🦠 microbiology

Defining the molecular interaction between influenza hemagglutinin and MHC-II

This study elucidates the molecular interface between influenza hemagglutinin and MHC-II by combining deep mutational scanning and cryo-EM to reveal that diverse influenza subtypes utilize MHC-II for cell entry, with specific structural determinants on both the viral protein and the host receptor governing this interaction.

Original authors: Dadonaite, B., Dosey, A., Ahn, J. J., Yu, T. C., Sunshine, S. A., Farrell, A. G., King, N. P., Bloom, J. D.

Published 2026-07-22

📖 5 min read🧠 Deep dive

CC BY 4.0

Original authors: Dadonaite, B., Dosey, A., Ahn, J. J., Yu, T. C., Sunshine, S. A., Farrell, A. G., King, N. P., Bloom, J. D.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Imagine the human body as a bustling city, and the immune system as a highly organized police force. To keep the city safe, the police need to spot intruders. They do this using special "wanted posters" called MHC-II molecules, which are displayed on the surface of certain cells. These posters hold up tiny fragments of proteins (like a piece of a criminal's uniform) to show the immune system what to look for. Usually, these posters are just for the police; they aren't meant to be used as front doors by the criminals themselves.

Now, imagine a virus as a master thief trying to break into the city. Most flu viruses have a specific key called Hemagglutinin (HA) that fits into a very common lock found on almost all cells: a molecule called sialic acid. It's like a universal master key that opens almost any door. But recently, scientists discovered something strange: some flu viruses have learned to pick a different lock. Instead of just using the common sialic acid lock, they can also use the police's "wanted posters" (MHC-II) as a backdoor to sneak inside cells. This is a bit like a thief realizing they can trick the police into opening the door for them by pretending to be part of the wanted poster. The big question has always been: How exactly does the thief's key fit into the police poster? Is it a perfect match, or just a lucky accident? And does this trick work for all types of flu thieves, or just a few?

This paper sets out to solve that mystery for a specific group of flu viruses, including the dangerous H5 subtype (often called "bird flu"). The researchers used a clever combination of digital mapping and high-tech photography to figure out exactly how the virus key and the immune poster fit together. They started by creating a massive library of tiny, fake viruses, each with a slightly different version of the HA key. They tested millions of these keys against cells that had the "wanted posters" on them but lacked the common sialic acid locks. By seeing which keys still worked and which ones broke, they could map out the exact shape of the interaction.

The team found that for many H5 flu viruses, the HA key fits into a specific spot on the MHC-II poster, but it's not the same spot where the virus usually grabs onto sialic acid. It's like the virus has a second, secret handle on its key that only fits the police poster. To see this in action, they engineered a version of the virus key that stuck to the poster even tighter. Using a powerful microscope called cryo-EM, they took a 3D picture of the virus key locked onto the MHC-II poster. The image, which is a bit fuzzy (about 4.8 Ångströms resolution, which is like seeing the outline of a building but not the bricks), revealed that the virus grabs onto the "alpha chain" part of the poster, specifically a section that usually holds the wanted fragment.

The researchers also tested if this trick works for other types of flu. They found that H7 flu viruses (another bird flu strain) use the same secret handle on the MHC-II poster. They even checked H1, H2, H3, and H9 strains. Some of these could use the poster as a door, but others couldn't, and the ability to do so varied wildly depending on where the virus came from (birds, humans, or pigs). Interestingly, they discovered that the "beta chain" part of the poster, which usually holds the wanted fragment, also matters. Even though the virus doesn't touch this part directly, the shape of the fragment held there seems to change how well the virus can grab on.

One of the most surprising findings was that the virus doesn't just use the poster to get in; it might get stuck there. When the researchers made viruses that could only use the poster and not the common sialic acid lock, these viruses had trouble spreading. They would enter a cell but then get trapped on the surface of the cell that made them, unable to break free to infect neighbors. This suggests that while the virus can use the poster as a door, it's a risky strategy that might trap it if it doesn't have its usual tools to escape.

The study also looked at whether this trick works better with bird MHC-II or human MHC-II. They found that for most of the bird flu viruses they tested, the bird version of the poster was a much better door than the human version. However, some human flu strains had evolved to use the human poster, while others lost the ability to use any poster at all. This suggests that as flu viruses jump between different animals, they constantly tweak their keys to see which doors are open.

In short, this paper provides the first clear map of how flu viruses can trick the immune system's "wanted posters" into opening the door. It shows that this isn't just a fluke for one virus; it's a widespread strategy used by many flu strains, though the effectiveness depends heavily on the specific virus and the specific host. While the researchers didn't prove that this trick makes the virus more dangerous or helps it spread better in real life, they did show that it's a real, physical interaction that happens across many different flu types. They also noted that understanding this could help design better vaccines, perhaps by making virus keys that don't fit the poster, forcing the virus to rely only on the common lock and making it easier for the immune system to spot. But for now, the main takeaway is that flu viruses are surprisingly adaptable, capable of using the very tools the body uses to fight them as a way to sneak inside.

Technical Summary: Defining the Molecular Interaction between Influenza Hemagglutinin and MHC-II

Problem Statement
While it is established that certain influenza hemagglutinin (HA) proteins can utilize major histocompatibility complex class II (MHC-II) as entry receptors—distinct from or in addition to sialic acid binding—the molecular basis of this interaction remains poorly defined. No high-resolution structure of HA bound to MHC-II exists, the specific HA surface involved has not been fully characterized, and the MHC-II residues critical for binding are unknown. Furthermore, the extent to which this interaction occurs across diverse influenza subtypes and host species is unclear.

