🦠 microbiology

Serine proteases are required to activate influenza D virus haemagglutinin-esterase fusion (HEF) protein

This study identifies the human airway trypsin-like (HAT) and swine airway trypsin-like (swAT) proteases, along with endogenous matriptase, as key activators of the influenza D virus HEF protein, suggesting that protease specificity is not the primary factor restricting IDV replication in the human upper respiratory tract.

Original authors: Maina, M., Zhang, J., Mayora Neto, M., da Costa, K. A., Bottcher-Friebertshauser, E., Hutchinson, E., Marotta, M. G., Trombetta, C., Scott, S. D., Temperton, N. J., Daly, J. M.

Published 2026-04-16

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CC BY 4.0

Original authors: Maina, M., Zhang, J., Mayora Neto, M., da Costa, K. A., Bottcher-Friebertshauser, E., Hutchinson, E., Marotta, M. G., Trombetta, C., Scott, S. D., Temperton, N. J., Daly, J. M.

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 ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed.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

The Big Picture: A Virus with a "Safety Lock"

Imagine the Influenza D virus (IDV) is like a spy trying to break into a fortress (a cell in your body). The spy has a special tool called the HEF protein (the virus's key). However, this key comes in a locked, useless state called HEF0. It's like a car key that is still encased in a block of concrete.

To break into the cell, the virus needs to smash that block of concrete off the key. It can't do this itself; it needs a host cell protease (a specific type of enzyme) to act as a "chisel" and chip away the concrete. Once the concrete is gone, the key works, the virus enters the cell, and infection begins.

The big mystery this paper solved was: Which specific "chisel" does the Influenza D virus need to work?

The Cast of Characters

To solve this, the scientists used a clever trick. Instead of using the dangerous, real virus, they built Pseudotyped Viruses (PVs).

The Analogy: Think of these as "training dummies." They look exactly like the virus on the outside (wearing the HEF coat) but are harmless on the inside. If the training dummy can get into a cell, it proves the "key" (HEF) was successfully unlocked by the "chisel" (protease).

They tested several potential "chisels" (proteases) found in humans and pigs:

HAT (Human Airway Trypsin-like protease): A common tool in human lungs.
TMPRSS2: Another famous tool often associated with flu viruses.
SwAT (Swine Airway Trypsin-like protease): The pig version of HAT.
Matriptase: A tool found in kidney cells.

The Investigation: Who is the Real Chisel?

The researchers set up a series of experiments to see which tool could unlock the Influenza D virus key.

1. The Human vs. Pig Test
They found that HAT (the human tool) and SwAT (the pig tool) were incredibly effective. They smashed the concrete off the key perfectly, allowing the virus to enter.

The Result: The virus works just as well in humans as it does in pigs, provided these specific tools are present.

2. The TMPRSS2 Disappointment
They tried using TMPRSS2, a tool that is famous for unlocking other flu viruses (like the seasonal flu).

The Result: It failed completely. It was like trying to open a door with a screwdriver when you needed a hammer. TMPRSS2 simply couldn't cut the Influenza D virus key.

3. The "Self-Unlocking" Kidney Cells
When they tried to grow the virus in MDCK cells (a type of kidney cell used in labs), they noticed something weird: the virus grew fine without adding any extra tools.

The Discovery: It turns out these kidney cells have their own built-in chisel called Matriptase. It's not as strong as HAT, but it's good enough to get the job done. This explains why the virus can replicate in these cells without help.

Why Does This Matter? (The "So What?")

1. The "Species Barrier" Myth
For a long time, scientists wondered why Influenza D virus (found mostly in cows and pigs) doesn't seem to infect humans easily. One theory was that humans simply lacked the right "chisel" to unlock the virus.

The Verdict: This theory is wrong. Since humans have HAT, and HAT unlocks the virus perfectly, the lack of a specific enzyme is not the reason humans don't get sick from it. There must be other reasons (perhaps the virus doesn't like our cells for other reasons), but it's not because we lack the key-turner.

2. The Pig Connection
Since the pig version of the tool (SwAT) works just as well as the human version, it confirms that pigs are a perfect "mixing pot" for this virus. If a human and a pig virus swapped genes, the resulting virus could likely jump between species easily because the "locks" in both species are the same.

3. Future Medicine
Because we now know exactly which "chisels" (HAT and Matriptase) the virus needs, scientists can start designing drugs that block these specific tools. If you can jam the chisel, the virus can't break in, and the infection stops.

Summary in One Sentence

This study discovered that the Influenza D virus needs a specific tool called HAT (found in both humans and pigs) to unlock its entry into cells, proving that the virus could theoretically infect humans if other barriers weren't in place, and suggesting new ways to block the virus by targeting that specific tool.

