Cell surface localization and intercellular transfer of the hantavirus nucleocapsid protein via a viral assembly-independent mechanism

Hantaviruses are segmented negative-sense RNA viruses that package their genome into helical ribonucleocapsids, by oligomerization of the nucleocapsid (N) protein along the viral RNA. Upon transmission to humans, hantaviruses can cause severe disease characterized by increased vascular permeability in the microvasculature, a key driver of pathogenesis. In patients and rodent reservoirs, robust and persistent antibody responses against the nucleocapsid (N) protein are consistently observed. Here we investigated whether the hantavirus N protein, described as a strictly intracellular protein, is released into the extracellular environment during viral infection. By using diverse experimental approaches, we demonstrate that in cells infected with Andes virus (ANDV) or the non-pathogenic Tula virus (TULV), the N protein localizes on the plasma membrane. Transfection experiments further revealed that the surface localization of N proteins from Murine, Arvicoline, Neotomine and Sigmodontine rodent-reservoir-associated hantaviruses did not require co-expression of other viral proteins indicating that its trafficking to the cell surface is mediated by a mechanism distinct from canonical virion assembly pathways. Moreover, we show that the N protein is secreted from cells into the extracellular milieu and is further transferred to neighboring cells. Interestingly, despite being present on the cell surface and secreted into the extracellular media, the TULV N protein transfer of the N protein to neighboring cells was significantly lower compared to that of ANDV N protein. The extracellular presence of hantavirus N proteins, and likely also from other clinically-relevant bunyaviruses, unveils new possible functions of the protein during the viral infection cycle, which is likely related to immune responses, immune modulation and viral pathogenesis.