The guanine nucleotide exchange factor GBF1 participates in rotavirus replication

José L. Martínez; Francesca Arnoldi; Elisabeth M. Schraner; Catherine Eichwald; Daniela Silva-Ayala; Eunjoo Lee; Elizabeth Sztul; Óscar R. Burrone; Susana López; Carlos F. Arias

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/619924 doi: bioRxiv preprint 15 and with plasmids encoding the non-glycosylated forms of VP7 or NSP4, or with both 308 plasmids simultaneously. At 24 hpt the cells were fixed and immunostained for NSP4 and 309 VP7. In cells in which the plasmid for VP7 was transfected alone, the monomeric form of 310 VP7 (MAb M60) was clearly visible, whereas there was no signal with MAb 159 that detect 311 the trimer; in contrast, when the plasmids for VP7 and NSP4 were co-transfected, trimeric 312 VP7 was detected and colocalized with NSP4 (Fig. 10) . These results confirm that the 313 presence of NSP4 is relevant (directly or indirectly) for the correct assembly of VP7 into 314 trimers. For these assays, Hek293 cells were transfected with the different constructs, and at 24 hpt, 321 the cells were infected with RRV. At 12 hpi the cells were harvested, and the viral yield was 322 determined. Figure 11B shows that the full-length GBF1/795 was able to rescue the 323 replication of rotavirus in the presence of BFA. In addition, the GBF1/795/1531t was able to 324 rescue, albeit partially, viral replication. In contrast, none of the other GBF1 mutants could 325 support virus replication. These findings indicate that the C-terminal domain of GBF1 is not 326 absolutely critical for replication of the virus, while all other domains of GBF1 are required. 327 We also tested a GBF1/795 mutant that encoded a protein with an amino acid substitution 328 E794K in the Sec7 domain (GBF1/795/E794K) that is inactive in Arf activation (59), and 329 All rights reserved. No reuse allowed without permission.