Article Title: A CRISPR screen defines a signal peptide processing pathway required by flaviviruses
Figure Lengend Snippet: SPCS1 is required for flavivirus protein processing and infection a . Western blotting of SPCS1 −/− 293T cells. b . Cells were transfected with YFV-luciferase replicon RNA (WT GDD or loss-of-function GVD). Firefly luciferase activity was measured and normalized to intracellular protein levels. The data reflects the average of two to three independent experiments performed in duplicate. c–h . Cells were infected with WNV ( c, h ), DENV-2 ( d ), JEV ( e ), YFV ( f ) or ZIKV ( g ), and viral yield measured. In h , cells were trans-complemented with an SPCS1 or control plasmid. Results are the average of two to three independent experiments performed in triplicate. i–k . Cells were infected with CHIKV (alphavirus), RVFV (bunyavirus), or VSV (rhabdovirus) and viral yield was measured. Results are the average of two to three independent experiments performed in triplicate. l . The polyprotein processing strategy of flaviviruses 13 . Red and blue arrows indicate sites of cleavage by host and viral (NS2B-NS3) proteases, respectively. m–o . Control or SPCS1 −/− 293T ( m , o ) or Huh7.5 ( n ) cells were infected with WNV ( m, o ) or JEV ( n ). Lysates were blotted with ( m ) anti-WNV E, ( n ) anti-JEV E, or ( o ) anti-WNV NS1 MAbs. Higher molecular weight bands (E hi , E med , and NS1 hi ) that react with anti-flavivirus MAbs are indicated. One experiment of three is shown. p . 293T cells were infected with CHIKV or RVFV. Lysates were blotted with anti-CHIKV E2 or anti-RVFV Gn mAbs. One experiment of two is shown. For gel source data, see Supplementary Figure 1 .
Article Snippet: Cells then were rinsed one additional time with Perm buffer, transferred to a U-bottom plate, and incubated for 1 h at 4°C with 1 µg/ml of the following virus-specific antibodies: WNV (human E16 ); DENV2 (mouse E18 ); JEV (mouse E18 ); YFV (mouse E60 ); CHIKV (CHK-11 ); SINV (ascites fluid, ATCC VR-1248AF), LACV (807-31 and 807-33, gift of A. Pekosz, Baltimore, MD).
Techniques: Infection, Western Blot, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Molecular Weight