A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC.

The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1WSN and H3N8JL89. We found that the promotion of self-replication could be mainly attributed to NS1pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines.Abbreviations: 293T: human embryonic kidney 293 cells; 293T_LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1JL89: non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1pdm09: non-structural protein 1 of A/(H1N1) pdm09; NS1SC09: non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1WSN: non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1JL89: WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1SC09: WSN recombinant strain in which NS1 was replaced with that of SC09.

Human genes with codon usage bias similar to that of the nonstructural protein 1 gene of influenza A viruses are conjointly involved in the infectious pathogenesis of influenza A viruses.

Molecular mechanisms of the non-structural protein 1 (NS1) in influenza A-induced pathological changes remain ambiguous. This study explored the pathogenesis of human infection by influenza A viruses (IAVs) through identifying human genes with codon usage bias (CUB) similar to NS1 gene of these viruses based on the relative synonymous codon usage (RSCU). CUB of the IAV subtypes H1N1, H3N2, H3N8, H5N1, H5N2, H5N8, H7N9 and H9N2 was analyzed and the correlation of RSCU values of NS1 sequences with those of the human genes was calculated. The CUB of NS1 was uneven and codons ending with A/U were preferred. The ENC-GC3 and neutrality plots suggested natural selection as the main determinant for CUB. The RCDI, CAI and SiD values showed that the viruses had a high degree of adaptability to human. A total of 2155 human genes showed significant RSCU-based correlation (p < 0.05 and r > 0.5) with NS1 coding sequences and was considered as human genes with CUB similar to NS1 gene of IAV subtypes. Differences and similarities in the subtype-specific human protein-protein interaction (PPI) networks and their functions were recorded among IAVs subtypes, indicating that NS1 of each IAV subtype has a specific pathogenic mechanism. Processes and pathways involved in influenza, transcription, immune response and cell cycle were enriched in human gene sets retrieved based on the CUB of NS1 gene of IAV subtypes. The present work may advance our understanding on the mechanism of NS1 in human infections of IAV subtypes and shed light on the therapeutic options.

Mutation W222L at the Receptor Binding Site of Hemagglutinin Could Facilitate Viral Adaption from Equine Influenza A(H3N8) Virus to Dogs.

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., α2,3-linkage or α2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses.IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., α2,3-linked and α2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers.

Recent H3N2 Viruses Have Evolved Specificity for Extended, Branched Human-type Receptors, Conferring Potential for Increased Avidity.

Human and avian influenza viruses recognize different sialic acid-containing receptors, referred to as human-type (NeuAcα2-6Gal) and avian-type (NeuAcα2-3Gal), respectively. This presents a species barrier for aerosol droplet transmission of avian viruses in humans and ferrets. Recent reports have suggested that current human H3N2 viruses no longer have strict specificity toward human-type receptors. Using an influenza receptor glycan microarray with extended airway glycans, we find that H3N2 viruses have in fact maintained human-type specificity, but they have evolved preference for a subset of receptors comprising branched glycans with extended poly-N-acetyl-lactosamine (poly-LacNAc) chains, a specificity shared with the 2009 pandemic H1N1 (Cal/04) hemagglutinin. Lipid-linked versions of extended sialoside receptors can restore susceptibility of sialidase-treated MDCK cells to infection by both recent (A/Victoria/361/11) and historical (A/Hong Kong/8/1968) H3N2 viruses. Remarkably, these human-type receptors with elongated branches have the potential to increase avidity by simultaneously binding to two subunits of a single hemagglutinin trimer.

Comparing the functions of equine and canine influenza H3N8 virus PA-X proteins: Suppression of reporter gene expression and modulation of global host gene expression.

The influenza PA-X protein is translated from the PA open reading frame from frameshifting and suppresses cellular gene expression due to its ribonuclease activity. We further defined the functional roles of PA-X by comparing PA-X proteins from two related viruses - equine influenza (EIV) and canine influenza (CIV) H3N8 - that differ in a C-terminal truncation and internal mutations. In vitro reporter gene assays revealed that both proteins were able to suppress gene expression. Interestingly, EIV PA-X demonstrated ~50% greater activity compared to CIV PA-X, and we identified the mutations that caused this difference. We used RNA-seq to evaluate the effects of PA-X on host gene expression after transfection into cultured cells. There were no significant differences in this property between EIV and CIV PA-X proteins, but expression of either resulted in the up-regulation of genes when compared to controls, most notably immunity-related proteins, trafficking proteins, and transcription factors.

