By Budi Oetomo Narendra 
Scientists at EMBL Hamburg, in close collaboration with researchers from the Leibniz Research Institute for Molecular Pharmacology (FMP) and Charité – Universitätsmedizin Berlin, have unveiled an extraordinarily detailed map of how the influenza A virus profoundly reshapes and commandeers human cells during infection. This pioneering work, published in Nature Microbiology, introduces a sophisticated new workflow that allowed researchers to directly observe and map protein interactions within intact, infected cells, circumventing the limitations of traditional methods that often require cellular disruption. This innovative approach provides structural insights into the intricate dance between viral and host proteins, illuminating critical vulnerabilities that could be exploited for the development of more effective vaccines and antiviral therapeutics.
The Enduring Global Burden of Influenza A
Influenza A stands as a formidable and persistent global health threat. Annually, seasonal influenza epidemics are responsible for an estimated 3 to 5 million cases of severe illness worldwide, leading to a staggering 290,000 to 650,000 deaths. Beyond its seasonal toll, influenza A is notoriously responsible for multiple devastating pandemics throughout history, including the catastrophic 1918 Spanish Flu, which was caused by an H1N1 strain and claimed an estimated 50 million lives globally. More recently, the 2009 H1N1 pandemic underscored the virus’s unpredictable nature and capacity for rapid global spread. The constant threat of emerging avian influenza strains, such as H5N1, which possess high pathogenic potential, further highlights the urgent need for a deeper understanding of this virus at a molecular level to bolster pandemic preparedness.
Upon gaining entry into a human host cell, the influenza A virus initiates a rapid and aggressive takeover. It releases its segmented RNA genome, which contains the genetic blueprints for a small but potent arsenal of viral proteins. These proteins then disperse throughout the host cell, systematically hijacking and re-engineering its complex molecular systems, transforming the cell into an efficient factory dedicated to mass-producing new virus particles. This intricate process of viral replication, while essential for the virus’s survival, represents a series of critical junctures where scientific intervention could potentially disrupt the infection cycle.
Unveiling the Viral Takeover: The Scientific Challenge
A clearer, more granular view of this cellular usurpation is paramount for scientists striving to develop superior flu vaccines and more potent antiviral drugs. Achieving this goal necessitates a precise understanding of which viral proteins interact directly with which human proteins, the exact cellular locations where these contacts occur, and, crucially, the specific mechanisms by which the virus leverages these interactions to facilitate its own replication. Until now, obtaining such detailed, context-specific information has presented a formidable experimental hurdle.
For decades, many studies aimed at mapping protein-protein interactions (PPIs) have relied on biochemical techniques that inherently require the physical lysis, or breaking open, of cells. While invaluable for identifying general interactions, this process carries significant drawbacks. The destruction of internal cellular compartments can distort the native biological reality. Proteins that were strictly separated in a living cell might come into artificial contact once cellular integrity is compromised in a laboratory setting. Conversely, weak, transient, or highly localized interactions – often the most biologically relevant ones – can be lost or undetectable. Consequently, researchers have often struggled to definitively determine which protein connections genuinely existed and functioned during the dynamic process of an active infection. This limitation has represented a major bottleneck in comprehensively understanding the precise strategies viruses employ to manipulate their hosts.
A Methodological Revolution: Specialized XL-MS and Adapted AlphaFold
The breakthrough achieved by the EMBL and FMP teams addresses this fundamental experimental problem head-on. The new study marks a significant leap forward as the first to map direct contacts between influenza and human proteins on a large scale inside intact infected cells. Furthermore, the structural resolution achieved was precise enough to allow researchers to computationally model how these interacting proteins likely fit together, providing unprecedented architectural insight.
"Our work provides a new way to study flu-host interactions in their native context and with structural insight," explained Jan Kosinski, a Group Leader at EMBL Hamburg and the Centre for Structural Systems Biology (CSSB). He emphasized the forward-looking aspect of the research, stating, "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, providing a dynamic view of viral manipulation."
The innovative solution emerged from a specialized version of cross-linking mass spectrometry (XL-MS), a well-established technique for mapping protein contacts, which was specifically tailored for virus-infected cells by Boris Bogdanow and Fan Liu at FMP Berlin. XL-MS works by using chemical cross-linkers to covalently "lock" proteins that are in close proximity within the living cell. After cell lysis (performed after cross-linking) and enzymatic digestion, mass spectrometry is used to identify the cross-linked peptides, revealing which proteins were interacting. The customization for infected cells allowed the team to capture interactions that are often brief, transient, or confined to specific subcellular regions – precisely the type of interactions crucial for viral pathogenesis.
"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," elaborated Bogdanow, now a Junior Research Group Leader at the Institute of Virology, Charité – Universitätsmedizin Berlin. He underscored the therapeutic potential: "This gives us insight into the interface between the virus and the human cell and may, through structural modeling, help identify actionable targets for future pharmaceutical interventions."
To further enhance their findings, the researchers ingeniously paired their XL-MS results with computational structural modeling. This allowed them to not only identify viral and human proteins that interact but also to estimate the precise three-dimensional positioning of these proteins when they connect. A critical component of this modeling was a modified version of AlphaFold, the revolutionary artificial intelligence algorithm developed by DeepMind that has transformed protein structure prediction.
