VP40 octamers are essential for Ebola virus replication.
Matrix protein VP40 of Ebola virus is essential for virus assembly and budding. Monomeric VP40 can oligomerize in vitro into RNA binding octamers, and the crystal structure of octameric VP40 has revealed that residues Phe125 and Arg134 are the most important residues for the coordination of a short single-stranded RNA. Here we show that full-length wild-type VP40 octamers bind RNA upon HEK 293 cell expression. While the Phe125-to-Ala mutation resulted in reduced RNA binding, the Arg134-to-Ala mutation completely abolished RNA binding and thus octamer formation. The absence of octamer formation, however, does not affect virus-like particle (VLP) formation, as the VLPs generated from the expression of wild-type VP40 and mutated VP40 in HEK 293 cells showed similar morphology and abundance and no significant difference in size. These results strongly indicate that octameric VP40 is dispensable for VLP formation. The cellular localization of mutant VP40 was different from that of wild-type VP40. While wild-type VP40 was present in small patches predominantly at the plasma membrane, the octamer-negative mutants were found in larger aggregates at the periphery of the cell and in the perinuclear region. We next introduced the Arg134-to-Ala and/or the Phe125-to-Ala mutation into the Ebola virus genome. Recombinant wild-type virus and virus expressing the VP40 Phe125-to-Ala mutation were both rescued. In contrast, no recombinant virus expressing the VP40 Arg134-to-Ala mutation could be recovered. These results suggest that RNA binding of VP40 and therefore octamer formation are essential for the Ebola virus life cycle.
Hoenen
Thomas
T
Institut für Virologie, Robert-Koch-Str. 17, 35037 Marburg, Germany.
Volchkov
Viktor
V
Kolesnikova
Larissa
L
Mittler
Eva
E
Timmins
Joanna
J
Ottmann
Michelle
M
Reynard
Olivier
O
Becker
Stephan
S
Weissenhorn
Winfried
W
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D019281:Dimerization; D029043:Ebolavirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication / Q000502:physiology*
D012367:RNA, Viral; C080981:VP40 protein, virus; D014763:Viral Matrix Proteins