Influenza Virus Research

The influenza viral Hemagglutinin (HA) protein is a homo trimer with a receptor binding pocket on the globular head of each monomer. Hemagglutinin (HA) protein is translated in cells as a single protein, HA0, or hemagglutinin precursor protein. For viral activation, hemagglutinin precursor protein (HA0) must be cleaved by a trypsin-like serine endoprotease at a specific site, normally coded for by a single basic amino acid (usually arginine) between the HA1 and HA2 domains of the protein. After cleavage, the two disulfide-bonded protein domains produce the mature form of the protein subunits as a prerequisite for the conformational change necessary for fusion and hence viral infectivity.

Influenza Hemagglutinin Structure

The hemagglutinin (HA) membrane glycoprotein of influenza virus is a trimer of identical subunits that are formed of two disulfide-linked polypeptides:
• Membrane-distal, HA1
• Membrane-proximal, HA2

The two domain are also called a triple-stranded coiled-coil of alpha-helices extends 76 A from the membrane and a globular region of antiparallel beta-sheet, which contains the receptor binding site and the variable antigenic determinants, is positioned on top of this stem. Each subunit has an unusual loop-like topology, starting at the membrane, extending 135 A distally and folding back to enter the membrane.
Influenza type A viruses into 16 HA (H1–H16) subtypes. Phylogenetically, there are two groups of HAs: group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and group 2 contains H3, H4, H7, H10, H14, and H15

Influenza neuraminidase is on the surface of influenza viruses that enables the virus to be released from the host cell. Neuraminidases are enzymes that cleave sialic acid groups from glycoproteins and are required for influenza virus replication. In addition to the mutations that arise due to antigenic drift, the NA of influenza A viruses (IAVs) can exist in different forms.Based on HA and NA antigenicity using serologic tests with hyperimmune sera, there have been a total of 16 HA (H1-16) and 9 NA (N1-9) subtypes identified in birds.Nine subtypes of influenza A NA are divided into two phylogenic groups. The first group consists of the neuraminidases of N1, N4, N5 and N8 subtypes, and the second one consists of N2, N3, N6 N7 and N9 subtypes.These are expressed in numerous combinations of viruses isolated from aquatic avian species, and an additional two combinations, H17N10 and H18N11, have been identified in bats

Influenza Neuraminidase Structure

The neuraminidase (NA) assembles as a tetramer of four identical polypeptides.The four monomers fold into four distinct structural domains:
• cytoplasmic tail
• transmembrane region
• stalk
• catalytic head

NA tetramer exists in local clusters on the virion surface or as isolated spikes surrounded by HA. Reduced stalk length may impact the ability of NA to contact sialic acids on mucins or cellular receptors as neighboring HA may sterically hinder its approach. Depending on the length of the stalk region, the NA may protrude slightly more or less above the viral envelope than the HA, which may influence the overall enzymatic activity of the virus.

Influenza virus NP is a major structural protein in Influenza virus particles and has multiple functions in the viral infectious cycle. Nucleoprotein (NP) is major component of the ribonucleoprotein complex and a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. NP binds RNA with high affinity in a sequence-independent manner. Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers while the wild type (wt) nucleoprotein is trimers. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes.

Influenza Nucleoprotein Structure

In Influenza A, Nucleoprotein is composed of a head and a body domain and a tail loop/ linker region. The head domain is more conserved than the body domain. NP proteins assume the overall shape of a crescent with a head and a body domain. In between the two domains is a deep groove enriched for basic amino acid residues and thus may function as the RNA-binding site.
Structure of Influenza D NP is a tetramer.

Here is the illustration of functional domains of NP.

Sub-fragments of NP identified as capable of binding RNA (blue), NP (green) or PB2 (yellow) are indicated on a linear representation of the NP molecule. Numbers refer to the amino acid co-ordinates. Also indicated is a C-terminal acidic region (red), which acts as a repressor of PB2 and NP binding. Black bars indicate regions shown to be important for binding the cellular polypeptides actin, BAT1/UAP56, importin α (NLS I) and/or function as nuclear localization signals (NLS I and II) or as a cytoplasmic accumulation signal (CAS).

The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins. The various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways.

