Mapping how influenza virus rewires human host cells

Researchers at EMBL Hamburg and collaborators at the Leibniz Research Institute for Molecular Pharmacology (FMP) have mapped how the influenza A virus rewires infected human cells in unprecedented detail. To do this, the researchers used a customised experimental workflow to directly observe how proteins interact inside intact infected cells.

Every year, seasonal influenza kills up to 650,000 people globally and causes serious illness for 3–5 million individuals. The influenza A virus, in particular, has been responsible for several pandemics, including the 1918 Spanish Flu pandemic. When this virus infects cells, it releases its genetic material, called RNA, which contains blueprints for a handful of proteins. These proteins then spread throughout the host cell and repurpose its molecular machinery to make more viruses.

Scientists want to understand this process in detail, as it would help in designing better drug therapies and vaccines against the flu virus. That's why it's crucial to figure out how proteins of the flu virus interact with proteins of host cells and subvert them to meet the virus's needs. This is 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. 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."

Jan Kosinski, Group Leader at EMBL Hamburg and Centre for Structural Systems Biology (CSSB)

Finding a way into the interactome

Studying protein-protein interactions in action during infection is easier said than done. Most previous studies relied on biochemical methods that shared one limitation: the cell had 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 specialised 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."

Peeking deeper into structures

By combining the results obtained through XL-MS with computational structural modelling, the researchers could not only identify which viral and human proteins interact, but also predict how they physically fit together. For this, they used a modified version of AlphaFold, the Nobel prize-winning protein structure prediction algorithm.

"The key advantage of the modified AlphaFold approach is that it allowed us to feed our experimental cross-linking data directly into the structural modelling," 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."

Understanding how a virus takes over a cell

Published in Nature Microbiology, the study results uncovered two important ways in which the virus hijacks the cell. The first one involves haemagglutinin, a protein on the virus's surface that it uses to bind and enter host cells. The researchers traced how haemagglutinin moves through the cell's internal transport and processing system. This is a network of compartments that modifies and prepares proteins before they are shipped to their final destination. This revealed how host proteins, some with previously unknown functions, helped the virus correctly fold and modify haemagglutinin during infection. 

The second one 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. 

"What surprised us most was the paraspeckles," said Iuliia Kotova, former predoctoral fellow at the Kosinski Group at EMBL Hamburg, currently at ETH Zurich and first author of the publication. "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," 

"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 defence response," added Kosinski.

The work relied on shared infrastructure across three institutions. The cross-linking mass spectrometry experiments were run at Charité in Berlin, the glycoproteomics analyses at the EMBL Proteomics Core Facility, the AlphaFold modelling on the EMBL Compute Cluster, and the microscopy imaging at CSSB's Advanced Light and Fluorescence Microscopy (ALFM) Facility.

The road ahead

The work highlights a powerful new way to study influenza and shows how analysing molecular contacts inside living infected cells can reveal both where and how viruses take control of host machinery. The researchers believe that this '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 modelling, and targeted cell-biology follow-up to map native virus-host interactions at specific stages of infection – remains broadly applicable," Kosinski said.

Bogdanow agrees: "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."