Macropinocytosis and Clathrin-Dependent Endocytosis Play Pivotal Roles for the Infectious Entry of Puumala Virus

Viruses from the taxonomic family Hantaviridae are encountered as emerging pathogens causing two life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with case fatalities of up to 50%. Here we comprehensively investigated entry of the Old-World Hantavirus, Puumala virus (PUUV), into mammalian cells, showing that upon treatment with pharmacological inhibitors of macropinocytosis and clathrin-mediated endocytosis, PUUV infections are significantly reduced. We demonstrated that the inhibitors did not interfere with viral replication and that RNA interference, targeting cellular mediators of macropinocytosis, is able to decrease PUUV infection levels significantly. Moreover, we established lipophilic tracer staining of PUUV virus particles and showed co-localization of stained virions and markers of macropinocytic uptake. Cells treated with lysosomotrophic agents were shown to exhibit an increased resistance to infection, confirming previous data suggesting that a low pH-dependent step is involved in PUUV infection. Finally, we observed a significant increase in the fluid-phase uptake of cell infected with PUUV, indicative of a virus-triggered promotion of macropinocytosis. Author Summary The Hantaviridae family comprises a very diverse group of virus species and is considered an emerging global public health threat. Human pathogenic hantaviruses are primarily rodent-borne. Zoonosis is common with more than 150,000 annually registered cases and a case fatality index of up to 50%. Individual hantavirus species differ significantly in terms of their pathogenicity, but also 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 virions into mammalian cells. We showed that both, clathrin-mediated endocytosis and macropinocytosis, are cellular pathways exploited by the virus to establish productive infections and demonstrated that pharmacological inhibition of macropinocytosis or its targeted knockdown using RNA interference significantly reduced viral infections. We also found indications for an increase of macropinocytic uptake upon PUUV infections, suggesting that the virus triggers specific cellular mechanisms in order to promote its own internalization and facilitate infections.