Supplemental information contains 7 movies and 4 supplemental Figures
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1 Supplemental information contains 7 movies and 4 supplemental Figures Movies: Movie 1. Single virus tracking of A4-mCherry-WR MV colocalization with GFP-Rab5. Movie 2. 3D trajectory profile of an A4-CFP MV particle co-trafficking with a Rab5-positive endosome after internalization. The movie records infection time at 1-14 min after virus addition and is played at 3 frames per sec. Movie 3. Single virus tracking of A4-mCherry-WR MV colocalization with GFP-Rab11. Movie 4. 3D trajectory profile of an A4-CFP MV particle colocalizing with a Rab5-positive endosome and a Rab11-positive endosome sequentially. The movie records infection time at min after virus addition and is played at 3 frames per sec. Movie 5. Single virus tracking of an A4-mCherry-WR MV colocalization with GFP-Rab22. Movie 6. 3D trajectory profile of an A4-CFP MV particle colocalizing within a Rab5-positive and Rab22-positive endosome sequentially. The movie records infection time at min after virus addition and is played at 3 frames per sec. Movie 7. Single virus tracking of time-lapsed epi-fluorescence image showed that there is no colocalization of A4-mCherry-WR MV particle with GFP-Rab7 endosome. Supplemental figures: Figure S1. Immunoblot and RT-PCR data regarding gene knock-down efficiency in HeLa cells. Figure S2. Knocking-down individual components of the WASH protein complex does not reduce WRΔA26 virus entry into HeLa cells. Figure S3. Vaccinia virus intracellular trafficking is Rab34-independent. Figure S4. Inhibition of vaccinia virus core uncoating by GTP-bound and GDP-bound mutants of Rab11 and Rab22 proteins in HeLa cells.
2 Movie 1. Single virus tracking of A4-mCherry-WR MV colocalization with GFP-Rab5. Time-lapsed epi-fluorescence image acquired at 37 C showed that the endocytic trafficking behavior of a A4- mcherry-wr MV particle co-localized with a GFP-Rab5 early endosome. Movie 2. 3D trajectory profile of an A4-CFP MV particle co-trafficking with a Rab5-positive endosome after internalization. Spinning-disk confocal image of an A4-CFP MV particle inoculated into mcherry-rab5 expressing HeLa cells was acquired at 37 C. The time-lapsed images were recorded in one-minute interval. The trajectories of a single virus particle and the Rab-5 positive endosomes were showed in red and blue, respectively. Movie 3. Single virus tracking of A4-mCherry-WR MV colocalization with GFP-Rab11. Timelapsed epi-fluorescence image acquired at 37 C revealed that the endocytic trafficking behavior of a A4- mcherry-wr MV particle co-localized with a GFP-Rab11 recycling endosome. Movie 4. 3D trajectory profile of an A4-CFP MV particle colocalizing with a Rab5-positive endosome and a Rab11-positive endosome sequentially. Spinning-disk confocal image of an A4-CFP MV particle inoculated into mcherry-rab5 and GFP-Rab11 expressing HeLa cells was acquired at 37 C. The time-lapsed images were recorded in one-minute interval. The trajectories of an individual virus particle, the rab5-positive endosome and the Rab11-recycling endosome were shown in red, blue and green, respectively. Movie 5. Single virus tracking of A4-mCherry-WR MV colocalization with GFP-Rab22. Timelapsed epi-fluorescence image acquired at 37 C revealed that the endocytic trafficking behavior of a A4- mcherry-wr MV particle co-localized with a GFP-Rab22 recycling endosome. Movie 6. 3D trajectory profile of an A4-CFP MV particle colocalizing within a Rab5-positive and Rab22-positive endosome sequentially. Spinning-disk confocal image of an A4-CFP MV particle traffics in the infected HeLa cells expressing both mcherry-rab5 and GFP-Rab22 at 37 C. The timelapsed images were recorded with one minute interval. The trajectories of an individual virus particle, the Rab5-positive endosome and the Rab22-positive recycling endosome were shown in red, blue and green, respectively. Movie 7. Single virus tracking of time-lapsed epi-fluorescence image showed that there is no colocalization of A4-mCherry-WR MV particle with GFP-Rab7 endosome
