Enhanced interaction of IFI16 with BRCA1 but not with other DNA damage response proteins during <i>de novo</i> KSHV infection.

<p><b>(A-C)</b> Analysis of <i>de novo</i> infection with BrdU or EdU genome labeled KSHV. (A) HMVEC-d cells were infected with BrdU genome labeled KSHV (30 DNA copies/cell) for 0.25 h and processed for IFA. Cells were fixed, permeabilized, treated with 4 N HCl and reacted with anti-BrdU antibodies followed by Alexa Fluor-488 secondary antibodies. Green spots represent BrdU KSHV genomes of representative virus infected cells. Yellow arrows: nuclear KSHV genome; white arrows: cytoplasmic KSHV genomes. (B) HFF cells uninfected or infected with EdU genome labeled virus for the indicated time points were processed for detection of EdU viral genome by Click-reaction with Alexa 594 labeled azide. Red spots and arrows indicate cytoplasmic or nuclear viral genome. Nuclei were stained with DAPI (blue). (C) Nuclear and cytoplasmic distribution of IFI16 during EdU KSHV infection (4 h p.i.) as shown in (B). HFF cells infected with EdU genome labeled KSHV (30 DNA copies/cell) were processed for IFA to detect IFI16 with anti-IFI16 antibodies followed by Alexa Fluor-488 secondary antibodies and then EdU labeled viral genomes were detected with Alexa 594 labeled azide by Click-reaction. Green staining represents IFI16; white arrows indicate cytoplasmic IFI16 in EdU (red) virus infected cells but not in uninfected cells. Nuclei were stained with DAPI (blue). <b>(D and E)</b> HMVEC-d cells infected with KSHV (30 DNA copies/cell) for 4 and 24 h. Lysates from uninfected (UI) and infected cells immunoprecipitated (IP-ed) with (D) anti-IFI16 and (E) BRCA1 antibodies and western blotted (WB) for BRCA1, IFI16, CHK2 and H2AX proteins. <b>(F)</b> Input controls for IP reactions in D and E. Whole cell-lysates (WCL) were blotted with anti-BRCA1, IFI16, CHK2, H2AX or β-actin antibodies. <b>(G)</b> Lysates from the above experiments (D and E) were used to IP with species specific IgG antibodies and WB for BRCA1, IFI16, CHK2 and H2AX for specificity controls (Ga). IP inputs were assessed by BRCA1, IFI16, CHK2, H2AX and β-actin WBs (Gb).