The family Hantaviridae comprises a diverse group of virus species and is considered an emerging global public health threat. Individual hantavirus species differ considerably in terms of their pathogenicity but also in their cell biology and host-pathogen interactions. In this study, we focused on the most prevalent pathogenic hantavirus in Europe, Puumala virus (PUUV), and investigated the entry and internalization of PUUV into mammalian cells. We show that both clathrin-mediated endocytosis and macropinocytosis are cellular pathways exploited by the virus to establish productive infections and demonstrate that pharmacological inhibition of macropinocytosis or a targeted knockdown using RNA interference significantly reduced viral infections. We also found indications of an increase of macropinocytic uptake upon PUUV infection, suggesting that the virus triggers specific cellular mechanisms in order to stimulate its own internalization, thus facilitating infection.

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological processes. Whether eukaryotes achieve similar regulation by selectively assembling and translating monocistronic messages derived from different chromosomes is unknown. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify yeast mRNAs that co-precipitate as ribonucleoprotein (RNP) complexes. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans, forming discrete messenger ribonucleoprotein (mRNP) complexes (called transperons). Chromatin capture and allele tagging experiments reveal that genes encoding multiplexed mRNAs physically interact; thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon histone H4, and its depletion leads to defects in RNA multiplexing, decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical histone H4 functions contribute to transperon formation in eukaryotic cells and regulate cell physiology.

Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

The methyltransferase nsp16 is a key enzyme that catalyses coronavirus replication. In this study, we virtually screened 1577 FDA-approved drugs against nsp16 of SARS-CoV-2, SARS-CoV-1, and MERS-CoV to identify compounds potentially serving as pan-coronavirus inhibitors. Microscale thermophoresis (MST) was used to verify the in-silico results obtained by virtual drug screening, followed by molecular docking and molecular dynamics simulation to test the binding affinities between the target and the candidates. Finally, the candidates were tested against a clinical isolate of SARS-CoV-2 in cell culture. The MST binding assay and molecular docking results showed that four of the candidates showed strong binding affinities to nsp16 of one or two coronaviruses. Nilotinib and simeprevir interacted with nsp16 protein of all three coronaviruses, viz., SARS-CoV-2, SARS-CoV-1, and MERS-CoV, suggesting their potential to act as pan-coronavirus inhibitors. The drugs inhibited the virus with IC₅₀ values ranging between 8.34 and 36.1 µM when tested against a clinical isolate of SARS-CoV-2 in cell culture.