclear mechanism of TREX complex function.

An influenza virus vector candidate vaccine stably expressing SARS-CoV-2 receptor-binding domain produces high and long-lasting neutralizing antibodies in mice.

Viral infectious pathogens, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, can cause extremely high infection rates and mortality in humans. Therefore, it is urgent to develop an effective vaccine against coronavirus and influenza virus infection. Herein, we used the influenza virus as a vector to express the SARS-CoV-2 spike receptor-binding domain (RBD) and hemagglutinin-esterase-fusion (HEF) protein of the influenza C virus. We then evaluated the feasibility and effectiveness of this design strategy through experiments in vitro and in vivo. The results showed that the chimeric viruses could stably express the HEF protein and the SARS-CoV-2 spike RBD at a high level. BALB/c mice, infected with the chimeric virus, exhibited mild clinical symptoms, yet produced high specific antibody levels against RBD and HEF, including neutralizing antibodies. Importantly, high neutralizing antibodies could be retained in the sera of mice for at least 20 weeks. Altogether, our data provided a new strategy for developing safe and effective COVID-19 and influenza virus vaccines.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

DDX5/METTL3-METTL14/YTHDF2 Axis Regulates Replication of Influenza A Virus.

DEAD-box helicase 5 (DDX5), a member of the DEAD/H-box helicases, is known to participate in all aspects of RNA metabolism. However, its regulatory effect in antiviral innate immunity during replication of influenza virus remains unclear. Herein, we found that human DDX5 promotes replication of influenza virus in A549 cells. Moreover, our results further revealed that DDX5 relies on its N terminus to interact with the nucleoprotein (NP) of influenza virus, which is independent of RNA. Of course, we also observed colocalization of DDX5 with NP in the context of transfection or infection. However, influenza virus infection had no significant effect on the protein expression and nucleocytoplasmic distribution of DDX5. Importantly, we found that DDX5 suppresses antiviral innate immunity induced by influenza virus infection. Mechanistically, DDX5 downregulated the mRNA levels of interferon beta (IFN-ß), interleukin 6 (IL-6), and DHX58 via the METTL3-METTL14/YTHDF2 axis. We revealed that DDX5 bound antiviral transcripts and regulated immune responses through YTHDF2-dependent mRNA decay. Taken together, our data demonstrate that the DDX5/METTL3-METTL14/YTHDF2 axis regulates the replication of influenza A virus. IMPORTANCE The replication and transcription of influenza virus depends on the participation of many host factors in cells. Exploring the relationship between viruses and host factors will help us fully understand the characteristics and pathogenic mechanisms of influenza viruses. In this study, we showed that DDX5 interacted with the NP of influenza virus. We demonstrated that DDX5 downregulated the expression of IFN-ß and IL-6 and the transcription of antiviral genes downstream from IFN-ß in influenza virus-infected A549 cells. Additionally, DDX5 downregulated the mRNA levels of antiviral transcripts via the METTL3-METTL14/YTHDF2 axis. Our findings provide a novel perspective to understand the mechanism by which DDX5 regulates antiviral immunity.

Antigenic Evolution Characteristics and Immunological Evaluation of H9N2 Avian Influenza Viruses from 1994-2019 in China.

The H9N2 subtype avian influenza viruses (AIVs) have been circulating in China for more than 20 years, attracting more and more attention due to the potential threat of them. At present, vaccination is a common prevention and control strategy in poultry farms, but as virus antigenicity evolves, the immune protection efficiency of vaccines has constantly been challenged. In this study, we downloaded the hemagglutinin (HA) protein sequences of the H9N2 subtype AIVs from 1994 to 2019 in China-with a total of 5138 sequences. The above sequences were analyzed in terms of time and space, and it was found that h9.4.2.5 was the most popular in various regions of China. Furthermore, the prevalence of H9N2 subtype AIVs in China around 2006 was different. The domestic epidemic branch was relatively diversified from 1994 to 2006. After 2006, the epidemic branch each year was h9.4.2.5. We compared the sequences around 2006 as a whole and screened out 15 different amino acid positions. Based on the HA protein of A/chicken/Guangxi/55/2005 (GX55), the abovementioned amino acid mutations were completed. According to the 12-plasmid reverse genetic system, the rescue of the mutant virus was completed using A/PuertoRico/8/1934 (H1N1) (PR8) as the backbone. The cross hemagglutination inhibition test showed that these mutant sites could transform the parental strain from the old to the new antigenic region. Animal experiments indicated that the mutant virus provided significant protection against the virus from the new antigenic region. This study revealed the antigenic evolution of H9N2 subtype AIVs in China. At the same time, it provided an experimental basis for the development of new vaccines.

SRSF3 facilitates replication of influenza A virus via binding and promoting the transport of viral mRNA.

Many host factors were involved in regulating the polymerase activity of influenza A virus. To fully explore the role of polymerase complex-related host factors, we combined high-throughput transcriptome data to analyze the changes in mRNA levels of these factors during viral infection. Transcriptome data showed that viral infection caused down-regulation of MYH9, HNRNPU, SRSF3 and RPS24 mRNA levels. We confirmed the changes in mRNA and protein levels of MYH9, HNRNPU and SRSF3 by qPCR and WB. Then their effects on virus replication were tested through overexpression and knockdown experiments. We emphatically explained the mechanism of SRSF3 during influenza virus replication. Results showed that SRSF3 promoted influenza virus replication and regulated viral protein expression at the post-transcriptional level. Further analysis found that SRSF3 regulated viral replication depends on the 88th amino acid. RIP and FISH experiments further proved that SRSF3 bound to viral mRNA and participated in the nuclear and cytoplasmic transport of viral mRNA. Collectively, these findings suggested that virus infection regulated the expression of many host factors and SRSF3 positively regulated virus replication.

