Influenza A virus (IAV) readily mutates to form new strains that are not recognized by the immune system’s memory. The virus relies on the cellular machinery within the cells that it infects to create new copies of its genetic and other components. Studies by researchers at the Instituto Gulbenkian de Ciência (IGC) in Portugal have now revealed both where in the infected cells and how the genomes of the new influenza A virus particles are assembled. Their findings could help researchers develop new treatments for treating or preventing infection by new strains of flu. “Our results pave the way for alternative therapies that could target genome formation, or the place where the genome is formed,” said IGC research lead Maria João Amorim, PhD. The team reported its findings in Nature Communications, in a paper titled, “Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites.”

Influenza A virus infections are serious threats to human health, and cause annual epidemics as well occasional pandemics, the authors wrote. When the virus infects a cell it releases its genome and proteins, which the cell then replicates and reconstructs into new virus particles. Influenza A is unusual in that its RNA genome is segmented into eight distinct parts. “The virus contains an eight-partite RNA genome, with each segment encapsidated as an individual viral ribonucleoprotein (vRNP) complex,” the authors wrote.

During virus replication the eight parts are replicated many times, but until now, it hasn’t been understood how the right molecules are selected to correctly reconstruct this eight-part viral genome. “The advantages of having a segmented genome are evident for viral evolution and for better gene expression control, but increase the complexity of the assembly of fully infectious virions,” the team wrote. “For an influenza particle to be fully infectious, the eight vRNPs must be packaged in a virion. Virions do not usually package more than eight segments and each segment generally occurs once per virion.”

The IGC team has now demonstrated that selection of genetic material is made in viral-induced compartments known as viral inclusions, “which are sites of accumulation of viral proteins, nucleic acids, and selected host proteins and can include (or not) viral factories.” The research also found that these compartments are not bounded by membranes, which typically delimit cellular organelles. Rather, viral inclusions are separated from their surroundings by liquid-liquid phase separation, a process that is similar to what happens when oil and vinegar are put together in a glass or jar. This effectively segregates the viral genetic material and confines it to a dedicated space in which the eight genome sections can be assembled.

Viral inclusions that emerge during IAV infection have been linked to genome assembly, the authors wrote. “Herein, we report that IAV forms viral inclusions with liquid-like properties.” The scientists say their evidence suggests that the viral inclusions are formed when cells express a single vRNP type. “We propose that viral inclusions allow the spatio-temporal control of the genome assembly process,” they continued. “Such a mechanism would require concentrating material in the cytosol, with a constant influx of vRNPs and efflux of assembled genomes.”

Interestingly, the research showed that formation of liquid viral inclusions is spatially regulated, and developed near the endoplasmic reticulum (ER). This provides a link between the classical, membrane-bound organelles, and liquid organelles. “We observe that movement of IAV inclusions matches that of the ER and, in some cases, inclusions seem to slide on or with the ER membranes,” the team stated. “This raises the possibility that viral inclusions move using the ER.”

Together with the finding that the vRNPs attach the ER, the results “might help to answer the unresolved question of vRNP transport to the plasma membrane,” the team continued. The combined evidence indicates that the vRNPs are transported out of the nucleus, move using the ER, and concentrate near to the ER exit site to assemble the viral genomes, which are then delivered to the plasma membrane, where assembly of the complete virus particles is carried out. “In summary, we propose that the role of liquid viral inclusions is to spatially restrict vRNPs, increasing their concentration at specific sites to boost the kinetics of genome assembly.”

Phase separation is an area of biological research that is gaining interest. Changes to the process can cause but also occur as consequences of diseases, and particularly neurological disorders. The authors suggest that identifying key features of viral inclusions could offer up new insights that could help in the future development of antiviral strategies. “Future experiments should detail the internal organization of the viral inclusions and factors affecting liquid properties, such as molecular crowding, solubility, affinity, or the valency of phase-separating proteins,” they wrote. “We are just beginning to understand the involvement of phase separation in virology, but we anticipate that, given the ancient co-evolution between viruses and eukaryotic cells and the diversity of host strategies used by viruses, the next years will provide an interesting overlap between the two fields.”

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