Extended Data Fig. 3: KSHV ORF45 interacts with hNLRP1.
a-c Two-hybrid interaction assay in yeast transformed with expression vectors for indicated combinations of KSHV-encoded proteins and inflammasome components. d Nuclear and cytoplasm fractions were generated from HeLa-hNLRP1-Flag stable cells infected with lentiviruses carrying ORF45 or vector for 48 h, followed by immunoprecipitation (IP) and immunoblot (IB) with indicated antibodies. e,f Interaction assay between ORF45 and indicated hNLRP1 mutants in yeast by two-hybrid interaction assay (e) or in HEK293T cells transfected with indicated expression vectors by co-IP (f). g Two-hybrid interaction assay in yeast transformed with expression vectors for indicated combinations of ORF45 and Linker1 mutants. h,i ASC spike microscopy images (h) and its quantification (i) in GFP-ASC HeLa cells infected by lentiviruses carrying vector, ORF45, or ORF45 mutants for 48 h. Scale bar, 50 μm. n = 6. j Schematic diagram of ORF45-hNLRP1 specific interaction. The left panel shows a phylogenetic tree of the analyzed species with hominoids in black text, Old World monkeys in green text, and New World monkeys in blue text. The right panel shows the alignment of a portion of the Linker1 region. Amino acid labelled in red color differ from hNLRP1. All data represent three independent experiments and are presented as mean ± s.d. For statistical analysis, i one-way ANOVA.
