Nipah (NiV) P gene editing, and the establishment of a NiV reverse genetic system.

<p>(<b>A</b>) Schematic of Nipah (NiV) genome with the name of each gene indicated. Black segments represent non-coding regions of the virus genome, while the white regions indicate open reading frames (ORF). Inset magnifies the P gene and indicates the accessible ORFs in the NiV P gene due to mRNA editing (NiV V and W) and to a downstream alternative ORF (NiV C). Approximate location of the mRNA site is indicated by the black arrowhead. (<b>B</b>) Establishment of NiV reverse genetic system. Panel a: mock-infected Vero cells. Panel b: Wild-type recombinant NiV infection of Vero cells at 48 h post infection (PI) (MOI = 1). Panels c–d: light and fluorescence micrographs of Vero cells infected with recombinant red fluorescent (RED2AM) virus, 48 h PI (MOI = 1). Panels e–f: light and fluorescence micrographs of primary human microvascular lung endothelial cells (HMVEC-L) infected with RED2AM virus, 24 h PI (MOI = 2). (<b>C</b>) Generation of recombinant NiVs. Nucleotide changes (indicated by red arrowheads and letters) were incorporated into the NiV P gene to ablate expression of the C, V, and W ORFs, to alter the mRNA editing site, or to prevent STAT-1 binding in the shared N-terminal regions of the P, V, and W proteins. Numbers flanking the nucleotide sequences indicate plus-sense antigenomic position.