The diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction

Orthohantaviruses are rodent-borne emerging viruses known to cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses – Puumala virus (PUUV), responsible for a mild form of hemorrhagic fever in humans, Tula virus (TULV) with low pathogenicity, and nonpathogenic Prospect Hill virus (PHV) – with human and rodent host cell lines. We showed that the three orthohantaviruses were able to infect Vero E6 and HuH7 cells but only the former secreted infectious particles. In cells derived from the PUUV reservoir (Myodes glareolus), PUUV achieved complete viral cycling, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting some differences in host specificity. A search for mature virions by electron microscopy revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. The interaction of orthohantaviruses with cellular factors was investigated by studying the colocalization of viral nucleocapsids with markers of cell compartments, identifying interactors of viral proteins by mass spectrometry analysis, and measuring the regulation of gene expression by transcriptomic analysis in infected cells. Overall, our study highlighted the complexity of the host-virus relationship, demonstrated that orthohantaviruses were restricted at different levels of the viral cycle, and provided different levels of analysis to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how this depends on the tissue and host species.