Hemagglutinin and Neuraminidase: Key Roles in Influenza

Influenza viruses rely on two surface proteins, hemagglutinin (HA) and neuraminidase (NA), to infect host cells and spread. These proteins play essential roles in viral entry and release, making them key targets for immune responses and antiviral treatments.

Understanding HA and NA helps explain how influenza evolves, evades immunity, and challenges vaccine development.

Hemagglutinin Structure And Attachment Mechanism

Hemagglutinin (HA) is a glycoprotein embedded in the influenza virus envelope, playing a central role in infection. Structurally, HA exists as a homotrimer, with each monomer consisting of two subunits: HA1 and HA2. HA1 contains the receptor-binding domain responsible for attaching to host cell receptors, while HA2 facilitates membrane fusion. These subunits are initially synthesized as a single polypeptide (HA0) that must be cleaved by host proteases to become functional. The cleavage site varies among strains, influencing viral pathogenicity. Highly pathogenic avian influenza viruses possess a polybasic cleavage site that allows systemic spread, whereas human-adapted strains require trypsin-like proteases found in the respiratory tract.

Attachment begins when HA1 binds to sialic acid residues on host epithelial cells. The specificity of this interaction determines host range and tissue tropism. Human-adapted influenza viruses bind to α2,6-linked sialic acids, abundant in the upper respiratory tract, while avian strains recognize α2,3-linked sialic acids, found in bird intestines. This distinction plays a major role in cross-species transmission. Structural studies have shown that minor mutations in the receptor-binding site can alter binding affinity, enabling zoonotic transmission. The H1N1 pandemic strain of 2009 acquired mutations enhancing its ability to bind human-like receptors, facilitating human-to-human spread.

Once HA secures viral attachment, the virus is internalized via endocytosis. Inside the endosome, acidic conditions trigger a conformational change in HA2, exposing the fusion peptide. This segment inserts into the endosomal membrane, bringing the viral and host membranes together. A series of structural rearrangements then drive membrane fusion, allowing the viral genome to enter the cytoplasm. The pH threshold required for this transition varies among subtypes, influencing viral stability and adaptation. Studies have shown that avian influenza HA proteins typically require a lower pH for fusion compared to human-adapted strains, affecting transmissibility and pathogenic potential.

Neuraminidase Structure And Release Mechanism

Neuraminidase (NA) is a tetrameric glycoprotein that enables the release of newly formed virions from infected cells. Each NA monomer consists of a globular head, a stalk region, and a transmembrane anchor. The enzymatic activity resides in the globular head, which contains a conserved catalytic site responsible for cleaving sialic acid residues from glycoproteins on the host cell surface. This function prevents viral self-aggregation and ensures efficient dissemination of progeny virions. The stalk region, which varies in length among strains, influences enzymatic stability and host adaptation. Shorter stalks, often seen in avian influenza NA, can impact viral fitness in mammals by altering substrate accessibility.

NA activity is tightly coordinated with HA to balance viral attachment and detachment. While HA binds to sialic acid to initiate infection, NA counteracts this by cleaving terminal sialic acid residues, facilitating viral egress. This dynamic is crucial in the respiratory tract, where mucus contains sialylated glycoproteins that can trap virions. By hydrolyzing these moieties, NA enables the virus to evade entrapment and spread. The efficiency of this process depends on the structural compatibility between HA and NA, as mismatches can reduce transmissibility. Studies on pandemic and zoonotic strains show that adaptive mutations in NA enhance viral propagation by optimizing this balance.

Structural analyses have revealed that the NA catalytic site is highly conserved, making it an attractive target for antiviral drugs. Neuraminidase inhibitors, such as oseltamivir and zanamivir, block this active site, preventing sialic acid cleavage and halting viral release. Resistance mutations, however, can alter the active site, reducing drug binding while maintaining enzymatic function. The H275Y mutation in H1N1, for example, decreases oseltamivir susceptibility without significantly impairing viral fitness. Monitoring such mutations is crucial for guiding treatment strategies and drug development.

Influence On Host-Pathogen Interactions

The interaction between influenza viruses and their hosts is shaped by HA and NA, which regulate viral entry, replication, and dissemination. The balance between these glycoproteins influences viral fitness, host adaptation, and transmissibility. HA binds to sialic acid residues on host cells, but without complementary NA activity to cleave sialic acids and facilitate release, newly formed virions risk remaining tethered to the cell surface or trapped in mucus. Even minor modifications to HA or NA can impact viral spread, requiring adaptations that maintain functional compatibility.

