Structure Of Influenza Research Articles

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Locations and structures of influenza A virus packaging-associated signals and other functional elements via an in silico pipeline for predicting constrained features in RNA viruses.

Influenza A virus contains regions of its segmented genome associated with ability to package the segments into virions, but many such regions are poorly characterised. We provide detailed predictions of the key locations within these packaging-associated regions, and their structures, by applying a recently-improved pipeline for delineating constrained regions in RNA viruses and applying structural prediction algorithms. We find and characterise other known constrained regions within influenza A genomes, including the region associated with the PA-X frameshift, regions associated with alternative splicing, and constraint around the initiation motif for a truncated PB1 protein, PB1-N92, associated with avian viruses. We further predict the presence of constrained regions that have not previously been described. The extra characterisation our work provides allows investigation of these key regions for drug target potential, and points towards determinants of packaging compatibility between segments.

The association of previous influenza vaccination and coronavirus disease-2019

ABSTRACT Studies have shown similarities in the structure of influenza and coronaviruses, in their binding receptors and in patterns of immune responses; and that influenza vaccine can induce cross-immunity. We examined the association of previous influenza vaccination and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, resulting in coronavirus disease-2019 (COVID-19), among 715,164 members of a health maintenance organization. In a multivariate regression model, the odds ratios for SARS-CoV-2 infection among individuals vaccinated for influenza in 2018–2019, 2019–2020, and in both seasons, compared to non-vaccinated individuals, were 0.82 (95% CI 0.68–0.99, p = .048), 0.79 (95% CI 0.67–0.98, p = .005), and 0.76 (95% CI 0.61–0.97, p = .004), respectively. Based on our findings, administration of influenza vaccine before the influenza season is highly recommended to reduce the burden of influenza, which is critical in scenarios of outbreaks of both influenza and SARS-CoV-2 infections, and also regarding its association with reduced rate of COVID-19.

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Influenza A viruses (IAV) are responsible for seasonal epidemics, and pandemics can arise from novel zoonotic influenza A viruses transmitting to humans1,2. IAV contain a segmented negative sense RNA genome that is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the PB1, PB2, and PA subunits3–5. Although the high-resolution crystal structure of bat IAV polymerase (FluPolA) has been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of viral RNA (vRNA) replication, which proceeds through a complementary RNA (cRNA) replicative intermediate and requires polymerase oligomerisation7–10, remain largely unknown. Here we report 3.0 – 4.3 Å resolution structures of polymerases from human A/NT/60/1968 (H3N2) and avian A/duck/Fujian/01/2002 (H5N1) IAVs, obtained by crystallography and cryo-electron microscopy (cryo-EM), in the presence or absence of cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the PA C-terminal domain and the PB1 thumb and PB2 N1 subdomains. A cryo-EM structure of a monomeric FluPolA, bound to cRNA template, reveals a binding site for the 3′ cRNA at the dimer interface. Using a combination of cell-based and in vitro assays we show that the FluPolA dimer interface is required for initiation of vRNA synthesis during viral genome replication. Furthermore, we show that a nanobody, a single-domain antibody, which interferes with FluPolA dimerisation, inhibits vRNA synthesis and consequently virus replication in infected cells. Our study provides the first high-resolution structures of medically relevant FluPolA and offers novel insights into the replication mechanisms of the viral RNA genome. Furthermore, it identifies novel sites of FluPolA that could be targeted for antiviral drug development.

MSphere of Influence: Resolution of the Structure of an Influenza Virus Polymerase Is a Game Changer.

Mathilde Richard works in the field of virology, more specifically on the evolution and pathogenesis of influenza viruses. In this mSphere of Influence article, she reflects on how the two articles "Structure of Influenza A Polymerase Bound to the Viral RNA Promoter" by A. Pflug, D. Guilligay, S. Reich, and S. Cusack (Nature 516:355-360, 2014, https://doi.org/10.1038/nature14008) and "Structural Insight into Cap-Snatching and RNA Synthesis by Influenza Polymerase" by S. Reich, D. Guilligay, A. Pflug, H. Malet, I. Berger, et al. (Nature 516:361-366, 2014, https://doi.org/10.1038/nature14009) made an impact on her by providing new grounds to study the influenza virus polymerase and its role in virus biology and evolution.

Using common spatial distributions of atoms to relate functionally divergent influenza virus N10 and N11 protein structures to functionally characterized neuraminidase structures, toxin cell entry domains, and non-influenza virus cell entry domains.

