Molecular Biology and Pathogenesis of Influenza A Virus

Respiratory viruses transmit quickly through respiratory droplets and bodily fluids while causing acute illness and have brief incubation periods. The two most common respiratory viruses identified are the influenza A virus (IAV) and RSV.

Structure of Influenza A Virus

IAV belongs to the Orthomyxoviridae family. The IAV genome consists of eight negative-sense RNA segments which produce eight distinct proteins that form the RNA polymerase complex and also include hemagglutinin (HA), neuraminidase (NA), matrix protein (M), and nucleoprotein (NP). The eight genomic segments feature conserved nucleotide sequences at their 5' and 3' ends which function as non-coding promoter regions for genome transcription and replication. Virus particles' surface proteins HA and NA become primary targets for researchers developing vaccines and antiviral drugs. The nucleoprotein NP envelopes the viral RNA genome inside the virus particle which preserves its stability and maintains its integrity. Structural protein coordination enables IAV to penetrate host cells where it starts its replication process resulting in influenza virus infection.

IAV is divided into many subtypes based on the different HA and NA antigens, with 18 HA subtypes and 11 NA subtypes. As a type I transmembrane protein the HA forms a trimeric structure which establishes the transmissibility and pathogenicity of the influenza virus. The HA protein attaches to sialic acid residues on the surface of host cells to initiate membrane fusion with the host cell during the process of endocytosis. The precursor protein HA0 forms HA in the endoplasmic reticulum and transforms into two subunits HA1 and HA2 through host protease cleavage. The globular head domain of HA consists primarily of HA1 which houses the receptor-binding site (RBS) for receptor attachment. The stem domain of HA includes HA2 as its primary element which anchors HA to the envelope by utilizing its C-terminal transmembrane domain. Following endocytosis and exposure to low pH conditions the virus experiences structural alterations in HA2 that trigger fusion of the viral envelope with the host's endosomal membrane. The HA protein clusters at lipid raft-enriched areas to create budding sites during progeny influenza virus formation while changing membrane curvature to start the budding process. The host adaptive immune system specifically targets HA which in turn controls the antigenic drift of the influenza virus.

Figure 1. Structural similarity of HAs in the five clades
(Source: Gamblin SJ, et al. 2021)

The NP protein mainly regulates the transcription and replication of vRNA and virus growth. The NP protein sequence contains a nuclear localization signal and a nuclear retention signal. Its nuclear localization signal helps the vRNP complex enter the cell nucleus, while the nuclear retention signal keeps the progeny vRNP complex in the nucleus before it is produced.

NA is a type II transmembrane protein that forms a tetrameric structure and mainly regulates the virus budding and release process. NA has neuraminidase activity, which can cleave sialic acid residues on proteins covering the respiratory epithelial cell membrane. Throughout the influenza virus invasion phase the virus utilizes NA to break through the mucins and glycoproteins barrier for infection of respiratory epithelial cells below. The NA enzyme breaks down sialic acid that attaches progeny virus particles to the cell membrane during the final replication stage which enables virus release and stops their aggregation on cell surfaces.

Matrix protein 1 (M1) is the most abundant protein in the virus and is located beneath the viral envelope. The first function of M1 is to participate in the export of progeny vRNP complexes from the nucleus. M1 binds to the vRNP complex and the NS2 protein, using the host cell's nuclear export machinery to move the progeny vRNP complexes out of the nucleus. The second function of M1 is to influence the morphology of virus particles. The M1 protein forms connections between the cytoplasmic tails of HA, NA, and M2 proteins together with the NP protein of the vRNP complex to unite the internal viral structures and envelope proteins. The influenza virus uses M1 to attach to the complement molecule C1qA which blocks the activation of the complement pathway thus helping the virus to escape immune detection.

M2 protein functions as a type III transmembrane protein which builds into a homotetramer structure that serves as a proton channel. It plays a role in viral uncoating: The M2 proton channel gets activated by acidic endosomal conditions which enables protons to move into the virus particle while acidifying its interior leading to the separation of the vRNP complex from other viral components during uncoating. During progeny virus budding, M2 localizes to lipid raft-enriched regions at the top of the plasma membrane. The cytoplasmic tail of M2, with the help of cholesterol, induces membrane curvature to form a bud neck, cutting the connection between the viral envelope and the host cell membrane, thus completing virus particle release.

Influenza Virus Lifecycle

The influenza virus attaches to sialic acid receptors on host cell membranes through its HA protein before entering the cell by clathrin-mediated endocytosis as well as caveolin-mediated endocytosis and macropinocytosis which leads to early endosome formation. The acidification of the endosome during its nuclear transport creates irreversible structural changes in the HA protein which reveal the HA2 amino-terminal fusion peptide. The fusion peptide inserts into the endosome membrane, bringing the viral envelope and endosome membrane closer to form a hemifusion stalk, then opens a fusion pore in the membrane. Meanwhile, the acidic environment of the endosome activates the proton channel activity of the M2 protein, allowing protons to flow into the virus interior. After weakening the interactions between the M1 protein and internal viral components, viral uncoating occurs, releasing the eight vRNP complexes from the fusion pore into the cytoplasm.

The vRNP complexes enter the nucleus with the help of NP protein and first synthesize viral mRNA. After mRNA synthesis, the host's mRNA splicing machinery splices its exons. The newly synthesized mRNA's 5' cap structure separates from the PB2 protein and assembles into mRNP by binding with the host cell's cap-binding complex. With the help of the transcription-export complex TREX and the nuclear export complex NXF1/TAP, mRNP is exported from the nucleus. The translation of HA, NA, and M2 protein mRNAs occurs on ER ribosomes before these proteins enter the secretory pathway for folding and then proceed to the Golgi apparatus for modification before reaching lipid raft-enriched cell membrane regions which serve as budding sites. Ribosomes in the cytoplasm carry out the translation of other viral proteins.

After the progeny vRNP complexes are exported from the nucleus, they bind to the host protein YB1. YB1 binds to microtubule proteins in the cytoplasm, localizing the vRNP complexes at the MTOC (microtubule organizing center). The NS2 protein binds to the host protein HRB, promoting the entry of vRNP complexes into the cytoplasmic transport system. When the PB2 protein binds to the host protein Rab11, the vRNP complexes enter recycling endosomes. In the recycling endosomes, eight different vRNP complexes converge and interact, and these eight complexes must meet the requirements of equal numbers and single-copy selective assembly. Subsequently, Rab11 dissociates from the vRNP complexes, and the vRNP complexes move further to the apical plasma membrane of polarized cells. After M1 protein is recruited to the cell membrane, it undergoes multimerization. When the progeny vRNP complexes are recruited to the budding site, they assemble with other viral components. M2 protein clusters at the neck of the virus particle, and in a low-cholesterol environment, its amphipathic helix domain inserts into the cell membrane at the virus particle neck, applying positive curvature to cut the connection between the virus particle and the cell membrane. Finally, the NA protein cleaves the binding between HA and sialic acid receptors on the cell membrane surface to ultimately release the virus particles.

Figure 3. Virus and host-specific determinants of influenza virus replication
(Source: Long JS, et al. 2019)

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