1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 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 41-56 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 38-58 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.
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 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. 62 63
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 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%.
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 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 < 0.01. 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 < 0.05. **P < 0.01. 113 114
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
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 75 50 25 0
Supplemental Figure S3 A. GFP Core GFP- Rab34 Core GFP-Rab34Q11L Core GFP-Rab34T66N Core B. % Virus uncoating Rab34Q11L
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