Hydrophobic Residues at the Intracellular Domain of the M2 Protein Play an Important Role in Budding and Membrane Integrity of Influenza Virus.

M2 protein of influenza virus plays an important role in virus budding, including membrane scission and vRNP packaging. Three hydrophobic amino acids (91F, 92V, and 94I) at the intracellular domain of the M2 protein constitute a hydrophobic motif, also known as the LC3-interacting region (LIR), whereas the role of this motif remains largely unclear. To explore the role of the 91-94 hydrophobic motif for influenza virus, all three hydrophobic amino acids were mutated to either hydrophilic S or hydrophobic A, resulting in two mutant viruses (WSN-M2/SSS and WSN-M2/AAA) in the background of WSN/H1N1. The results showed that the budding ability of the M2/SSS protein was inhibited and the bilayer membrane integrity of the WSN-M2/SSS virion was impaired based on transmission electron microscopy (TEM), which in turn abolished the resistance to trypsin treatment. Moreover, the mutant WSN-M2/SSS was dramatically attenuated in mice. In contrast, the AAA mutations did not have a significant effect on the budding of the M2 proteins or the bilayer membrane integrity of the viruses, and the mutant WSN-M2/AAA was still lethal to mice. In addition, although the 91-94 motif is an LIR, knocking out of the LC3 protein of A549 cells did not significantly affect the membrane integrity of the influenza viruses propagated on the LC3KO cells, which suggested that the 91-94 hydrophobic motif affected the viral membrane integrity and budding is independent of the LC3 protein. Overall, the hydrophobicity of the 91-94 motif is crucial for the budding of M2, bilayer membrane integrity, and pathogenicity of the influenza viruses. IMPORTANCE M2 plays a crucial role in the influenza virus life cycle. However, the function of the C-terminal intracellular domain of M2 protein remains largely unclear. In this study, we explored the function of the 91-94 hydrophobic motif of M2 protein. The results showed that the reduction of the hydrophobicity of the 91-94 motif significantly affected the budding ability of the M2 protein and impaired the bilayer membrane integrity of the mutant virus. The mouse study showed that the reduction of the hydrophobicity of the 91-94 motif significantly attenuated the mutant virus. All of the results indicated that the hydrophobicity of the 91-94 motif of the M2 protein plays an important role in budding, membrane integrity, and pathogenicity of influenza virus. Our study offers insights into the mechanism of influenza virus morphogenesis, particularly into the roles of the 91-94 hydrophobic motif of M2 in virion assembly and the pathogenicity of the influenza viruses.

N-Linked Glycosylation Plays an Important Role in Budding of Neuraminidase Protein and Virulence of Influenza Viruses.

Neuraminidase (NA) has multiple functions in the life cycle of influenza virus, especially in the late stage of virus replication. Both of hemagglutinin (HA) and NA are highly glycosylated proteins. N-linked glycosylation (NLG) of HA has been reported to contribute to immune escape and virulence of influenza viruses. However, the function of NLG of NA remains largely unclear. In this study, we found that NLG is critical for budding ability of NA. Tunicamycin treatment or NLG knockout significantly inhibited the budding of NA. Further studies showed that the NLG knockout caused attenuation of virus in vitro and in vivo Notably, the NLG at 219 position plays an important role in the budding, replication, and virulence of H1N1 influenza virus. To explore the underlying mechanism, the unfolded protein response (UPR) was determined in NLG knockout NA overexpressed cells, which showed that the mutant NA was mainly located in the endoplasmic reticulum (ER), the UPR markers BIP and p-eIF2α were upregulated, and XBP1 was downregulated. All the results indicated that NLG knockout NA was stacked in the ER and triggered UPR, which might shut down the budding process of NA. Overall, the study shed light on the function of NLG of NA in virus replication and budding.IMPORTANCE NA is a highly glycosylated protein. Nevertheless, how the NLG affects the function of NA protein remains largely unclear. In this study, we found that NLG plays important roles in budding and Neuraminidase activity of NA protein. Loss of NLG attenuated viral budding and replication. In particular, the 219 NLG site mutation significantly attenuated the replication and virulence of H1N1 influenza virus in vitro and in vivo, which suggested that NLG of NA protein is a novel virulence marker for influenza viruses.

Pathogenicity of reassortant H9 influenza viruses with different NA genes in mice and chickens.

To better understand the influence of different NA genes on pathogenicity of H9 viruses, three reassortant H9 viruses (rH9N1, H9N2 and rH9N3) were generated and characterized. All three viruses replicated efficiently in eggs and MDCK cells, whereas the rH9N1 and rH9N3 replicated more efficiently than H9N2 in A549 cells. The rH9N3 replicated more efficiently than rH9N1 and H9N2 viruses in mice, however, rH9N3 replicated and shed less efficiently than the H9N2 virus in chickens. Further studies indicate that N3 had higher NA activity and released virus from erythrocytes faster, which may improve the adaptation of H9 influenza virus to mammals.

