All four morbillivirus V proteins have the ability to block the induction of the type I IFN-induced antiviral state.

<p>(a) Demonstration of expression of different morbillivirus V proteins in the stable cell lines used for the VSV CPE functional assays. A549-blank and stable cell lines expressing the indicated proteins were plated in 24-well plates. Next day, cells were lysed in SDS-PAGE sample buffer and the expressed proteins were detected in Western blots using mouse anti-V5 antibody. PCNA levels served as loading control. (b) VSV CPE assay for the type I IFN-induced antiviral state. Stable cell lines expressing the indicated proteins were plated in 24-well plates. Next day, cells were mock treated or treated with 10, 100, 1000 IU/ml of IFNα for 24 hours. The cells were then challenged with VSV at a MOI of 0.1 for 24 hours (or left untreated), fixed and stained with crystal violet. Shown are results from one of three independent experiments.</p

Abilities of morbillivirus V proteins to block type I and type II IFN induced gene transcription.

<p>(a) Vero human-SLAM cells were transfected with 0.5 µg of empty vector or plasmid driving the expression of the indicated protein. Twenty-four hours post transfection, cells were lysed in SDS-PAGE sample buffer and the expressed proteins were detected by Western blot using mouse anti-V5 antibody. PCNA levels served as loading control. (b), (c) Vero human-SLAM cells were transfected with 0.5 µg each of (b) pGL3-MX1P-luc or (c) pGAS-luc, together with pJATLacZ and either empty vector or plasmid driving the expression of the indicated protein. Twenty-four hours post-transfection, cells treated with either (b) 1000 IU/ml of IFNα or (c) 1000 IU/ml of IFNγ or left untreated, lysed and assayed for luciferase and β-galactosidase activities as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#s4" target="_blank">Methods</a>. Letters above the bars for the IFN-treated samples indicate the results of statistical analysis: results which were not statistically different from one other have the same letter.</p

All four morbilliviruses efficiently blocks IFNα-induced STAT2 phosphorylation, but show varied abilities to block IFNα and INF-γ induced STAT1 phosphorylation.

<p>A549 cells were mock infected or infected with the indicated morbillivirus as in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#s4" target="_blank">Material and methods</a>”. Eighteen hours post-infection cells were mock treated or treated with 1000 IU/ml of IFNα (a), (b) or 1000 IU/ml of IFNγ (c) for 30 minutes, fixed with PFA and methanol and stained with (a) rabbit anti-STAT2 plus goat anti-PPRV or (b, c) mouse anti-phospho-STAT1 (STAT1P) plus rabbit anti-RPV. Representative confocal images from three independent experiments are shown.</p

All morbillivirus V proteins co-precipitate Jak1 and Tyk2.

<p>(a) Vero human-SLAM cells (b), (c) HEK 293FT cells were co-transfected with 1 µg of empty vector or plasmids encoding the indicated proteins along with 2 µg pFLAG-Jak1 or pFLAG-Tyk2. Forty-eight hours post-transfection, cells were lysed with NP-40 lysis buffer (pH 7.5) and the lysates were immunoextracted with mouse anti-V5 or mouse anti-FLAG antibodies as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#s4" target="_blank">Materials and methods</a>”. The immunoprecipitates and the total cell lysate (1/10) were analyzed in Western blots for the presence of FLAG-Jak1 or FLAG-Tyk2 and the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse IgG antibody.</p

RPV-Sa infection blocks IFNα-induced phosphorylation of Jak1/Tyk2.

<p>A549 cells were infected with RPV-Sa at a MOI of 5 or left uninfected. Eighteen hours post-infection cells were mock treated or treated with 1000 IU/ml of IFNα for 15 minutes, lysed in SDS-PAGE sample buffer containing protease and phosphatase inhibitors and the levels of phosphorylated Jak1 and Tyk2 were analyzed in Western blots using rabbit anti-Jak1P and rabbit anti-Tyk2P antibodies respectively. The blots were also probed with rabbit anti-Jak1, rabbit anti-Tyk2 and rabbit anti-RPV P protein (MB18) to analyze the total Jak1 and Tyk2 and to confirm RPV infection. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody. PCNA levels served as loading control.</p

RPV-Sa V but not RPV-Sa P or W, nor the PIV5 V, blocks Jak1 and Tyk2 phosphorylation.

<p>(a), (c) Vero human-SLAM cells were co-transfected with pFLAGJak1 and plasmids encoding the indicated viral proteins, and Jak1 phosphorylation determined, as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#pone-0057063-g008" target="_blank">Fig. 8 (a)</a>. (b), (d) Vero dog-SLAM cells were co-transfected with pFLAGTyk2 and plasmids encoding the indicated viral proteins, and Tyk2 phosphorylation determined, as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#pone-0057063-g008" target="_blank">Fig. 8 (b)</a>.</p

Morbillivirus V proteins show varied abilities to co-precipitate STAT1 and STAT2.

<p>Vero human-SLAM cells were transfected in duplicate wells with 1 µg of empty vector or plasmid driving the expression of the indicated protein. Forty-eight hours post-transfection, one member of each pair was lysed with lysis buffer at pH 7.5 and second with lysis buffer at pH 8.0. The lysates were immunoextracted with mouse anti-V5 antibody as in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057063#s4" target="_blank">Materials and methods</a>”. The immunoprecipitates (Co-IP) and a fraction of total cell lysate (1/10th) were analyzed in Western blots for the presence of STAT1 or STAT2 using rabbit anti-STAT1 and rabbit anti-STAT2 antibodies respectively. The blots from total cell lysates were also probed with mouse anti-V5 antibody for analyzing the expression levels of the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody.</p