Influenza Nonstructural protein 1 / NS1 Structure

NS1 is notionally divided into two distinct functional domains: an N-terminal RNA-binding domain (residues 1–73), which in vitro binds with low affinity to several RNA species in a sequence independent manner, and a C-terminal ‘effector’ domain (residues 74–230), which predominantly mediates interactions with host-cell proteins, but also functionally stabilizes the RNA-binding domain. Full-length NS1 likely exists as a homodimer, with both the RNA binding and effector domains contributing to multimerization.

NS1 has a strain-specific length of 230–237 aa, and an approximate molecular mass of 26 kDa. The N-terminal 73 amino acids form a functional RNA-binding domain, whilst the effector domain predominantly mediates interactions with host-cell proteins. The final C-terminal ~20 amino acids may be natively unstructured. NS1 contains two nuclear localization sequences (NLS1 and NLS2), and a nuclear export sequence (NES). A nucleolar localization sequence (NoLS) has been reported for some strains, and is concomitant with NLS2.

NS1 is composed of N-terminal RNA-binding domain (RBD, amino acids 1–73) and C-terminal effector domain (ED, amino acids 74–207).

Schematic ribbon diagrams of the RNA-binding domain (PDB ID,2N74) and effector domain (PDB ID, 3EE8).

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NS2 is also described as nuclear export protein (NEP). In Influenza A virus and Influenza B virus, the NS2 protein (nuclear export protein) is proposed to mediate the nucleocytoplasmic trafficking of viral ribonucleoprotein (vRNP) by forming NS2-vRNP complexes. NS2 protein is a structural component of the viral particle and it associates with the viral matrix M1 protein. New and unexpected roles for NEP during the influenza virus life cycle have started to emerge. These recent studies have shown NEP to be involved in regulating the accumulation of viral genomic vRNA and antigenomic cRNA as well as viral mRNA synthesized by the viral RNA-dependent RNA polymerase.

Influenza Nonstructural protein 2 / NS2 Structure

NS2/NEP can be divided into a protease-sensitive N-terminal domain (amino acids 1–53) and a protease-resistant C-terminal domain (amino acids 54–121). The NES is proposed to interact with the cellular nuclear export protein Crm1 and is unusual in the sense that three of the five critical hydrophobic residues are methionines rather than the canonical leucine.NEP is phosphorylated during the influenza replication cycle. The phosphorylation of a highly conserved serine-rich motif (S23, S24, and S25) proximal to the NES has recently been demonstrated in virion-associated NEP.
The highly structured C-terminal domain consists of two ahelices C1 (amino acids 64–85) and C2 (amino acids 94–115) that are connected by a short interhelical turn. The two a-helices are comparable in length and interact extensively, forming an almost perfectly antiparallel hairpin. N-terminal domain effectively buries the hydrophobic face of the C-terminal hairpin.

Organisation and structure of influenza virus A NEP

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Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The matrix protein M1 protein is membrane associates and forms a rigid matrix layer under the viral envelope utilizing multiple protein-lipid and protein-protein interactions. M1 protein is a candidate antigen for a broad-spectrum influenza virus vaccine and the adjuvant chitosan significantly improved the efficacy of the M1 vaccine.

Influenza Matrix 1 Structure

M1 is a structurally polarized molecule with a highly-ordered NM-fragment and a potentially unstructured C-terminal domain. The M1 protein consists of 252 amino acid residues with a calculated molecular mass (MM) based on the amino acid sequence of ,28 kDa. Numerous attempts to crystallize and solve the structure of full length M1 have been unsuccessful and consequently no high resolution model exists for the entire protein. The crystal structure of the 18 kDa NM domain (amino acids 1-164) has been determined at pH 4. Unfortunately, crystallographic studies have not yet provided structural information about the C-terminal domain of M1.

The matrix 2 M2 protein is a proton-selective ion channel protein, integral in the viral envelope of the influenza A virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds. It is activated by low pH. M2 protein is encoded on the seventh RNA segment together with the matrix protein M1. Proton conductance by the M2 protein in influenza A is essential for viral replication

Influenza Matrix 2 Structure

The influenza virus M2 protein is a 97-amino-acid integral membrane protein that forms disulfide-linked tetramers. M2 is predominantly associated with its wellcharacterized proton channel activity. During the virus entry process, this activity allows for the acidification of the virion interior, which permits vRNP release from M1.

