A research team from EMBL Hamburg, in collaboration with scientists from the Leibniz Institute for Molecular Pharmacology (FMP), has created a detailed map of how the influenza A virus alters infected human cells. Their individualized approach allowed them to observe protein interactions directly within intact cells, rather than only in disrupted samples.

The Impact of Influenza A on Cells

Seasonal influenza causes between 3 and 5 million severe cases worldwide each year and is linked to up to 650,000 deaths. Influenza A has also triggered several pandemics, including the one in 1918. Once inside a cell, the virus releases RNA containing instructions for synthesizing a limited set of viral proteins. These proteins spread throughout the host cell, redirecting its molecular machinery and turning the cell into a factory for producing new viral particles.

Innovative Research Methods

To develop more effective vaccines and antiviral drugs, it is crucial to understand which viral proteins interact with human proteins, where these interactions occur, and how the virus exploits them for replication. This new study is the first to comprehensively map direct contacts between influenza and human proteins inside whole infected cells. By detailing the structures, researchers were able to model how interacting proteins might connect with each other.

Tracking protein interactions during active infection is challenging. Previously, biochemical methods requiring cell disruption were used, which can distort the actual processes inside living cells. After breaking down internal compartments, proteins that were previously separated may interact in laboratory conditions, while weak or location-specific interactions may disappear. This complicates the identification of real connections that existed during infection.

The team used a specialized version of cross-linking mass spectrometry (XL-MS), adapted for virus-infected cells. This method enabled them to capture short-lived or localized protein interactions within intact infected cells.

Structural Modeling and Key Discoveries

The XL-MS results were combined with computational structural modeling, allowing the team to determine which viral and human proteins interact and to assess their spatial arrangement when bound together. A modified version of the AlphaFold algorithm was used to build these models.

The study identified two main strategies that influenza A uses to take control of the cell:

1. Hemagglutinin — a surface protein of the virus used for attachment and entry into host cells. The research found that several human proteins assist in the proper folding and modification of hemagglutinin during infection. Some of these proteins had not been well studied before.
2. Paraspeckles — small droplet-like compartments inside the cell nucleus. Influenza A infection leads to their dissolution, releasing RNA-binding proteins that were previously held inside. The virus may use these proteins for its own replication. Disruption of paraspeckles can weaken the cell’s defense mechanisms, as they are involved in stress responses and antiviral gene regulation.

Technologies and Future Perspectives

The project brought together the technologies and expertise of three institutes: cross-linking mass spectrometry was performed at Charité (Berlin), glycoproteomic analysis at the EMBL Proteomics Core Facility, modeling with AlphaFold on the EMBL computational cluster, and microscopy at CSSB on the Advanced Light and Fluorescence Microscopy (ALFM) platform.

The results show that studying molecular contacts within whole infected cells reveals where and how the virus takes control of human cellular mechanisms. This approach can be applied to study other viruses and to identify potential targets for future pharmaceutical interventions.

Although the research was conducted on a laboratory strain of influenza, it is believed that similar methods could be used to study viruses with high pandemic potential, such as H5N1, and to uncover interaction networks that support their replication in human cells.