1. Problem Statement

Influenza D virus (IDV) is a recently identified member of the Orthomyxoviridae family, with cattle serving as the primary reservoir. Like Influenza C virus (ICV) and Influenza A virus (IAV), IDV possesses a surface glycoprotein called Haemagglutinin-Esterase-Fusion (HEF). This protein is synthesized as an inactive precursor (HEF0) that must be proteolytically cleaved by host cell proteases into HEF1 and HEF2 subunits to trigger membrane fusion and enable viral infectivity.

While the proteases responsible for activating IAV Hemagglutinin (HA) are well-characterized (primarily TMPRSS2 and HAT), the specific host proteases required to activate IDV HEF were unknown. Identifying these proteases is critical for understanding:

The early virus-host interactions.
The mechanisms of host restriction (why IDV infects cattle/swine but not efficiently humans).
The potential for interspecies transmission.

2. Methodology

The researchers employed a combination of pseudotyped virus (PV) assays, cell culture replication studies, and sequence analysis to investigate protease activation.

Pseudotyped Virus (PV) System:
- Lentiviral vectors encoding a firefly luciferase reporter were pseudotyped with IDV HEF (from strains D/swine/Italy/2015 and D/bovine/France/2014) and ICV HEF.
- Co-transfection: HEK293T producer cells were co-transfected with the PV envelope plasmids and plasmids expressing specific Type II transmembrane serine proteases:
  - Human: HAT, TMPRSS2, Matriptase.
  - Swine: swAT (swine airway trypsin-like protease), swTMPRSS2.
- Controls: Included VSV-G (positive control), $\Delta$ envelope (negative control), and exogenous TPCK-treated trypsin.
- Assay: The resulting PVs were used to transduce Swine Testicular (ST) cells. Transduction efficiency was quantified via luciferase activity (Relative Light Units, RLU).
Viral Replication Assays:
- Wild-type IDV and IAV (A/Puerto Rico/8/34) were cultured in MDCK II, A549, and MDBK cells.
- Replication was monitored with and without exogenous trypsin to determine if endogenous proteases could support multiple replication cycles.
Sequence Analysis:
- Full-length HEF sequences of IDV isolates were aligned with ICV and IAV sequences to analyze the amino acid composition of the cleavage site (monobasic vs. multibasic).

3. Key Contributions

Identification of Activating Proteases: The study definitively identifies Human Airway Trypsin-like protease (HAT) and its swine homologue (swAT) as the primary activators of IDV HEF.
Exclusion of TMPRSS2: The study demonstrates that, contrary to some previous hypotheses regarding ICV, TMPRSS2 does not efficiently activate IDV HEF.
Role of Matriptase: The authors provide evidence that matriptase can also cleave IDV HEF, explaining why IDV can replicate in MDCK II cells without exogenous trypsin.
Cross-Species Protease Compatibility: The study confirms that human and swine orthologues of HAT activate IDV HEF with similar efficiency, suggesting protease specificity is not a barrier to interspecies transmission.

4. Key Results

Protease Specificity in PVs:
- PVs produced with HAT (human) or swAT (swine) showed significantly higher transduction titres (approx. 1 log higher than controls) for both IDV and ICV.
- PVs produced with TMPRSS2 or swTMPRSS2 showed transduction titres comparable to the negative control ( $\Delta$ env), indicating no significant activation.
- Matriptase co-transfection resulted in transduction titres higher than the "no protease" control but lower than HAT, confirming it has partial activation capability.
Replication in Cell Lines:
- MDCK II cells: Supported robust IDV replication without exogenous trypsin. This is attributed to the endogenous expression of matriptase in these cells.
- A549 (human) and MDBK (bovine) cells: Required exogenous trypsin for efficient replication; without it, viral titres were 3–4 logs lower.
Sequence Conservation:
- The IDV HEF cleavage site contains a monobasic motif (single Lysine/Arginine), similar to low-pathogenicity IAV and ICV.
- The amino acid sequence downstream of the cleavage site is highly conserved between ICV and IDV.

5. Significance and Conclusion

Mechanism of Activation: The study clarifies that IDV HEF activation relies on Type II serine proteases, specifically HAT and swAT, rather than TMPRSS2. This refines the understanding of IDV entry mechanisms.
Host Restriction: The finding that swAT activates IDV HEF as efficiently as human HAT suggests that the lack of IDV infection in humans is not due to a lack of compatible proteases in the human upper respiratory tract. Other factors (e.g., receptor availability, specifically 9-O-acetylated sialic acids) are likely the primary drivers of host restriction.
Therapeutic Implications: Identifying matriptase as a potential activator in MDCK cells and its role in viral replication highlights it as a potential target for host-directed antiviral therapies. Selective inhibitors of matriptase could potentially block influenza replication in specific cell types.
Methodological Insight: The study highlights the importance of cell line selection (e.g., MDCK II vs. MDCK I) and the utility of pseudotyped viruses in dissecting specific protease requirements without the need for high-containment live virus work.

In summary, this paper resolves the proteolytic requirements for IDV activation, identifying HAT/swAT as the primary activators and matriptase as a secondary activator, while ruling out TMPRSS2 as a primary driver for IDV.