Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses
Figure 2
Cell cycle progression into early mitosis is required for HPV16 entry.
(A) Depicted are images from representative time lapses for untreated (first row), aphidicolin-treated (second row), and monastrol-treated (last row) HeLa H2B-mCherry cells infected with HPV16-GFP (30vge/cell, see also Videos S1–S3). Time stamps (hh:mm) indicate the time after virus addition. Mitoses in untreated cells are marked by arrows/arrowheads. Note that monastrol-treated cells failed to separate chromosomes but underwent chromosome condensation, aberrant metaphase plate formation, and decondensation of chromosomes (arrows). (B) as in (A). To relate the timing of mitosis and the onset of GFP expression (i.e. successful HPV16 entry), HeLa H2B-mCherry cells were infected with HPV16 (30 and 300 vge/cell), and cells were imaged by time-lapse microscopy. The end of mitosis was defined by visible chromosome decondensation (i.e. telophase), whereas the onset of GFP expression was defined as 10% signal above background. Given is the relative timing of GFP onset post mitosis ± standard deviation (SD). (C) Schematic depiction of the cell cycle. The effects of the inhibitors on cell cycle transitions are indicated. (D) HeLa (black bars) or HaCaT (gray bars) cells were infected with HPV16 PsV after 16 h of preincubation in the presence of inhibitors or solvent control (unperturbed) at indicated concentrations. The number of infected cells was determined 48 h p.i. by flow cytometry. Depicted are infected cells relative to control in percent ± SD. (E) as in (D). To test the reversibility of the S-phase arrest for infection, cells were treated with aphidicolin, and the drug was washed out at the time of HPV16 infection (0 h p.i.) or 24 h p.i.. (F) as in (D) using PHK cells. (G) HeLa cells were infected with HPV6, HPV16, or HPV18 PsV after 16 h preincubation aphidicolin. The number of infected cells was determined 48 h p.i. by flow cytometry. Depicted are infected cells relative to the DMSO treated control in percent ± SD. (H) HaCat cells were infected with raft-derived HPV16 after 16 h preincubation with aphidicolin. Total RNA was extracted 48 h p.i., and infection was measured by the viral splice transcript E1∧E4 normalized to TBP. Infection is depicted relative to the DMSO treated control in percent ± SD.