Mutations of 127, 183 and 212 residues on the HA globular head affect the antigenicity, replication and pathogenicity of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild-type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r-76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor-binding assays showed that all the recombination viruses were prone to bind the human-like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N-glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild-type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Evolution of the PB1 gene of human influenza A (H3N2) viruses circulating between 1968 and 2019.

One avian H3N2 influenza virus, providing its PB1 and HA segments, reassorted with one human H2N2 virus and caused a pandemic outbreak in 1968, killing over 1 million people. After its introduction to humanity, the pandemic H3N2 virus continued adapting to humans and has resulted in epidemic outbreaks every influenza season. To understand the functional roles of the originally avian PB1 gene in the circulating strains of human H3N2 influenza viruses, we analyzed the evolution of the PB1 gene in all human H3N2 isolates from 1968 to 2019. We found several specific residues dramatically changed around 2002-2009 and remained stable through to 2019. Then, we verified the functions of these PB1 mutations in the genetic background of the early pandemic virus, A/Hong Kong/1/1968(HK/68), as well as a recent seasonal strain, A/Jiangsu/34/2016 (JS/16). The PB1 V709I or PB1 V113A/K586R/D619N/V709I induced higher polymerase activity of HK/68 in human cells. And the four mutations acted cooperatively that had an increased replication capacity in vitro and in vivo at an early stage of infection. In contrast, the backward mutant, A113V/R586K/N619D/I709V, reduced polymerase activity in human cells. The PB1 I709V decreased viral replication in vitro, but this mutant only showed less effect on mice infection experiment, which suggested influenza A virus evolved in human host was not always consisted with highly replication efficiency and pathogenicity in other mammalian host. Overall, our results demonstrated that the identified PB1 mutations contributed to the viral evolution of human influenza A (H3N2) viruses.

Analysis of the circRNAs expression profile in mouse lung with H7N9 influenza A virus infection.

Influenza A virus is a single-stranded RNA virus that can cause great mortality and economic loss worldwide. Circular RNAs (circRNAs) are non-coding RNAs that have been shown to have important functions in the regulation of biological processes. However, their functions during the influenza A virus infection process remain unclear. Herein, RNA sequencing technology was used to identify circRNAs expressed in mouse lungs during infection with H7N9/PB2-627 K/701D (H7N9/Wild-type) virus and PB2 mutant viruses (H7N9/PB2-627E/701D and H7N9/PB2-627E/701 N). We identified 7126 circRNAs at different genomic locations during H7N9 influenza virus and its mutant virus infections, of which 186 were differentially expressed. Enrichment analysis revealed that the differentially expressed circRNAs were associated with the viral infection process. Our study shows that circRNA expression profiles were altered following H7N9 influenza A virus infection and the differentially expressed circRNAs may have an important immune-regulating function during viral infection.

Hemagglutinin stalk-based monoclonal antibody elicits broadly reactivity against group 1 influenza A virus.

BACKGROUND: Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. METHODS: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments. RESULTS: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What's more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy. CONCLUSIONS: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

Integrated Analysis of microRNA-mRNA Expression in Mouse Lungs Infected With H7N9 Influenza Virus: A Direct Comparison of Host-Adapting PB2 Mutants.

MicroRNAs (miRNAs) are important regulators involved in the antiviral response to influenza virus infection, however, an analytical comparison of miRNA and mRNA expression changes induced by several H7N9 host-adapting PB2 mutants remains undone. Here, miRNA microarray and transcriptome sequencing of BALB/c mouse lungs infected with A/Anhui/1/2013 (H7N9) [hereafter referred to as H7N9/AH1-PB2-627K(WT)] and mutant variants with PB2 amino acid substitutions (avian-like H7N9/AH1-PB2-627E and mammalian-adapted H7N9/AH1-PB2-627E/701N) were directly compared. The results showed that influenza virus infection induced dysregulation of numerous host cell processes. In a miRNA-mRNA network associated with immunity, changes in the expression of 38 miRNAs and 58 mRNAs were detected following influenza virus infection. Notably, the miRNAs of mmu-miR-188-5p, mmu-miR-511-5p, mmu-miR-483-5p, and mmu-miR-690 were specifically associated with the replication of the avian-like virus H7N9/AH1-PB2-627E. Likewise, the miRNAs of mmu-miR-691, mmu-miR-329-3p, and mmu-miR-144-3p were specifically associated with the mammalian-adapted virus H7N9/AH1-PB2-627E/701N. Finally, the miRNAs of mmu-miR-98-5p, mmu-miR-103-3p, mmu-miR-199a-5p, and mmu-miR-378a-3p were specifically associated with H7N9/AH1-PB2-627K(WT) virus replication. This is the first report of comparative integration analysis of miRNA-mRNA expression of these three H7N9 influenza viruses with different host-adapting PB2 mutations. Our results highlight potential miRNAs of importance in influenza virus pathogenesis.