Fig. 1. Properties and retrieval of SFV Cp using mAVI tag.

a Partial structure of Eastern Equine encephalitis virus Cp dimer encompassing (on the left) Cp residues 85–261 (PDB: 6MX7)⁷⁴. Arrowhead indicates the homologous location where the mAVI tag was inserted in SFV Cp. The highlighted lysine residue within the mAVI tag sequence is the target for BirA biotinylation. b Vero parental or Vero BirA (+BirA) cells were mock infected, infected with SFV WT, or infected with SFV mAVI. At 7.5 hpi, lysates were harvested, subjected to SDS-PAGE, and analyzed by western blot using a polyclonal antibody against Cp or a Streptavidin Alexa-680 probe. Arrowhead indicates Cp WT, and arrows indicate Cp-mAVI. The images are representative examples from n = 2 biological replicates. c Specific infectivity was measured as the ratio of the number of infectious particles (determined by plaque assay) to the number of total particles (determined by quantitative western blot of purified virus using an E2-specific antibody, see Methods section). Individual data points from two biological replicates are shown and the black line represents their mean. Units represent ratio of PFU/E2 signal. d Vero BirA cells were grown in biotin-depleted media for 3 days (-biotin) or grown in normal media (+biotin). Cells were infected with SFV mAVI, and lysates were harvested at 7.5 hpi, retrieved with Streptavidin DynaBeads (SA-DB), and analyzed by SDS-PAGE and western blot using a monoclonal antibody against Cp or a Streptavidin Alexa-680 probe. The images are representative examples of n = 2 biological replicates. e Autoradiogram of Cp-RNA crosslinked adducts retrieved with SA-DB from Vero BirA cells infected with SFV mAVI (representative of n = 2 biological replicates). Asterisk indicates an infection- and UV-independent band (Supplementary Fig. 1e). See also Supplementary Fig. 1.