Memerald sec61b c1

ER-mRFP (Addgene no. 62236), mTagRFP-T2-Mito-7 (Addgene no. 58041) (referred to as mitoRFP in the text), mTagBFP2-N1 (Addgene no. 54566), mEGFP-N1 (Addgene no. 54767), mEGFP-C1 (Addgene no. 54759) and mEmerald-Sec61b-C1 (Addgene no. 90992) have been described previously, and were gifts from Erik Snapp, Michael Davidson, or generated in house. EGFP-VAPB^{59 (link)}, HA-PTPIP51 (ref. ^{16 (link)}) and pHAGE-Tet-STEMCCA^{60 (link)} have been previously described and were gifts from Pietro De Camilli, Kurt De Vos and Robert Tijan, respectively.
All insertions and cassette changes were performed using the NEBuilder implementation of Gibson Assembly (New England Biolabs) unless specified otherwise, taking care to leave appropriate restriction sites for later changes. All constructs were sequenced before use and will be available on Addgene unless prohibited by copyright. Specific strategies and resulting plasmid maps are linked in the Supplementary Information.

IF was performed after growing BHK-15 cells or Huh-7.5 cells on glass coverslips. Briefly, cells were either transfected with mEmerald-SEC61B-C1 (Addgene plasmid# 90992) and infected with Flag-6K SINV, or Flag-tagged chimeric SINV, or infected with WT SINV, Δ6K SINV, or Chimeric SINV. Cells were then fixed for 15 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 6 or 12 hpi. The fixed cells were permeabilized with 0.1% Triton-X100 in PBS for 5 min. a mouse monoclonal anti-E2 antibody and a rabbit polyclonal anti-CP antibody were used to detect E2 and CP. A mouse monoclonal anti-FLAG antibody (Sigma) was used to detect the FLAG tag. A rabbit polyclonal anti-Giantin antibody (Abcam, ab80864) was used to image the Golgi apparatus. The secondary antibodies used were fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit and goat anti-mouse antibodies (Thermo Fisher Scientific). Nuclei were stained with Hoechst stain (Invitrogen), and images were acquired using a Nikon AIR confocal microscope with a 60X oil objective. The NIS Elements software (Nikon) was used for processing images.