NA’s structural variations, particularly in the stalk region, affect enzymatic stability and accessibility to sialylated substrates. Shorter NA stalks, common in avian influenza strains, can limit the enzyme’s ability to cleave sialic acids in mammalian hosts, reducing transmissibility unless compensatory mutations arise. The 1997 H5N1 outbreak in Hong Kong showed how changes in NA stalk length and HA binding affinity influenced human infection. Similarly, the 2009 H1N1 pandemic strain exhibited NA adaptations that improved viral release and transmission in humans, highlighting evolutionary pressures on these glycoproteins.

Beyond viral fitness, HA-NA dynamics also affect tissue tropism and pathogenicity. Some strains prefer infecting the upper respiratory tract, where α2,6-linked sialic acids dominate, leading to efficient person-to-person transmission. Others, particularly avian-origin viruses, target deeper respiratory regions, where α2,3-linked sialic acids are more prevalent. This deep-lung infection is often associated with severe disease, including viral pneumonia and acute respiratory distress syndrome (ARDS). The 1918 H1N1 pandemic strain replicated in both upper and lower respiratory tissues, contributing to its high mortality rate.

Subtype Variations

Influenza A viruses are classified into subtypes based on genetic and structural differences in HA and NA, with 18 HA and 11 NA variants identified. These combinations result in diverse viral subtypes with distinct host preferences, transmission dynamics, and pathogenic potential. While some subtypes, such as H1N1 and H3N2, circulate in humans, others, like H5N1 and H7N9, primarily infect birds but have caused sporadic zoonotic outbreaks. The genetic flexibility of these glycoproteins allows influenza to adapt to new hosts through reassortment and mutations, contributing to the emergence of novel strains with pandemic potential.

Human-adapted H1 and H3 subtypes have undergone extensive antigenic drift, accumulating mutations that enhance receptor binding and transmission. In contrast, avian influenza subtypes, such as H5 and H7, retain features that facilitate replication in birds but limit their spread among humans. The 2009 H1N1 pandemic strain demonstrated how reassortment between swine, avian, and human influenza viruses can generate a novel subtype capable of sustained human-to-human transmission. These events underscore the unpredictable nature of influenza evolution and the role of HA-NA compatibility in shaping viral emergence.

Significance In Immune Recognition

The immune system recognizes influenza viruses primarily through HA and NA, as these surface glycoproteins are the most exposed viral antigens. Antibodies generated during infection or vaccination target specific epitopes on HA and, to a lesser extent, NA, neutralizing the virus before it can establish infection. Since HA mediates viral entry, it is the primary focus of antibody-mediated immunity. However, influenza evades immune recognition through antigenic drift, where gradual mutations alter HA and NA antigenic properties. This necessitates frequent updates to seasonal influenza vaccines, as antibodies against prior strains may have reduced efficacy against new variants.

Antigenic shift, a more abrupt change, occurs when influenza viruses undergo reassortment, leading to novel HA and NA combinations that the immune system has not encountered. This mechanism drove past pandemics, such as the 1918 and 2009 H1N1 outbreaks. While HA is the dominant target of neutralizing antibodies, NA-specific immunity can reduce viral replication and disease severity. NA-targeting antibodies do not prevent infection but limit viral spread by inhibiting sialic acid cleavage, reducing viral load. This has spurred research into NA-based vaccines, which could complement HA-targeted approaches for broader and more durable protection.

Diagnostic And Research Tools

HA and NA are focal points in influenza diagnostics and research. Laboratory detection often relies on HA-based assays, such as hemagglutination inhibition (HI) tests, which measure the ability of patient-derived antibodies to prevent viral agglutination of red blood cells. This method is widely used for vaccine efficacy studies and serological surveillance. Reverse transcription-polymerase chain reaction (RT-PCR) is another key technique, targeting HA and NA gene sequences to confirm infection with high sensitivity and specificity. These molecular assays help identify circulating subtypes and track viral mutations, informing public health responses and vaccine strain selection.

Structural studies using cryo-electron microscopy and X-ray crystallography have guided the design of antiviral treatments and vaccines. Neuraminidase inhibitors, such as oseltamivir and zanamivir, were developed based on detailed NA structural analyses. Additionally, reverse genetics techniques allow researchers to manipulate HA and NA genes, enabling recombinant influenza virus generation for vaccine production. These advancements highlight the critical role of HA and NA in influenza control.