Methodology
The authors employed a multi-faceted approach combining deep mutational scanning (DMS), structural biology, and virology:

Pseudovirus Deep Mutational Scanning (HA): Using libraries containing nearly all single amino-acid mutations in H5 HA (from A/American Wigeon/South Carolina/USDA-000345-001/2021), the authors measured how mutations affected cell entry in cells lacking sialic acid but expressing tufted duck MHC-II versus cells expressing sialic acids. This allowed the identification of HA residues specific to MHC-II binding.
Pseudovirus Deep Mutational Scanning (MHC-II): Using "inverted pseudotyping," where lentiviral particles expressed tufted duck MHC-II (with all possible single amino-acid mutations in the ectodomains of the alpha and beta chains) and infected cells expressing H5 HA, the authors mapped MHC-II residues critical for HA binding.
Binding Assays: To directly measure binding affinity, the authors generated MHC-II-coated virus-like particles (MHCII-VLPs) and soluble HA trimers. They used these to neutralize pseudoviruses, correl neutralization potency with binding strength.
Structural Biology: To stabilize the complex for imaging, the authors selected four HA mutations (I80L, A137S, T219E, R227G) identified by DMS to increase MHC-II binding. They produced soluble, stabilized H5 HA trimers with these mutations and solved a 4.8 Å cryo-electron microscopy (cryo-EM) structure of the complex with tufted duck MHC-II.
Validation: Findings were validated using conditionally replicative influenza virions (lacking PB1 polymerase, requiring engineered cells for replication) and by testing HAs from other subtypes (H1, H2, H3, H7, H9) and species (human, avian, swine).

Key Results

MHC-II as a Receptor: Diverse H5 HAs can use MHC-II for cell entry. Avian MHC-II (tufted duck) generally supports more efficient entry than human MHC-II alleles, though most H5 HAs can utilize both.
HA Interface Mapping: DMS revealed that mutations affecting MHC-II entry cluster in a specific region on the HA globular head, distinct from the sialic acid receptor-binding pocket. Key sites include residues 77–80, 120, and 141–150 (H3 numbering). Mutations at these sites (e.g., D77E, R149T) ablate MHC-II entry while sparing sialic acid entry, and vice versa for sialic acid-specific mutations (e.g., S136H, I155H).
Structural Model: The 4.8 Å cryo-EM model shows the tufted duck MHC-II binds to the side of the HA head. The interaction is primarily mediated by the $\alpha$ 1 domain of the MHC-II alpha chain, specifically involving residues in the $\beta$ -sheet (22–24), a loop (37–44), and the peptide-binding groove-forming helix (59–74). The MHC-II peptide-binding groove faces the HA.
MHC-II Determinants: DMS of MHC-II showed that the alpha chain is the dominant determinant of HA binding, with critical sites in the $\alpha$ 1 domain. Mutations in the beta chain also affect binding, likely indirectly by altering the peptide bound in the groove, which faces the HA.
Cross-Subtype and Cross-Species Specificity:
- H7 HA: Interacts with MHC-II similarly to H5 HA, utilizing the same region on the HA head.
- H1, H2, H3, H9: These subtypes also show MHC-II mediated entry, but specificity varies widely. For example, early human H1 (1918) and avian H1 use avian MHC-II efficiently, while later human H1 strains do not. H2 strains generally use both avian and human MHC-II, whereas H3 strains tested could use human but not avian MHC-II.
- Species Variation: MHC-II from different species (e.g., ferret, chicken, mouse) shows varying abilities to support H5 HA entry, correlating with the DMS-predicted effects of mutations found in their sequences.

Significance and Claims
The paper claims to provide the first molecular definition of the interface between influenza HA and MHC-II. By combining DMS with cryo-EM, the authors define a specific binding site on the HA head and identify the corresponding residues on the MHC-II alpha chain. The study demonstrates that MHC-II usage is not limited to rare bat or avian strains but is a widespread capability across multiple influenza subtypes (H1, H2, H3, H5, H7, H9), though with significant strain-to-strain and host-to-host variability.

The authors propose that while the biological role of this interaction remains unclear, it could influence viral tropism or immunogenicity. Specifically, they speculate that HA binding to MHC-II might sterically interfere with T-cell receptor (TCR) engagement of the peptide-MHC-II complex, potentially dampening CD4 T-cell activation and neutralizing antibody production. However, they explicitly state that further work is required to determine the full biological relevance of this interaction in authentic infections. The study also highlights the potential for using this knowledge to design vaccine antigens with reduced MHC-II binding to enhance immunogenicity, though this remains a speculative application.

Biosafety Note
The authors emphasize that their experiments utilized pseudoviruses and conditionally replicative influenza viruses that cannot replicate in humans or animals, ensuring the work was conducted at Biosafety Level 2. They explicitly avoided systematic characterization of mutations designed to increase binding to human MHC-II to mitigate potential information hazards, focusing instead on naturally occurring sequences and binding to avian MHC-II.

Technical Summary: Defining the Molecular Interaction between Influenza Hemagglutinin and MHC-II

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