Revelation of Influencing Factors in Overall Codon Usage Bias of Equine Influenza Viruses.

Equine influenza viruses (EIVs) of H3N8 subtype are culprits of severe acute respiratory infections in horses, and are still responsible for significant outbreaks worldwide. Adaptability of influenza viruses to a particular host is significantly influenced by their codon usage preference, due to an absolute dependence on the host cellular machinery for their replication. In the present study, we analyzed genome-wide codon usage patterns in 92 EIV strains, including both H3N8 and H7N7 subtypes by computing several codon usage indices and applying multivariate statistical methods. Relative synonymous codon usage (RSCU) analysis disclosed bias of preferred synonymous codons towards A/U-ended codons. The overall codon usage bias in EIVs was slightly lower, and mainly affected by the nucleotide compositional constraints as inferred from the RSCU and effective number of codon (ENc) analysis. Our data suggested that codon usage pattern in EIVs is governed by the interplay of mutation pressure, natural selection from its hosts and undefined factors. The H7N7 subtype was found less fit to its host (horse) in comparison to H3N8, by possessing higher codon bias, lower mutation pressure and much less adaptation to tRNA pool of equine cells. To the best of our knowledge, this is the first report describing the codon usage analysis of the complete genomes of EIVs. The outcome of our study is likely to enhance our understanding of factors involved in viral adaptation, evolution, and fitness towards their hosts.

Recent evolution of equine influenza and the origin of canine influenza.

In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222 → Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30 → Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.

Formation of raft-like assemblies within clusters of influenza hemagglutinin observed by MD simulations.

The association of hemagglutinin (HA) with lipid rafts in the plasma membrane is an important feature of the assembly process of influenza virus A. Lipid rafts are thought to be small, fluctuating patches of membrane enriched in saturated phospholipids, sphingolipids, cholesterol and certain types of protein. However, raft-associating transmembrane (TM) proteins generally partition into Ld domains in model membranes, which are enriched in unsaturated lipids and depleted in saturated lipids and cholesterol. The reason for this apparent disparity in behavior is unclear, but model membranes differ from the plasma membrane in a number of ways. In particular, the higher protein concentration in the plasma membrane may influence the partitioning of membrane proteins for rafts. To investigate the effect of high local protein concentration, we have conducted coarse-grained molecular dynamics (CG MD) simulations of HA clusters in domain-forming bilayers. During the simulations, we observed a continuous increase in the proportion of raft-type lipids (saturated phospholipids and cholesterol) within the area of membrane spanned by the protein cluster. Lateral diffusion of unsaturated lipids was significantly attenuated within the cluster, while saturated lipids were relatively unaffected. On this basis, we suggest a possible explanation for the change in lipid distribution, namely that steric crowding by the slow-diffusing proteins increases the chemical potential for unsaturated lipids within the cluster region. We therefore suggest that a local aggregation of HA can be sufficient to drive association of the protein with raft-type lipids. This may also represent a general mechanism for the targeting of TM proteins to rafts in the plasma membrane, which is of functional importance in a wide range of cellular processes.

A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus.

A latex agglutination test (LAT) was developed for detecting antibodies against avian influenza virus. The recombinant avian influenza virus nucleoprotein expressed in Escherichia coli was purified, coupled with latex beads, and used as an antigen for the LAT. The LAT was capable of detecting anti-avian influenza virus antibodies irrespective of the avian-influenza subtype, and in most cases, the results correlated with the results of an agar gel precipitation test (AGPT). However, in comparison with the AGPT, the LAT could detect the anti-avian influenza virus antibodies for a longer period of time after the infection. The nonspecific agglutination observed in uninfected chicken sera was resolved by pretreating the sera with dried chicken-liver powder for 1 h. The LAT is easy to perform, and even after considering the time required for pretreatment of the serum, the total time required for obtaining the results is reduced in comparison to the time required in the case of the AGPT. This easy and rapid LAT is considered to be useful for monitoring avian influenza virus infection in the field.

Acquisition of a polybasic hemagglutinin cleavage site by a low-pathogenic avian influenza virus is not sufficient for immediate transformation into a highly pathogenic strain.

