Figure 4.

VSV M protein impairs nuclei formation and chromatin decondensation. (A) HeLa cells were synchronized with double thymidine block at the G1/S boundary and then released into fresh medium. At 6 h post-release, cells in G2 phase were mock infected or infected with VSV or VSV M(D) at MOI 1. Cells that progressed through mitosis and entered G1 were stained with VSV antibodies, which detect cytoplasmic VSV proteins, and DAPI. (B) Nuclear area, for each condition shown in (A), was quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, USA). (C) Interphase nuclei were formed on sperm chromatin in the presence of GST, GST–M or GST–M(D) proteins. Their growth was monitored by microscopy and the presence of the nuclear envelope was confirmed using Nup160 antibodies. (D) Representative samples were taken at the end of the experiments, as in (C), and analysed by using SDS–PAGE. Integrity of the GST, GST–M and GST–M(D) proteins was assessed by immunoblot using GST and γ-tubulin antibodies. γ-Tubulin was used as a loading control. (E) Nuclear area, for each condition shown in (C), was quantified by using the ImageJ program. (F) Cell viability was assessed in parallel experiments to those in (A), 10 h post-infection, by using the Cell Titre-Glo Luminescent cell viability assay kit (Promega, Madison, WI, USA). CSF, cytostatic factor; DAPI, 4′,6-diamidino-2-phenylindole; GST, glutathione-S-transferase; M, matrix; MOI, multiplicity of infection; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.