RNA-Seq Analysis of Influenza A Virus-Induced Transcriptional Changes in Mice Lung and Its Possible Implications for the Virus Pathogenicity in Mice.

The influenza A virus (IAV) is an important cause of respiratory disease worldwide. It is well known that alveolar epithelial cells are the target cells for the IAV, but there is relatively limited knowledge regarding the role of macrophages during IAV infection. Here, we aimed to analyze transcriptome differences in mouse lungs and macrophage (RAW264.7) cell lines infected with either A/California/04/2009 H1N1 (CA09) or A/chicken/SD/56/2015 H9N2 (SD56) using deep sequencing. The uniquely differentially expressed genes (UDEGs) were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; the results showed that the lungs infected with the two different viruses had different enrichments of pathways and terms. Interestingly, CA09 virus infection in mice was mostly involved with genes related to the extracellular matrix (ECM), while the most significant differences after SD56 infection in mice were in immune-related genes. Gene set enrichment analysis (GSEA) of RAW264.7 cells revealed that regulation of the cell cycle was of great significance after CA09 infection, whereas the regulation of the immune response was most enriched after SD56 infection, which was consistent with analysis results in the lung. Similar results were obtained from weighted gene co-expression network analysis (WGCNA), where cell cycle regulation was extensively activated in RAW264.7 macrophages infected with the CA09 virus. Disorder of the cell cycle is likely to affect their normal immune regulation, which may be an important factor leading to their different prognoses. These results provide insight into the mechanism of the CA09 virus that caused a pandemic and explain the different reactivities of monocytes/macrophages infected by H9N2 and H1N1 IAV subtypes.

Getting insight into the influence of coexisting airborne nanoparticles on gas adsorption performance over porous materials.

Adsorption as one of the most important air cleaning methods has been extensively applied during which the coexisting airborne nanoparticles (NPs) with sizes close to adsorbent pore sizes could inevitably influence gas adsorption processes. In this work, the influence of sub-20 nm NPs on toluene adsorption on ZSM-5 zeolites exchanged with different cations (Li+, Na+ and K+) were studied based on gas-and-particle coexisting adsorption/filtration tests. Affinities for both toluene and NPs on adsorbents follow Li-ZSM-5 > Na-ZSM-5 > K-ZSM-5 regarding the orders of charge density, pore size, and internal and external specific surface areas. The toluene adsorption was shown to be impaired by coexisting NPs from perspectives of thermodynamics and kinetics. For Li-ZSM-5, Na-ZSM-5 and K-ZSM-5, significant relative reductions of 10.4 %, 10.5 % and 16.0 % in toluene adsorption capacity at the lower feed concentration, and of 20.3 %, 15.2 % and 2.3 % in mass transfer coefficient at the higher feed concentration were observed, respectively. The influential mechanisms regarding competitiveness between toluene and NPs in interaction with cationic and porous surfaces were accordingly proposed, which are of practical significance for selecting robust adsorbents under realistic harsh air conditions.

Limited adaptation of chimeric H9N2 viruses containing internal genes from bat influenza viruses in chickens.

Influenza virus-like sequences of H17N10 and H18N11 were identified in bats, despite there has been no live virus isolated. The genetic analysis indicated that they have distinct but relatively close evolutionary relationships to known influenza A viruses. However, the infectivity and adaptation of bat influenza viruses in avian species remain unclear. In this study, two modified bat influenza viruses cH9cN2/H17 and cH9cN2/H18 containing HA and NA coding regions replaced with those of H9N2 influenza A virus were generated in the background of the H17N10 or H18N11 viruses. These two modified viruses replicated less efficiently than wild type H9N2 virus in cultured chicken cells. The mini-genome assay showed that viral ribonucleoproteins (vRNPs) of H9N2 has significantly higher polymerase activity than that of bat influenza viruses in avian cells. In chicken study, compared with H9N2 virus, which replicated and transmitted efficiently in chickens, the cH9cN2/H17 and cH9cN2/H18 viruses only replicated in chicken tracheas with lower titers. Pathological examination showed that the H9N2 caused severer lesions in lung and trachea than the modified bat influenza viruses. Notably, the cH9cN2/H18 transmitted among chickens, but not cH9cN2/H17, and chicken IFN-ß antagonism results showed that H18N11 NS1 protein inhibited chicken IFN-ß response more efficiently than H17N10 NS1 protein in avian cells. Taken together, our data indicated that the internal genes of bat influenza viruses adapted poorly to chickens, while the internal genes of H18N11 seemed to adapt to chickens better than H17N10.