"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," Kosinski clarified. "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 from sequence alone, as their interactions can be highly specific and context-dependent." By integrating empirical data from intact cells, the team was able to refine AlphaFold’s predictions, generating highly accurate models of these complex interaction interfaces.
The Discoveries: Two Key Strategies of Viral Hijacking
The groundbreaking findings, detailed in Nature Microbiology, brought to light two particularly notable strategies employed by influenza A to assert control over human cellular machinery.
The first discovery centers on hemagglutinin (HA), a crucial glycoprotein found on the surface of the influenza virus. Hemagglutinin is indispensable for the virus’s lifecycle, as it mediates the initial attachment to host cells and facilitates viral entry. The researchers meticulously tracked HA as it navigated through the cell’s sophisticated internal transport and processing network – a system comprising various compartments, including the endoplasmic reticulum and Golgi apparatus, responsible for folding, modifying, and dispatching proteins to their correct final destinations.
The in-depth analysis revealed that several human proteins actively participate in helping hemagglutinin fold and undergo necessary modifications correctly during infection. Crucially, some of these identified host proteins had previously possessed poorly understood or uncharacterized functions. This finding suggests that influenza A doesn’t merely exploit pre-existing cellular pathways; it actively recruits and repurposes host factors, some of which might not be immediately obvious targets, to ensure the proper maturation and functionality of its own viral components. Understanding this precise host assistance could open new avenues for antiviral drug development by targeting these specific host chaperones or modification enzymes, thereby disrupting the production of functional hemagglutinin.
The second significant discovery involved paraspeckles, small, dynamic, droplet-like compartments located within the cell nucleus. These non-membranous organelles are known to play vital roles in regulating gene expression, sequestering RNA-binding proteins, and participating in cellular stress responses and innate immunity. The team made the striking observation that influenza A infection consistently caused these paraspeckle structures to dissolve.
When paraspeckles disintegrated, they released a variety of RNA-binding proteins that had been sequestered within them. The researchers hypothesize that the virus then strategically utilizes these liberated proteins to support its own replication processes, effectively turning host regulatory machinery against the host itself.
"What surprised us most was the paraspeckles," remarked Iuliia Kotova, a former predoctoral fellow at the Kosinski Group at EMBL Hamburg and first author of the publication, now at ETH Zurich. She underscored the robustness of the finding: "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." This consistency across different experimental conditions strongly implies a deliberate viral maneuver rather than a mere consequence of cellular stress.
The disruption of paraspeckles may offer the influenza virus more than one strategic advantage. As 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." By dismantling these critical components of the host’s innate immune system, influenza A potentially disarms the cell’s ability to mount an effective antiviral response, further facilitating its unhindered replication. This dual benefit – acquiring host factors for replication and simultaneously suppressing host defenses – highlights the sophisticated evolutionary adaptations of the influenza virus.
A Collaborative Effort Across Three Institutions
The success of this ambitious project was intrinsically linked to the shared technology and expertise provided by three leading research institutions, exemplifying the power of interdisciplinary collaboration in modern science. The intricate cross-linking mass spectrometry work, crucial for capturing in-cell protein interactions, was expertly carried out at Charité – Universitätsmedizin Berlin, leveraging their specialized facilities and expertise. Glycoproteomics analyses, essential for understanding protein modifications like those on hemagglutinin, were meticulously completed at the state-of-the-art EMBL Proteomics Core Facility.
The complex computational structural modeling, particularly the modified AlphaFold approach, relied heavily on the robust infrastructure of the EMBL Compute Cluster, which provided the necessary processing power for these intensive simulations. Concurrently, high-resolution microscopy imaging, vital for visualizing the dissolution of paraspeckles and other cellular changes, took place at the CSSB’s Advanced Light and Fluorescence Microscopy (ALFM) Facility, providing crucial visual confirmation of the molecular findings. This intricate network of specialized facilities and expert teams underscores the collaborative spirit driving cutting-edge biomedical research today.
A New Way to Study Potential Pandemic Viruses
The findings from this study represent more than just a deeper understanding of influenza A; they demonstrate a powerful new paradigm for studying molecular contacts inside intact infected cells. This methodology can reveal not only where a virus exerts control over human cellular machinery but also how it achieves this manipulation at a structural level. The researchers are confident that this type of ‘mapping in context’ can also illuminate the operational strategies of other pathogenic viruses.
"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 asserted. This broad applicability is a critical aspect, suggesting that the methodology developed here could serve as a blueprint for rapidly characterizing emerging or high-priority viral threats.
Although the current study focused on a laboratory-adapted strain of influenza, the researchers firmly believe that the same strategic approach can, and should, be applied to investigate viruses with far greater pandemic potential. Bogdanow echoed this sentiment, stating, "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."
This innovative research offers a potent new tool for pandemic preparedness. By providing unprecedented structural detail of virus-host interactions within their native cellular environment, it opens critical avenues for identifying novel drug targets and informing the design of more broadly effective vaccines. In an era where new viral threats constantly emerge, the ability to rapidly and comprehensively map how pathogens hijack human cells is an invaluable asset in the ongoing global fight against infectious diseases.