The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell entry has been supplemented or replaced by sialidase-independent receptor binding to an expanded cell population that may include neurons and T-cells.

Structure of influenza A polymerase bound to the viral RNA promoter.

The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3' and 5' extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5' extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design.

A recommended numbering scheme for influenza A HA subtypes.

Comparisons of residues between sub-types of influenza virus is increasingly used to assess the zoonotic potential of a circulating strain and for comparative studies across subtypes. An analysis of N-terminal cleavage sites for thirteen subtypes of influenza A hemagglutinin (HA) sequences, has previously been described by Nobusawa and colleagues. We have expanded this analysis for the eighteen known subtypes of influenza. Due to differences in the length of HA, we have included strains from multiple clades of H1 and H5, as well as strains of H5 and H7 subtypes with both high and low pathogenicity. Analysis of known structures of influenza A HA enables us to define amino acids which are structurally and functionally equivalent across all HA subtypes using a numbering system based on the mature HA sequence. We provide a list of equivalences for amino acids which are known to affect the phenotype of the virus.

Structure of influenza B nucleoprotein and its functional characterization

Structure of influenza B nucleoprotein and its functional characterization

Structures of influenza A proteins and insights into antiviral drug targets

The world is currently undergoing a pandemic caused by an H1N1 influenza A virus, the so-called 'swine flu'. The H5N1 ('bird flu') influenza A viruses, now circulating in Asia, Africa and Europe, are extremely virulent in humans, although they have not so far acquired the ability to transfer efficiently from human to human. These health concerns have spurred considerable interest in understanding the molecular biology of influenza A viruses. Recent structural studies of influenza A virus proteins (or fragments) help enhance our understanding of the molecular mechanisms of the viral proteins and the effects of drug resistance to improve drug design. The structures of domains of the influenza RNA-dependent RNA polymerase and the nonstructural NS1A protein provide opportunities for targeting these proteins to inhibit viral replication.

The Influenza-A mystery: insight from Bioinformatics resources and analysis.

Sir, Bioinformatics is a tool which can excavate any information, one wants to obtain. I found its implication in handling an emerging and highly pathogenic strain of Influenza virus, H5N1. The first influenza pandemic was happened in 1918 “Spanish flu” followed by Asian flu in 1957 caused by H2N2. Moreover, one million deaths were reported in Hong Kong due to H3N2 outbreak during 1968. The antigenic shift and drift of influenza A virus led to a new strain H1N1 in 1977 “Russian flu”. After two decades a new strain, H5N1 was evolved, called avian influenza virus. The outbreaks of this strain were continued till 2008 with the yearly epidemic in the world. In 2009, WHO has declared Influenza pandemic due to the outbreak of H1N1. The disease has infected over 35928 people in 80 countries, with 163 deaths. But it has been most severe in Mexico, which has reported the highest number of fatalities. The influenza virus is mysterious pathogen known for severe respiratory illness. It causes yearly epidemics in tropical and subtropical countries with epidemic and even pandemic flu, a major cause of morbidity and mortality. Recurrent appearance of influenza in different parts of the world is due to frequent mutations which may lead to either antigenic drift or shift. The structure of influenza A viral genome composed of eight segments of negative-sense single stranded RNA. Surface glycoprotein of influenza A virus, Hemagglutinin (HA) and Neuraminidase (NA) are the main components of viral genome to undergo reassortment and may cause to develop a new subtype or strain of Influenza virus. There are 16 different H antigens (H1 to H16) and nine different N antigens (N1 to N9). The latest H type (H16) was isolated from black-headed gulls found in Sweden and Netherlands during 1999 and was published in 2005. The history on influenza epidemic revealed circulation of these strains (H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H5N2 and H10N7) among human, pig and bird population of different regions on the globe [1]. The strains appeared during outbreaks in different parts of the world and with the gap of different time periods could be analyzed for their phylogeny to understand their origin or source of spread. Phylogenetic trees are built by Neighbour-joining program with Kimura 2 parameter available at phylip3.65 package [2]. It is drawn by using TreeView1.6 program [3]. We can further analyze genome using GOR IV method [4]. Different domains/motifs were searched by “Composite” available at Expasy server [5]. Moreover, Amino acids sequences are also compared by MAP MUTATION program [6] to analyze the mutations among these isolates. Homology modeling of template structure related to proteins was searched using Blast algorithms against PDB. Amino acids sequence of PB1-F2 protein of A/HK/156/97 was obtain from Swiss-Prot [ID: {type:entrez-protein,attrs:{text:P0C0U0,term_id:83288374,term_text:P0C0U0}}P0C0U0]. Homology modeling of PB1-F2 from A/HK/156/97 (H5N1) based on crystal structure of PB1-F2 protein [PDB id: 2HN8] from H1N1 subtypes with ≫69% identity, was built by modeler [7]. Rigid body docking of PB1-F2 protein with VDAC1 and ANT3 was performed by HEX5.1 [8,9]. Each docking solution was minimized by energy minimization technique. Details of interaction derived from refined structures were analyzed using the LIGPLOT program [10]. It is trouble-free as well as time saving to understand the insight of microbial genome to answer several hidden mysteries and therefore, the origin of viral strains during an outbreak, by exploring these tools can easily be identified. The viral genome databases, available on different sources, NCBI [11] and IVDB [12] could be used to retrieve the sequences for analysis.

Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

Structural basis for receptor specificity of influenza B virus hemagglutinin.

Receptor-binding specificity of HA, the major surface glycoprotein of influenza virus, primarily determines the host ranges that the virus can infect. Influenza type B virus almost exclusively infects humans and contributes to the annual "flu" sickness. Here we report the structures of influenza B virus HA in complex with human and avian receptor analogs, respectively. These structures provide a structural basis for the different receptor-binding properties of influenza A and B virus HA molecules and for the ability of influenza B virus HA to distinguish human and avian receptors. The structure of influenza B virus HA with avian receptor analog also reveals how mutations in the region of residues 194 to 196, which are frequently observed in egg-adapted and naturally occurring variants, directly affect the receptor binding of the resultant virus strains. Furthermore, these structures of influenza B virus HA are compared with known structures of influenza A virus HAs, which suggests the role of the residue at 222 as a key and likely a universal determinant for the different binding modes of human receptor analogs by different HA molecules.

Biology of Influenza A Virus

The outbreaks of avian influenza A virus in poultry and humans over the last decade posed a pandemic threat to human. Here, we discuss the basic classification and the structure of influenza A virus. The viral genome contains eight RNA viral segments and the functions of viral proteins encoded by this genome are described. In addition, the RNA transcription and replication of this virus are reviewed.

Structure of influenza a virus promoter and its implications for viral RNA synthesis.

Since the worst worldwide pandemic ever recorded — the 1918 Spanish influenza outbreak that killed more than 20 million people — we have achieved significant advances in understanding the influenza virus. However, the fear of such a pandemic remains strong. For example, in 1997, when a lethal influenza variant afflicted eight people in Hong Kong, contributing to the death of six, officials feared the next wave had begun. They managed to solve the problem quickly, however, by destroying all of the poultry in Hong Kong[1].

Effects of site-specific mutation on structure and activity of influenza virus B/Lee/40 neuraminidase

A cDNA copy of the neuraminidase (NA) gene of a high-growing reassortant influenza virus which has the hemagglutinin of B/Hong Kong/8/73 and the NA of B/Lee/40 was cloned into plasmid pUC 9 and subcloned into a late-replacement SV40 vector so that the NA gene could be expressed in eukaryotic cells. The expressed protein was antigenically and enzymatically active. To study structure-function relationships in the B/Lee NA, particularly in comparison to the known structure of influenza A (N2) NA, specific mutations were introduced using synthetic oligonucleotides. Mutation of an apparently unpaired cysteine residue to serine at position 251 had no effect on protein transport or folding as judged by cell-surface reactivity with monoclonal antibodies, and the NA retained enzyme activity, confirming that this Cys is not essential for correct folding of the polypeptide. Mutation of Trp 364 to Leu abolished detectable enzyme activity, while mutation of Thr 368 to Val reduced enzyme activity to less than 25% of wild-type levels. Neither mutation affected antigenic properties. Therefore it is likely that both these side chains extend into the NA active site pocket. The results of these experiments are in accord with similarities in the structure of the B/Lee NA compared with that of influenza A (N2) NA. Although there is about 70% amino acid sequence difference between influenza A and B NAs, residues in the active site are highly conserved. Our in vitro mutagenesis experiments help to confirm the tentative alignment of sequences and have identified conserved amino acid side chains involved in enzyme activity.