Transcriptome analysis of MyglaSWRecB bank vole cells infected with PUUV and PHV.

Graphs in (A) represents the upregulated genes in MyglaSWRecB cells infected with PUUV (left panel) and PHV (right panel), as compared to non-infected (NI) cells, which were identified by RNA-Seq using the Mus musculus reference genome, GRCm38/mm10. Graphs were generated using GraphPad Prism software by plotting the -log10 of the adjusted p-value against the log2 of the fold change in gene expression. Genes colored in blue were up regulated under both viral conditions, while PUUV- and PHV-specifically induced genes are shown in orange and green, respectively. In (B) M. musculus transcriptome data for PHV-infected bank vole cells were analyzed using the on line-software STRING. The up-regulated genes are represented as an interaction network, with the nodes involved in the immune response colored in red, according to the GO terminology. In (C), the most significantly enriched pathways, compared to non-infected cells, are listed based on Reactome classification. The number of genes identified in RNA samples of PHV-infected cells (entities found, light green), is shown relative to the total amount of genes involved in the specific corresponding functions that are indicated (total entities, dark green).</p

Co-localization of viral N protein with markers of the secretory pathway.

Individual and merged fluorescence of N labelled in green and, ER (A), ERGIC (B), Golgi (C) and Trans-Golgi network (D) in red, is shown for Vero E6 cells infected with PUUV, TULV or PHV. Nuclei are stained in blue with DAPI. Co-localizing proteins appear in yellow in the merge panel. (TIF)</p

Co-localization of viral N protein with markers of the endocytic pathway.

Individual and merged fluorescence of N protein labelled in green and, early endosomes (EE), late endosomes (LE), and recycling of transferrin (tf) in red, in Vero E6 cells infected with PUUV, TULV or PHV, are shown in panel (A), (B) and (C), respectively. Nuclei are stained in blue with DAPI. (TIF)</p

Human proteins found in complex with PHV-N in HEK293T cells.

Human proteins found in complex with PHV-N in HEK293T cells.</p

Co-localization of viral RNA with filamentous structures of N protein.

Vero E6 cells infected with PUUV, TULV or PHV were treated for in situ hybridization by incubation with fluorescent RNA probes either complementary of the viral genomic (-)RNA (green) or of the antigenomic (+)RNA (red). The viral nucleocapsids were detected by immunofluorescence staining with the A1C5 antibody but using secondary antibodies conjugated to Alexa 555 (red) or Alexa 488 (green) according to the fluorescence of the RNA probes. Colocalization of the N filaments with viral RNA appears in yellow in the merge panels. White arrows indicate co-localisation of N dots with PUUV RNA probes.</p

Maturation of viral particles assessed by electron microscopy and immunofluorescence assay.

Panels A-G show thin sections of fixed cells processed by transmission EM. Vero E6 cells were either infected with TULV (A-C) or remained non-infected (D). The lower panels in A and B correspond to a magnification of the plasma membrane area framed by a square in the upper panels. Viral particles (insets), at the plasma membrane and in the extracellular space, are marked with large black arrows and tubular structures are indicated with thin black arrows. In (C), the white asterisk highlights the filamentous structure induced by TULV infection. HuH7 cells infected by TULV (E, F) only show tubular structures at the plasma membrane and the extracellular space (thin black arrows). Multivesicular vacuoles containing viral particles (black arrowheads) and their magnification are seen in MyglaSWRecB cells infected by PHV (G). Immunofluorescent staining of membrane (Mb) and intracellular (IC) viral glycoprotein Gn is shown in panel H for Vero E6 cells infected with PUUV, TULV and PHV. The scale bar corresponding to 20 μm indicates the magnification.</p

Immunostaining validation of identified interactions of N protein with human proteins.

Immunofluorescence co-staining of infected Vero E6 cells was performed as in Fig 4. The localization of viral N protein (green) with markers of P bodies, DDX6, and lipid droplets is shown in (A) and (B) respectively. In (B), the distribution of lipid droplets in non-infected cells (NI) is shown for comparison with infected cells. The co-staining of N and tubulin is shown in (C). Cellular markers are labelled in red while nuclei are counterstained in blue (DAPI).</p

Interaction of the nucleocapsid of PUUV, TULV and PHV with cellular proteins.

Panel (A) shows the different organizations of PUUV-N, TULV-N or PHV-N, identified by intracellular detection at dpi 3 in infected Vero E6 cells (green staining). Small dots of N protein are marked with yellow arrows, filaments with red arrows, bigger granules with white arrows and aggregates are circled in pale purple. Nuclei are counterstained with DAPI (blue staining). In (B), kinetics of TULV-N distribution is shown at dpi 1, 2 and 3. In (C), interactors of the N of PUUV, TULV and PHV identified by mass spectrometry analysis are shown. Cellular partners, statistically significant (p-value<0.05), are colored in orange, blue and green for PUUV, TULV and PHV, respectively. Further explored interactors are colored in violet. In (D), HuH7 cells were infected with TULV or PHV at a MOI of 0.5 and stained for ribophorin I (red) and viral N (green). Co-localization appears in yellow in the merge panel. In (E), HuH7 infected with PHV were stained for NKRF (red) and viral N (green). White arrows highlight the distribution of small dots of NKRF in the cytoplasm of infected cells while NKRF mostly localized in the nucleus of non-infected cells.</p

RNA-Seq identification of bank vole genes induced by orthohantavirus infection.

Up- and down-regulated genes in MyglaSWRecB cells infected by PUUV (Tables A and C) and PHV (Tables B and D) were identified by alignment of the RNA sequences either with M. musculus (Tables A and B) or M. glareolus (Tables C and D) genome. (DOCX)</p

Schematic representation of the different interactions of orthohantaviruses with human and rodent cells.

The cell susceptibility to PUUV- (orange), TULV- (blue) and PHV- (green) infection, viral replication and production of infectious particles in human A549 and HuH7 cells and bank vole MyglaSWRecB cells, are schematically summarized. Colored arrows, as indicated for each of the three viruses, indicate the main steps of the viral cycle i.e. entry, replication and release. Levels of expression of IFNs and ISGs are visualized as black RNA strands and their relative amount by grey arrows next to their name.</p