• The C-terminal 54 amino acids of M2: Form the highly conserved cytoplasmic tail, which is important for both the assembly and budding processes but has little effect on the M2 proton channel activity. The membrane-distal region of the cytoplasmic tail has been shown to be critical for the incorporation of vRNPs into budding particles.
• The membrane-proximal region of M2 : Induce membrane curvature and has been implicated in ESCRT-independent membrane scission and budding of IAV particles.

PA is a key protein in the influenza virus polymerase complex, although its role was only partly outlined until recently. PA contains an N-terminal endonuclease domain (PAN) that interacts with PB26 and a C-terminal domain (PA-CTD) that interacts with PB1. There have been reports of peptide inhibitors and small molecule inhibitors that bind to PA and disrupt the PA-PB1 interaction.

Influenza polymerase acidic / PA Structure

PA can be cleaved by limited tryptic digestion into two domains: a smaller N-terminal domain of ~25 kD and a larger C-terminal of ~55 kD. The two domains are separated by a long linker peptide. Neither the N-terminal nor C-terminal domains of PA could ensure a stable interaction with PB1 without the presence of the linker.

The crystal structure of the N-terminal domain of PA, which has been implicated in a diverse range of functions, such as endonuclease and protease activities.The PAN structure has an α/β architecture with five mixed β-strands (β1-5) forming a twisted plane surrounded by seven α-helices (α1-7). A strongly negatively charged cavity, formed by a concentration of acidic residues, is surrounded by helices α2−α5 and strand β3 and houses a metal binding site. Structural comparison of PAN revealed a close match with other endonucleases, pointing towards PAN as a new member of the (P)DXN(D/E)XK endonuclease superfamily.

Organisation and structure of influenza virus A NEP

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PB1 is the RNA-dependent RNA polymerase (RdRP) subunit, which carries out viral mRNA and genomic RNA synthesis in reactions with the cap-binding subunit PB2 and the endonuclease subunit PA. The influenza virus PB1 protein is the core subunit of the heterotrimeric polymerase complex (PA, PB1 and PB2) in which PB1 is responsible for catalyzing RNA polymerization and binding to the viral RNA promoter.

Influenza Polymerase basic 1/ PB1 Structure

There is no crystal structure available for PB1 protein except its N terminal 25 amino acids and C terminal about 80 amino acids. This is mainly due to its low solubility when expressed alone in bacteria or the low productivity of the active heterotrimeric polymerase complex when expressed in insect cells and mammalian cells.

PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. These four conserved motifs form a large functional domain with at least one ‘invariant’ amino acid per motif.

The PB2 subunit of the influenza virus RNA polymerase is a major determinant of viral pathogenicity. The molecular mechanisms of how PB2 determines pathogenicity remain poorly understood. PB2 is responsible for binding to the 5΄ cap of nascent host pre-mRNAs to facilitate cleavage by PA into short capped RNA fragments, which are used as primers for viral transcription. Meanwhile, PB2 has been implicated in virulence and host adaptation. PB2 associates with mitochondria and inhibits the function of the mitochondrial antiviral signaling protein MAVS, implicating PB2 in the regulation of innate immune responses.

Influenza polymerase basic 2/ PB2 Structure

PB2 is an 87-kDa basic cap-binding protein involved in the initiation of viral transcription as well as viral replication. The N- and C-terminal egions of PB2 contain independent NP binding sites, located between residues 1–269 and 580–683, respectively. The interaction of PB2 with NP affected the activity of RNPs, and may be involved in regulating the switch from viral transcription to replication. Two regions of PB2 (aa 448–496 and aa 736–739) were required for the nuclear localization of influenza virus PB2.

An N-terminal mitochondrial targeting sequence (MTS) was found to be responsible for the mitochondrial localization of PB2. Despite high sequence conservation in this region, position 9 shows variations in influenza A viruses of different hosts and is a determinant of the mitochondrial localization of PB2. Most human seasonal influenza virus pre-2009 H1N1, H2N2, and H3N2 strains encode mitochondrial PB2 with asparagine at position 9 (N9-PB2).

