Figure 1

Role of the coiled coil region in NP for NP self interaction, inclusion body formation and MARV-specific transcription/replication. (A) In silico analysis of NP predicted two coiled coil motifs at aa position 315 to 400 which are separated by a linker of 23 aa. (B) Schematic presentation of the constructed mutants of NP with deletions in the coiled coil region. (C) The constructed plasmids were in vitro translated, metabolically labeled, separated by SDS-PAGE and analyzed using a BioImager. (D) In vitro translated and metabolically labeled mutants of NP were incubated with bacterially expressed GST-NP or GST (negative control). Complexes were pulled down with glutathion-sepharose, separated on SDS-PAGE and analyzed using a BioImager. Binding of NP to GST-NP was set to 100%. (E) Quantification of 3 separate experiments as shown under (D). (F) Intracellular distribution of NP and NPΔC1. HUH7 cells were transfected with plasmids encoding NP or NPΔC1 together with a plasmid encoding T7 polymerase. Cells were fixed with 4% paraformaldehyde at 18 h post transfection and incubated with a rabbit anti-NP antiserum. Bound antibodies were detected with a rhodamine-coupled donkey anti-rabbit antibody (NP), and a FITC-coupled donkey anti-rabbit antibody (NPΔC1). (G) Impact of coiled coils on the function of NP in a MARV-specific minigenome transcription/replication system. MARV nucleocapsid proteins were expressed in HUHT7 cells together with a MARV specific minigenome. NP was replaced by the indicated mutants of NP and reporter gene activity (CAT) was measured. Below the CAT assay, expression of NP and the NP mutants was confirmed in Western Blot analysis. +: presence of L. -: absence of L (negative control). (H) Results of a transcription/replication analysis using different internal deletion mutants of NP in a minigenome-based assay (G). +: minigenome system active (transcription and replication monitored by CAT assay). -: minigenome system inactive.