HSV-1 (A–H), HSV-2 (G) or VZV capsids (G) were incubated with cytosol at ATP/GTPhigh for 1 hr or the indicated time (E,F) at 37 °C, and classified into intact, punched and flat capsids by electron microscopy (A–G), or the amount of released viral DNA was measured by qPCR (H). (A) Quantification of punched and flat D capsid shells in cytosol prepared from rested Mφ or IFN-induced MφIFN macrophages, or from control A549 (mock) or A549-MxB(1-715) cells. (B) Increasing amounts of MxB(1-715) [%] were added to control A549 cytosol, and the amounts of punched and flat capsids were quantified after incubation in these mixtures. (C) Cytosols of A549 cells expressing MxB(1-715) and Mx(25-715) or MxB(1-715)-FLAG and MxB(26-715)-FLAG were incubated with anti-FLAG antibodies coupled to magnetic beads, the flow-through fractions (FT) were harvested, capsids were treated with anti-FLAG treated or control cytosols, and the amount of punched and flat capsids were quantified. (D) Capsids were incubated in cytosols prepared from A549 cells expressing full-length (FL) MxB(1-715), MxB(26-715), MxB(K131A), MxB(T151A), or MxB(M574D) at ATP/GTPlow or ATP/GTPhigh levels. (E) Time-course of MxB-induced disassembly of capsids pre-adsorbed onto EM grids, incubated with cytosol from A549-MxB(1-715). (F) Analysis of D, V0.5, or V0.1 capsids treated with MxB(1-175) cytosol for broken (punched +flat) capsids after negative stain and EM as described for panel E. (G) Quantification of MxB cytosol disassembly of D capsids of HSV-1(17+)Lox, HSV-1(KOS), or HSV-2(333), or nuclear C capsids of VZV, after incubation in cytosol from A549-MxB(1-715) cells. (H) D capsids were incubated with different cytosols for 1 hr at 37 °C or treated with 1% SDS and 10% Tx-100 only, and the released DNA not protected by capsid shells was quantified by qPCR. Error bars: SEM from 100 capsids in three biological replicates. One symbol of *or § denotes p < 0.05, two p < 0.01, and three p < 0.001 as determined in One-way analysis of variance with a Bonferroni post-test, and comparing the relative amounts of (*) punched and (§) flat capsids, or indicating the differences with the mock-treated samples (*).