have developed mechanisms to alter mitochondrial functions. Herein we show that arenavirus 25 nucleoprotein (NP) enters the mitochondria of infected cells and affects their morphological 26 integrity. We initially demonstrate electron-dense inclusions within mitochondria of 27 reptarenavirus infected cells and hypothesized that these represent viral NP. Software 28 predictions then serve to identify a putative N-terminal mitochondrial targeting signal (MTS) 29 in arenavirus NPs; however, comparisons of wild-type and N-terminus mutated NPs suggest 30 MTS-independent mitochondrial entry. NP does not enter isolated mitochondria, indicating 31 that translocation requires additional cellular factors or conditions. Immune electron 32 microscopy finally confirms the presence of NP within the mitochondria both in vitro and in 33 infected animals. We hypothesize that mitochondria targeting might complement the known 34 interferon antagonist functions of NP or alter the cell's metabolic state. 35 36 Viruses are obligate cell parasites that have adapted specific strategies to either promote or 37 inhibit various host cell functions for their own benefit. To hijack the host cell, viruses can 38 target specific subcellular structures and alter their morphology, or can affect host protein 39 levels and/or their trafficking 1 . To counteract the host defense, viruses can interfere with the 40 functions of cellular compartments that contribute to antiviral signalling, such as 41 mitochondria, peroxisomes, endoplasmic reticulum (ER), lipid droplets and the nucleus 1,2 .
8 reptarenavirus infected cells, whereas the MTS-mutated NPs induced a diffuse staining 153 pattern ( Fig. 2c-f ). In addition, V/4 Br cells transfected with UHV1-NP-FLAG bearing the 154 putative MTS (Table 1) consistently exhibited the punctate NP staining pattern (Fig. 2c,d) . In 155 contrast, LCMV NP, which also bears a putative MTS (Table 1) , yielded a diffuse staining 156 pattern (Fig. 2e,f) . Interestingly, HISV-1 NP, devoid of the MTS-like sequence (Table 1) , 157 was either diffusely distributed in the cells or formed perinuclear tubular structures ( Fig.   158 2c,d), confirming our earlier IF, TEM and immune EM findings in Boa constrictor I/1 Ki 159 cells 16 . These findings suggest that the putative MTS of many arenavirus NPs plays a role in Fig. 2a-d) . 168 Exceptionally, LCMV NP did neither produce larger cellular aggregates (Fig. 2e ,f) nor induce 169 staining with anti-MTS ( Supplementary Fig. 2c,d) , leading us to hypothesize that it is rather 170 the tertiary or quaternary structure of NPs than aggregate formation per se that renders the N-171 terminus inaccessible to epitope recognition. When we included the mitochondrial marker 172 mtCO2 to the IF analyses to locate the arenavirus NPs in relation to mitochondria, there was 173 again no clear evidence of co-localization, neither for wt nor mutNP variants (Fig. 2c-f ). 174 However, the NP staining occasionally appeared to follow the mitochondrial network ( Fig.   175 2c-f). These results are in line with the ultrastructural observations that not all mitochondria 176 develop inclusions, and that mitochondria containing NP often lose their structural integrity. Hartmani NP antiserum (Fig. 3d ). To demonstrate that the N-terminus remains intact, we 188 performed immunoblotting using the anti-MTS antiserum, thereby confirming that the NPs 189 remain non-cleaved (Fig. 3a-c) . Furthermore, the immunoblotting revealed that both non- Table 2 ). The Table 2 ). The findings indicate that cytosolic ribosomes mediate NP 201 translation, and that the mitochondrial translocation occurs post-translationally. 
Both subcellular fractionation and mitochondria isolation methods rely on differential 220 centrifugations, which could lead to co-purification of IBs and mitochondria. To exclude the 221 latter and to confirm that a fraction of NP indeed resides inside mitochondria, we performed 222 protease-based submitochondrial localization analyses 48 . We compared the effect of suggesting that some cytosolic NP co-purifies with the mitochondria. However, sonication 243 resulted in slight NP degradation, while almost complete loss of NP occurred following TX-244 100 treatment (Fig. 4c) . The results indicate similar degradation patterns for MRPS35 and NP, 245 adding to the evidence that NP localizes in the mitochondrial matrix. 
Immunoblotting of whole-cell lysates at three dpt using anti-FLAG or anti-HA antibodies 255 showed that all wt and mutNP constructs produced a protein of approximately 63-68 kDa in 256 molecular weight at both temperatures (Figs. 5a,b, 6a) . For HISV-1 NP we confirmed the 257 result also by using the anti-Hartmani NP antibody (Fig. 5b) . Re-probing of the membranes 258 with anti-MTS antiserum showed that the putative MTSs remained uncleaved (Figs. 5a, 6a), 259 as observed for the reptilian cells (Fig. 3a-c) . Curiously, the expression of both wt and 260 mutUGV1 NPs as well as LCMV NP was stronger at 30°C than at 37°C (Figs. 5a, 6a). The 261 phenomenon was most evident for HISV NP which was barely detectable after incubation at 262 37°C (Fig. 5b) .
At three dpt, after incubation at 37°C, IF analyses showed that wtUGV1, UHV1 and 264 wtJUNV NP yielded a punctate staining pattern, while both mutUGV1 and mutJUNV NPs as 265 well as LCMV NP yielded a diffuse staining pattern (Figs. 5c, 6b), as previously described for 266 reptilian cells incubated at 30°C (Fig. 2c-f ). The IF analysis for HISV-1 NP concurred with 267 the immunoblot, a weak and diffuse staining pattern was seen in cells incubated at 37°C 268 whereas cells kept at 30°C presented more intense staining with tubular structures around the 269 perinuclear area (Fig. 5c ). At 30°C, while wtUGV1, wtJUNV and UHV1 NP showed mainly a 270 punctate staining pattern, and mutUGV1, mutJUNV and LCMV NP a diffuse pattern, wtNPs 271 also yielded some diffuse staining and mutUGV1 and mutJUNV NP formed occasional 287 To understand the mechanism behind the mitochondrial translocation of arenavirus NPs, 288 we performed an in vitro mitochondrial import assay utilizing mitochondria freshly isolated 289 from I/1 Ki, Python regius heart (VI/1 Hz) and Vero E6 cells. First, we compared the ability with intact (CCCP-untreated) mitochondria, while the mature form was protected from 301 degradation as it was located within the mitochondria; all bands were lost following proK 302 14 treatment of CCCP-treated samples (Fig. 7a-c) . The Su9-DHFR translocation occurred at both 303 30°C and 37°C with similar efficacy (Fig. 7a-c) . These results indicate that mitochondria 304 isolated from cultured snake cells remain intact, and that the mitochondrial import system 305 functions in a similar way in reptilian and mammalian cells.
Next we assessed the mitochondrial import of wtUGV1-NP, mutUGV1-NP, UHV-1, 
We set up a mitochondrial import assay with reptilian mitochondria, and used a control Table 3 . were separated on 14% SDS-PAGE gels and transferred to nitrocellulose as described above. Tables 1-2 are provided in a PDF file and Supplementary Table 3 