Researchers at EMBL Hamburg and collaborators at the Leibniz Research Institute for Molecular Pharmacology (FMP) have mapped in unprecedented detail how the influenza A virus (AIV) rewires infected human cells. The researchers developed a customized experimental workflow that used in-cell cross-linking mass spectrometry (XL-MS), combined with AlphaFold-based structural modeling and functional assays, to directly map protein-protein interactions (PPIs) in IAV-infected human cells.
They claim that the study marks the first time that scientists have mapped direct virus-host protein contacts at scale inside intact influenza-infected cells, with enough structural detail to model how the proteins fit together. “Our work provides a new way to study flu-host interactions in their native context and with structural insight,” said Jan Kosinski, PhD, group leader at EMBL Hamburg and the Centre for Structural Systems Biology (CSSB). “The current results are a snapshot of a moment during infection, and it opens the door to studying flu-host interactions across the entire infection cycle.”
Kosinski is co-senior and co-corresponding author of the team’s published paper in Nature Microbiology, titled “Mapping in-cell protein contact sites reveals hijacking of paraspeckles during influenza A virus infection,” stating that their findings “… uncover mechanisms by which IAV exploits and remodels host compartments during infection.”
Every year, seasonal influenza kills up to 650,000 people globally and causes serious illness for 3–5 million individuals. When IAV infects cells, it releases RNA that contains the blueprints for a handful of proteins that spread throughout the host cell and repurpose its molecular machinery to make more viruses. “Its replication relies on protein–protein interactions (PPIs) between up to 14 viral proteins and host factors, often confined to cellular compartments and organelles,” the team stated.
Scientists want to understand this process in detail, as it would help in designing better drug therapies and vaccines against the flu virus. “Understanding these host–IAV PPIs in context is essential for elucidating viral strategies and therapeutic targets,” they added.
Studying protein-protein interactions in action during infection is challenging. Most previous studies relied on biochemical methods that required the cell to be broken open before the interactions could be measured. Once the cell’s compartments were gone, proteins that were never in contact inside the cell could meet in the test tube, and fragile or location-specific contacts could be lost. It was then hard to know which interactions actually happened inside an infected cell.
“This is when we learned that our collaborators—Boris Bogdanow and Fan Liu—at FMP Berlin had developed a specialized version of cross-linking mass spectrometry (XL-MS), a long-established technique for mapping protein contacts, tailored specifically to virus-infected cells,” said Kosinski. This was the critical breakthrough. It allowed researchers to do what previous methods couldn’t, including capturing short-lived and location-specific interactions.
“XL-MS allows us to capture protein-protein interactions directly in infected intact cells, while also providing structural information about how these interactions are happening,” explained Bogdanow, who is now a junior research group leader at the Institute of Virology, Charité—Universitätsmedizin Berlin. “This gives us insight into the interface between the virus and the human cell and may, through structural modelling, help identify actionable targets for future pharmaceutical interventions.”
By combining the results obtained through XL-MS with computational structural modeling, the researchers could identify which viral and human proteins interact and also predict how they physically fit together. For this, they used a modified version of the protein structure prediction algorithm AlphaFold.
“The key advantage of the modified AlphaFold approach is that it allowed us to feed our experimental cross-linking data directly into the structural modeling,” explained Kosinski. “This tells the model which parts of the viral and host proteins are close to each other inside infected cells. This was especially useful for virus-host complexes, which are often difficult to predict reliably.”
The study findings revealed two important ways in which the virus hijacks the cell. One involves hemagglutinin, a protein on the virus’s surface that it uses to bind and enter host cells. Tracing how hemagglutinin moves through the cell’s internal transport and processing system revealed how host proteins, some with previously unknown functions, helped the virus correctly fold and modify hemagglutinin during infection.
The other involves paraspeckles, small droplet-like compartments in the nucleus. The researchers found that infection by the influenza A virus causes these organelles to dissolve, releasing the RNA-binding proteins bound within them, which the virus can then use to replicate. “We identified host factors linked to the maturation of distinct glycoforms of the viral surface glycoprotein haemagglutinin through the membrane-bound endoplasmic reticulum–Golgi system,” the scientists wrote in summary. “In the nucleus, we observed the progressive disassembly of paraspeckles (phase-separated membraneless compartments) across multiple cell lines.”
First author Iuliia Kotova, PhD, former predoctoral fellow at the Kosinski group at EMBL Hamburg, and currently at ETH, said, “What surprised us most was the paraspeckles. Watching these tiny organelles in the nucleus dissolve, consistently across every cell line and every flu strain we tested, told us this isn’t a side effect of infection—it might be a strategy.”
Kosinski added, “There may also be a second benefit for the virus: some evidence suggests paraspeckles contribute to cellular stress responses and antiviral gene regulation, so disrupting them could also weaken parts of the cell’s defense response.”
The researchers believe that their “mapping in context” approach can be used to understand the mechanism of action of other viruses that act similarly. “While the exact host factors and mechanisms often differ from virus to virus, we think our overall approach—combining in-cell cross-linking, structural modeling, and targeted cell-biology follow-up to map native virus-host interactions at specific stages of infection—remains broadly applicable,” Kosinski said.
Bogdanow further commented, “Although this study has focused on a lab-adapted strain, this study lays the groundwork to apply the methodology to viruses of potential pandemic relevance, such as H5N1, and for uncovering the interaction networks that support their multiplication in human cells.”
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