3 Figure S1. Immunoblot and RT-PCR data regarding gene knock-down efficiency in HeLa cells. (A) Immunoblot analyses of the CypB, VPEF, and CapZα1 proteins in control and sirnatreated HeLa cells. HeLa cells were either mock-transfected (Control) or transfected with sirna duplexes targeting cyclophilin B (Si-CypB), VPEF (Si-VPEF), and CapZα1 (Si-CapZα1), and then harvested for immunoblot analyses with indicated antibodies. (B) RT-PCR analyses of WASH, KIAA1033, KIAA0196, and CCD53 transcripts in control and sirna-treated HeLa cells. HeLa cells were either mock-transfected (Control) or transfected with sirna duplexes targeting WASH (Si-WASH), KIAA1033 (Si-KIAA1033), KIAA0196 (Si-KIAA0196), and CCD53 (Si-CCD53). We also included the Si-VPEF and Si-CypB cells described in A. These cells were harvested and total RNA was purified for RT-PCR analyses with gene-specific primers and control primers for the GAPDH transcript. The PCR products were separated on 2% agarose gel and the intensity of each transcript was first normalized with GAPDH (the internal control). Then the knock-down efficiency of each gene was calculated as [normalized PCR product remaining in the knock-down cells] [normalized PCR product of the same gene in the mock cells]. The experiments were repeated twice, and these sirna were effective in reducing each transcript to less than 30%. Figure S2. Knocking-down individual components of the WASH protein complex does not reduce WRΔA26 virus entry into HeLa cells. (A) HeLa cells were either mock-transfected (Control) or transfected with sirna duplexes targeting cyclophilin B (Si-CypB), VPEF (Si- VPEF), CapZα1 (Si-CapZα1), WASH (Si-WASH), KIAA1033 (Si-KIAA1033), KIAA0196 (Si- KIAA0196), HSP70A8 (Si-HSP70A8), HSP70A1A (Si-HSP70A1A), and CCD53 (Si-CCD53) and subsequently infected with WRΔA26 for virus uncoating assays. Ant-A4 antibody staining of viral cores (green) in cells were analyzed using a confocal laser scanning microscope with a 40 objective lens. (B) Quantification of viral core uncoating in the knockdown cells shown in A. The averaged viral core numbers in control HeLa cells were considered 100%.
4 Figure S3. Vaccinia virus intracellular trafficking is Rab34-independent. (A) HeLa cells were transfected with individual plasmids encoding GFP, GFP-Rab34, GFP-Rab34Q11L, and GFP-Rab34T22N and then infected with WT-WR MV for virus uncoating assays. Cells were stained with anti-a4 antibody and images were collected by confocal laser scanning microscope using a 63x objective. Green and white lines delineate the edges of GFP-expressing or nonexpressing cells, respectively. (B) Quantification of the viral core uncoating assays of the cells in A. The numbers of A4 cores in control GFP-expressing cell was considered 100%. The bars represent the standard deviations of three independent experiments. Statistical significance was determined by Student s t-test. **P < Figure S4. Inhibition of vaccinia virus core uncoating by GTP-bound and GDP-bound mutants of Rab11 and Rab22 proteins in HeLa cells. HeLa cells were transfected with a pair of plasmids that encode GFP and mcherry tagged proteins as shown at the bottom of the figure, respectively. Next day these cells were infected with WT-WR MV for 2h and viral core numbers in cells expressing both GFP and mcherry fusion proteins were determined as described in Figure S3. The numbers of A4 cores in each dual-fluorescent cells (GFP +mcherry; GFP+mCherry-Rab11Q70L;GFP-Rab22Q64L+mCherry; GFP-WTRab22+mCherry-WTRab11; GFP-Rab22Q64L+mCherry-Rab11Q70L; GFP-Rab22S19N+mCherry-Rab11S25N ) were determined. The A4 core number in control cells ( GFP+mCherry) was considered as 100%. The bars represent the standard deviations of three independent experiments. Statistical significance was determined by Student s t-test. *P < **P <
5 Supplemental Figure S1 A. Immunoblot VPEF CapZα1 CypB β-actin B. RT-PCR VPEF WASH KIAA1033 KIAA0196 CCD53 CypB GAPDH Each cellular RNA is Normalized by GAPDH
6 Supplemental Figure S2 A. Control Si- CypB Si- VPEF Si- CapZα1 Si- CapZα2 Si- WASH Si- KIAA1033 Si- KIAA0196 Si- HSP70A8 Si- HSP70A1A Si- CCD53 B. 100 % of uncoating
7 Supplemental Figure S3 A. GFP Core GFP- Rab34 Core GFP-Rab34Q11L Core GFP-Rab34T66N Core B. % Virus uncoating Rab34Q11L
8 Supplemental Figure S4 * ** ** ** % Virus uncoating GFP tag GFP GFP Rab22 Q64L Rab22 WT Rab22 Q64L Rab22 S19N mcherry tag mcherry Rab11 Q70L mcherry Rab11 WT Rab11 Q70L Rab11 S25N
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