Hantaviruses and SARS-CoV-2 : cell stress and novel antivirals

Hantaviruses and coronaviruses are zoonotic viral pathogens that can cause severe disease in humans. The global spread of SARS-CoV-2 demonstrated the full pandemic potential of viral infections and highlighted our need for novel antivirals as well as for a better understanding of the complex interactions between viral pathogens and their hosts.

In Paper I and Paper II, we studied the interactions between hantaviruses and SARS-CoV-2 and the integrated stress response (ISR), a cellular pathway through which cells react to stress factors including viral infections. The ISR is activated through the kinases PKR, PERK, GCN2 or HRI, which phosphorylate eIF2α, leading to a translational shutdown and the formation of stress granules (SGs) in the cytoplasm. We found that hantavirus infection triggered a transient PKR dependent formation of SGs in a small fraction of infected cells and that the SG prevalence varies based on cell type and hantavirus species. Moreover, we showed that hantavirus proteins and RNA do not co-localize with SGs. All studied hantaviruses suppressed PKR activation and our data also suggest that they can inhibit activation of PERK.

Like hantaviruses, SARS-CoV-2 triggered PKR activation and we detected high levels of phosphorylated eIF2α in infected cells, indicating ISR activation. However, SG formation was inhibited and despite a reduction in translational levels, the expression of the stress-responsive transcription factors ATF4 and CHOP was suppressed. Furthermore, we observed variant-specific differences in ISR activation in comparison to ancestral SARS-CoV-2: Infection with the Delta variant led to weaker PKR activation while infection with Omicron BA.1 induced higher levels of eIF2α phosphorylation and an increased SG prevalence.

In Paper III and Paper IV, we screened drug libraries for chemical compounds with antiviral effects against SARS-CoV-2 and Puumala virus (PUUV). By performing a high-throughput microscopy screen, we discovered a previously undescribed chemical, later named virapinib, which reduced the infectivity of a pseudovirus expressing the SARS-CoV-2 spike protein as well as the infectivity of live virus. Virapinib was characterized as an inhibitor of macropinocytosis and was shown to affect the cell entry of different SARS-CoV-2 variants and other viruses like TBEV and MPXV in vitro.

By screening the Specs drug repurposing library, we found a variety of drugs with antiviral effects against the hantavirus PUUV in both the lung epithelial cell line A549 and primary human umbilical vein endothelial cells (HUVECs). These PUUV antivirals include nucleotide analogues, HSP inhibitors and immunosuppressants and most of them have previously reached phase I clinical trials. Interestingly, we also found 19 compounds that instead enhanced PUUV infectivity including HDAC inhibitors, integrin inhibitors, and catenin activators.

Antivirals are essential tools in the research, prevention, and treatment of viral infections. Our findings can be a starting point in the development of new treatment strategies and can provide valuable insights into SARS-CoV-2 and hantavirus replication cycles.

List of scientific papers

I. Christ, W.*, Tynell, J.*, and Klingström, J. Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules. 2020. Journal of Virology, 94(3):e01168-19. *Contributed equally.
https://doi.org/10.1128/JVI.01168-19

II. Christ, W., Klingström, J., and Tynell, J. SARS-CoV-2 variant-specific differences in inhibiting the effects of the PKR-activated integrated stress response. 2024. Virus Research. 339:199271.
https://doi.org/10.1016/j.virusres.2023.199271

III. Porebski, B., Christ, W., Corman, A., Haraldsson, M., Barz, M., Lidemalm, L., Häggblad, M., Ilmain, J., Wright, S.C., Murga, M., Shlegel, J., Sezgin, E., Bhabha, G., Lauschke, V.M., Lafarga, M., Klingström, J., Huhn, D., and Fernandez-Capetillo, O. Discovery of a novel inhibitor of macropinocytosis with antiviral activity. 2023. bioRxiv. Oct 25. [Manuscript]
https://doi.org/10.1101/2023.10.25.563967

IV. Christ, W.*, Porebski, B.*, Fernandez-Capetillo, O., and Klingström, J. High-Throughput Drug Repurposing Screen for Antivirals Targeting Orthohantaviruses. *Contributed equally. [Manuscript]