FIG 5.

OR-FP proteins specifically bind to their target ANCH sequence, generating the spots observed in fluorescence microscopy. (a) ChIP experiment showing that chromatin extracted from TB40-ANCHOR3 HCM-infected MRC5 cells (but not from TB40-GFP-infected cells) and immunoprecipitated with anti-GFP antibody is enriched only in ANCH3 and GFP sequences; the hatched area corresponds to background noise, since no ANCH3 sequence is present in the TB40-GFP virus. (b) MRC5 cells were transfected with a single vector expressing OR2-GFP and then infected or not with TB40-ANCH2-Kana viruses; spots appear only when OR-FP and the corresponding ANCH target sequences on the viral genomes are present simultaneously in the same cell. The images were acquired 24 h posttransfection and 72 h postinfection with a wide-field Zeiss Axiovert Observer Z1 with a 1.4-NA 63× objective or a Zeiss LSM 510 NLO. (c) MRC5 cells were transfected with an expression vector for OR3-mCherry, infected with TB40-GFP, or transfected and infected simultaneously; no spots were observed in any situation, indicating that neither OR3 proteins nor TB40-GFP genomes form nonspecific spots, even when present simultaneously in the same cell. The images were acquired 24 h posttransfection and 72 h postinfection with a wide-field Zeiss Axiovert Observer Z1 with a 1.4-NA 63× objective or a Zeiss LSM 510 NLO.