Researchers at EMBL Hamburg, in partnership with the Leibniz Institute for Molecular Pharmacology (FMP), have successfully developed a highly detailed map illustrating how Influenza A virus alters infected human cells. This tailored workflow enabled scientists to directly observe protein interactions within intact cells, moving beyond reliance on disaggregated samples.
Seasonal influenza leads to 3 million to 5 million severe cases and approximately 650,000 deaths globally each year. Moreover, Influenza A has been responsible for several pandemics, including the infamous 1918 Spanish flu outbreak.
Upon invading a cell, the virus releases RNA containing instructions to synthesize a limited set of viral proteins. These proteins infiltrate the host cell, reprogramming its molecular machinery and transforming the cell into a factory for generating new virus particles.
Mapping Influenza Virus Interactions in Intact Cells
An enhanced understanding of these interactions can aid scientists in developing more effective vaccines and antiviral treatments for influenza. To achieve this, researchers must identify the viral proteins that bind to human proteins, understand where these interactions happen, and how the virus exploits them for replication.
This groundbreaking study is the first to map direct interactions between influenza proteins and human proteins on a large scale within intact, infected cells, providing detailed structural insights that allow researchers to model binding interactions accurately.
“Our study offers a new way to investigate influenza-host interactions in their natural environment, shedding light on structural details,” said Jan Kosinski, group leader at EMBL Hamburg and the Center for Structural Systems Biology (CSSB). “These findings represent a snapshot of infection processes and pave the way for exploring influenza-host interactions throughout the complete infection cycle.”
Addressing Key Experimental Challenges
Tracking protein-protein interactions during active infections is notoriously challenging. Much of the previous research relied on biochemical methods that necessitated cell disruption before measuring protein interactions.
This disassembly can distort the cellular environment, causing originally isolated proteins to interact in ways that wouldn’t occur in living cells, and potentially resulting in the loss of weak, transient, or location-specific interactions. Consequently, determining which connections existed during actual infection can be complicated.
“During this research, we discovered our collaborators Boris Bogdanou and Huang Liu from FMP Berlin had designed a specialized version of cross-linking mass spectrometry (XL-MS), specifically optimized for mapping protein contacts in virus-infected cells,” Kosinski shared.
This innovative method provided the breakthrough necessary to capture fleeting interactions occurring only in specific areas of infected cells.
“XL-MS empowers us to capture protein-protein interactions directly within intact infected cells and offers structural insights into how these interactions occur,” explained Bogdanow, currently a junior research group leader at the Charité Institute for Virology, University of Berlin. “This aids in understanding the interface between viruses and human cells, potentially identifying viable targets for future pharmaceutical interventions.”
Integrating Experimental Data with AlphaFold
The research team integrated XL-MS results with computational structural modeling, allowing them to pinpoint the interacting viral and human proteins and estimate their arrangement upon binding.
For this structural modeling, the researchers employed a modified version of AlphaFold, a Nobel Prize-winning protein structure prediction tool.
“The primary benefit of utilizing the modified AlphaFold approach lies in its ability to incorporate experimental cross-linking data directly into structural modeling,” Kosinski explained. “This informs the model about which regions of virus and host proteins are in proximity within infected cells, which is especially useful for predicting virus-host complexes.”
Two Strategies Used by Influenza to Hijack Human Cells
The results of the study revealed two significant strategies employed by Influenza A to manipulate host cells.
The first strategy centers around hemagglutinin, a surface protein of the virus. Influenza uses hemagglutinin to latch onto and penetrate host cells. The researchers monitored its journey through the cell’s internal transport and processing networks.
This network consists of compartments that modify and prepare proteins for transport to their final destination. The analysis revealed that several human proteins assist in the proper folding and modification of hemagglutinin during infection, many of which had poorly understood roles before.
Influenza Disrupts Structures Within the Nucleus
The second notable finding involved paraspeckles—small droplet-like structures found within the cell nucleus. The research team observed that infestation by Influenza A led to the dissolution of these structures.
As the paraspeckles disintegrated, RNA-binding proteins contained within were released. The virus may utilize these proteins to further its replication processes.
“The dissolution of paraspeckles surprised us significantly,” remarked Julia Kotova, a former predoctoral researcher in Kosinski’s group at EMBL Hamburg, now at ETH Zurich and lead author of the paper. “Observing this consistent disruption across all tested cell lines and influenza strains indicated that this was likely a calculated strategy rather than merely a side effect of infection.”
This disruption could offer multiple advantages to the virus.
“There’s also emerging evidence to suggest that paraspeckles play a role in cellular stress responses and the regulation of antiviral genes. Thus, their destruction could potentially weaken cellular defense mechanisms,” Kosinski added.
Collaborative Efforts Among Three Institutions
This project leveraged the expertise and technology shared among the three institutions. Cross-linking mass spectrometry work was conducted at Charité in Berlin. Glycoproteomic analyses were completed at the EMBL Proteomics Core Facility.
AlphaFold modeling was executed on the EMBL Compute Cluster, while microscopy imaging took place at the Advanced Light and Fluorescence Microscopy (ALFM) facility at CSSB.
A New Approach to Studying Potential Pandemic Viruses
The findings underscore the value of analyzing molecular contacts within intact, infected cells to elucidate how viruses manipulate human cellular machinery. Researchers believe this “contextual mapping” approach could also shed light on the behaviors of other viruses.
“Although host factors and mechanisms may vary from virus to virus, we propose that our overarching approach of mapping native virus-host interactions during specific infection phases—through intracellular cross-linking, structural modeling, and subsequent target cell biology studies—remains broadly applicable,” Kosinski stated.
While this study focused on lab-adapted strains of influenza, researchers believe the same methodology can be adapted to investigate viruses with potential pandemic implications.
Professor Bogdanow concurs, stating, “While our findings center around laboratory-adapted strains, this research lays the foundation for applying our methodology to potentially pandemic viruses like H5N1, unveiling the intricate networks that support virus replication in human cells.”
Source: www.sciencedaily.com