Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus

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1 SUPPLETARY INFORATION Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle Tanvi Agrawal, 1 Peter Schu 2 and Guruprasad R. edigeshi 1* 1 accine and Infectious isease Research Center, Translational Health Science and Technology Institute, Gurgaon INIA. 2 Abteilung Bihemie II, Humboldtallee 23, Georg-August-Universität Göttingen, Göttingen, GERANY

2 JE genome equivalents(log10) normalized to GAPH Figure S WT ha Figure S1. Transfection efficiency of JE RNA in EFs. Cells were transfected with JE RNA as described in the methods. Total RNA was isolated from cells at 8 h post-transfection and JE RNA copy numbers were measured by qrt-pcr.

3 Figure S2 a -C-AP-1-TGN 46 b -C and LAP C-AP-1 C-TGN46 AP-1 - TGN46 C-LAP Pearson's correlation coefficient c Figure S2. Colalization analysis of -C and AP-1 with the indicated proteins. a. Huh-7 cells were grown on coverslips and were infected with (OI of 5 pfu/cell). At 24 h pi cells were fixed and stained with the antibodies against -C (blue), γ-adaptin (red) and TGN46 (green) followed by Alexa-488 and Alexa-568 and CY5 -conjugated secondary antibodies. Overlap images are shown. b. Huh-7 cells infected as above and stained with Primary and secondary antibodies-c (Green) and LAP-1 (Red).The nuclei were stained by API. c. Quantitation of colalization between the indicated proteins using Image J software. and -infected images were quantitated using intensity correlation analysis. Error bars represent ean ± SE. Pearson s correlation coefficient is represented on a scale of -1 to 1 with -1 being no colalization and 1 being complete overlap.

4 Figure S3 a API δ-adaptin ds RNA erge JE Inset

5 Pearson's correlation coefficient b J2 and AP-3 colalization JE Figure S3. AP-3 partially colalizes with JE replication compartments. a. Wild type EFs were infected with JE at an OI of 5 pfu/cell. 8h pi, cells were fixed and stained with antibodies against dsrna and delta adaptin followed by Alexa-488 (green) and Alexa-546 (red)-conjugated secondary antibodies. Nuclei was stained by API. Scale 10 μ. A blow-up of the mared area in the merge image is shown as inset for visualizing colalization. b. Pearson s correlation co-efficient was determined from 5 images of infected and uninfected slices. Figure S4 API No stain ds RNA Figure S4. Testing bleed-through effects. Huh 7 cells were grown on coverslips and were infected with (OI of 5 pfu/cell). At 16 h pi cells were fixed and stained with anti-dsrna antibody followed by Alexa-546-conjugated secondary antibody. The nuclei were stained by API. Images were captured sequentially using the following lasers: 405, 473 and 559. Scale bar 10 µm.

6 Pearson's correlation coefficient Figure S5 J2 and AP-3 colalization Figure S5. AP-3 partially colalizes with replication compartments. Quantitation of dsrna and delta adaptin colalization from infected Huh7 cells shown in Figure 7. Pearson s correlation co-efficient was determined from 4 images each of infected and uninfected slices. Figure S6 API ds RNA δ-adaptin Figure S6. Immunostaining of dsrna and δ-adaptin in mha cells: Cells were infected with JE at an OI of 5 pfu/cell. 8h pi, cells were fixed and stained with antibodies against dsrna and delta adaptin followed by Alexa-488 (green) and Alexa-568 (red)-conjugated secondary antibodies. Nuclei was stained by API. Scale 10 μ.