Highly pathogenic avian influenza viruses (HPAIV) differ from all other strains by a polybasic cleavage site in their hemagglutinin. All these HPAIV share the H5 or H7 subtype. In order to investigate whether the acquisition of a polybasic cleavage site by an avirulent avian influenza virus strain with a hemagglutinin other than H5 or H7 is sufficient for immediate transformation into an HPAIV, we adapted the hemagglutinin cleavage site of A/Duck/Ukraine/1/1963 (H3N8) to that of the HPAIV A/Chicken/Italy/8/98 (H5N2), A/Chicken/HongKong/220/97 (H5N1), or A/Chicken/Germany/R28/03 (H7N7) and generated the recombinant wild-type and cleavage site mutants. In contrast to the wild type, multicycle replication of these mutants in tissue culture was demonstrated by positive plaque assays and viral multiplication in the absence of exogenous trypsin. Therefore, in vitro all cleavage site mutants resemble an HPAIV. However, in chicken they did not exhibit high pathogenicity, although they could be reisolated from cloacal swabs to some extent, indicating enhanced replication in vivo. These results demonstrate that beyond the polybasic hemagglutinin cleavage site, the virulence of HPAIV in chicken is based on additional pathogenicity determinants within the hemagglutinin itself or in the other viral proteins. Taken together, these observations support the notion that acquisition of a polybasic hemagglutinin cleavage site by an avirulent strain with a non-H5/H7 subtype is only one among several alterations necessary for evolution into an HPAIV.

Monitoring influenza virus content in vaccine production: precise assays for the quantitation of hemagglutination and neuraminidase activity.

Robust and precise quantitation of influenza virus is a premise for the efficient development of vaccine production processes. In this article, revised assays for the determination of hemagglutination (HA) and neuraminidase (NA) activity are presented. Bias of traditional discontinuous HA assays and operator dependency was overcome by introduction of a regression procedure. At little effort, a continuous assay result is obtained with repeatability as good as +29%/-22% in the best case (95% confidence intervals reported). Similarly, neuraminidase activity determined in microtiter plates resulted in repeatability better than +/-20%. NA activity decreased almost linearly for pH ranging from 5.8 to 7.8 and was enhanced by the addition of Ca2+. Non-linearity of the assay (due to unspecific adsorption) was overcome by addition of BSA. Using 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid as substrate, Michaelis-Menten constants of 30 and 460 microM were determined for strains A/PR/8/34 (H1N1) and A/Equi 2/NM/1/93 (H3N8), respectively. The error introduced by approximation of Michaelis-Menten kinetics (zero and first order) was minimized by limiting substrate consumption to about 10%. Linearity of both assays was verified in dilution experiments. Applicability was demonstrated in three cases: virus propagation in mammalian cell culture, ultrafiltration and precipitation of nucleic acids.

Localization of influenza virus sialoreceptors in equine respiratory tract.

This study was performed to identify the equine respiratory tract areas which express the specific receptor for equine influenza virus; findings may be useful to provide new ways to treat the infectious disease. The present work aims to visualize in situ the presence of sialoderivatives in the horse respiratory tract in order to localize sialoderivatives acting as influenza virus receptors. To this purpose, nasal mucosae, trachea, bronchus and lung parenchyma were removed from 8 mature horses of both sexes. We performed sialic acid characterization by means of mild and strong periodate oxidation and saponification, combined with lectin histochemistry and sialidase digestion, in addition to the direct evidentiation of sialic acid residues. No differences were shown between sexes. Sialic acid residues are present in the nasal mucous cell secretion, where they are linked to galactose by means of alpha2-3 linkage and are mainly C9 acetylated, and in the nasal and tracheal epithelial lining, where they are represented by periodate labile residues (alpha2-3)- and/or (alpha2-6)- linked to galactose. Specific receptors for equine influenza viruses are present at the nasal and tracheal epithelial lining cell coat levels, and in some trachea epithelial cells, but the horse possesses a preventive defence, which consists of the secretion of a mucous layer at nasal level, which could specifically inactivate the hemagglutinins of equine influenza virus; in addition, it expresses other sialoreceptors which can mask the influenza specific ones.

[Relationship of nuclear import of influenza virus nucleoprotein polymers to their conformational status].

It has been earlier shown that in the cells infected with influenza virus, the molecules of nucleoprotein (NP) are polymers that differ in their conformational maturity and stability. The present investigation has studied the ability of different conformational forms of NP polymers to migrate into the nucleus. Conformationally mature compact NP oligomers are shown to predominantly import into the nucleus. In contrast, unstable, loose, and conformationally immature NP multimers accumulate in the cytoplasm and do not migrate into the nucleus. The present investigation is the first evidence for that that the conformational maturity of influenza virus NPs is essential for their nuclear traffic and, hence, for participation in the transcription and replication of viral genomes.