H2N2 is also called Asian Flu which has emerged in East Asia, triggering a pandemic in 1957. H2N2 virus is originated from an avian influenza A virus, including the H2 hemagglutinin and the N2 neuraminidase genes. It was first reported in Singapore in February 1957, Hong Kong in April 1957, and in coastal cities in the United States in summer 1957. The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic.

H7N7 was firstly detected in chickens in Italy in 1902. Since then, H7N7 viruses have been widely detected in wild birds and domestic poultry around the world. There are highly pathogenic strains and low pathogenic strains of influenza H7N7. H7N7 avian influenza viruses can cross-species (birds, pigs, seals, and horses) and transmit to humans.
The first reported human infection of H7N7 virus occurred in England in1996, when a woman exposed to ducks developed conjunctivitis. In 2003, an outbreak of highly pathogenic H7N7 virus occurred in the Netherlands, resulting in the culling of more than 30 million birds. During this outbreak, 86 humans involved in the culling operation and three of their family members who did
not have direct contact with infected poultry were confirmed to have H7N7 virus infections, suggesting that limited human-to- human transmission of the H7N7 virus occurred. In 2013 in Italy, three poultry workers with conjunctivitis were diagnosed with highly pathogenic H7N7 avian influenza virus infections. This represents a pandemic threat.

Reference:
Pengfei Cui. New influenza A (H7N7) viruses detected in live poultry markets in China. Virology.2016

H7N9 has caused 5 epidemic waves of infection among humans in China, resulting in 1307 laboratory-confirmed clinical cases and 489 deaths among 2013 to 2017 since its first documentation. According to reports of H7N9 virus outbreaks among humans in China, the virus clustered into the Yangtze River Delta lineage and the Pearl River Delta lineage. Infections of H7N9 are associated with considerable morbidity and mortality, particularly within certain demographic groups. This rapid increase in cases over a brief time period is alarming and has raised concerns about the pandemic potential of the H7N9 virus.

Reference:
• Nianchen Wang. Avian Influenza A (H7N9) Viruses Co-circulating among Chickens, Southern China. Emerging Infectious Diseases.2017
• W. D. TANNER. REVIEW ARTICLE The pandemic potential of avian influenza A (H7N9) virus: a review. Epidemiol. Infect. 2015

H9N2 subtype influenza viruses have become endemic in various types of terrestrial poultry in Eurasian and African countries and have caused sporadic infections in humans and mammals. China is regarded as the epidemic center of H9N2 viruses.
In China, H9N2 subtype avian influenza outbreak is firstly reported in Guangdong province in 1992. Subsequently, the disease spreads into vast majority regions nationwide and has currently become endemic there. Over vicennial genetic evolution, the viral pathogenicity and transmissibility have showed an increasing trend as year goes by, posing serious threat to poultry industry.
H9N2 has demonstrated significance to public health as it could not only directly infect mankind, but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H5N1, H7N9, H10N8 and H5N6 viruses.

Reference:
Min Gu. Current situation of H9N2 subtype avian influenza in China. Gu et al. Vet Res.2017
Chong Li. Genetic evolution of influenza H9N2 viruses isolated from various hosts in China from 1994 to 2013. Emerging Microbes & Infections .2017
Trushar Jeevan. A(H9N2) influenza viruses associated with chicken mortality in outbreaks in Algeria 2017. Influenza Other Respi Viruses. 2019

Since 2004, the H10N7 subtype avian influenza virus (AIV) has caused sporadic human infections with variable clinical symptoms world-wide. Three H10N7 subtype avian influenza viruses were isolated from chickens in live poultry markets in Eastern China in 2014. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals.

Reference:
Bodewes R. Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe. J Virol. 2016
van den Brand JM. Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets. PLoS One. 2016

Influenza B viruses (IBVs) circulate annually along with influenza A (IAV) strains during seasonal epidemics. Influenza B viruses can dominate influenza seasons and cause severe disease, particularly in children and adolescents. Influenza B virus is only known to infect humans and seals with influenza.

Symptoms of Influenza B
• Fever, sometimes as high as 106 degrees F; children tend to experience higher fevers than adults
• Fatigue
• Body aches
• Respiratory ailments such as cough, runny nose, and sore throat; these become more pronounced as the fever subsides
• Stomach irritation, including loss of appetite, vomiting, and nausea.

Reference:
Koutsakos M. Knowns and unknowns of influenza B viruses. Future Microbiol. 2016