Varicella-Zoster Virus Immediate-Early 63 Protein Interacts with Human Antisilencing Function 1 Protein and Alters Its Ability To Bind Histones H3.1 and H3.3

Interaction of VZV IE63 with human ASF1a and ASF1b. (A) MeWo cells were transiently transfected with plasmids expressing myc-tagged human ASF1a (p408) or human ASF1b (p542). At 24 h posttransfection, the cells were infected with VZV ROka-T7, and 48 h postinfection, the cells were lysed and the clarified lysates were incubated with mouse anti-myc antibody (Ab). The immune complexes were precipitated and immunoblotted with rabbit anti-ASF1 or rabbit anti-VZV IE63 antibodies. IP, immunoprecipitation. (B) ASF1a, ASF1b, and chimeric ASF1 constructs used to identify the region of ASF1a important for binding to VZV IE63. (C) COS cells were transiently transfected with plasmids expressing myc-tagged, full-length ASF1a (p408), ASF1b (p542), and ASF1a and ASF1b (p601, p941, p942, p1051, or p1052) chimeras or pmaxGFP together with a plasmid expressing full-length VZV IE63 (pCI63). At 48 h posttransfection, the cells were lysed and equal amounts of clarified lysates were immunoblotted with mouse anti-myc monoclonal antibody (9E10), mouse anti-beta-actin antibody (clone AC-15; Sigma-Aldrich), or rabbit anti-VZV IE63 antibodies. (D) Equal amounts of lysates (comparable to those used in panel B) were incubated with mouse anti-myc antibody, and immune complexes were precipitated and immunoblotted with rabbit anti-VZV IE63 antibody or mouse anti-myc antibody. The numbers below the IE63 panel indicate the intensities of the IE63 bands relative to those in cells transfected with ASF1a (p408), normalized to the intensities of the corresponding ASF1 bands on the basis of densitometry by using ImageJ software (http://rsb.info.nih.gov/ij/ ). The IE63 doublets in Fig. 2D were seen when plasmids expressing IE63 were transfected into COS cells (also seen in Fig. 3C) but not into HeLa cells (Fig. 5C) and are likely due to differences in cell type used.

Amino acids 170 to 280 and putative phosphorylation sites of VZV IE63 are important for the interaction with ASF1. (A) Equal numbers of MeWo cells that were uninfected or infected with VZV ROka, ROka63-KpnI, or ROka63-AccI were lysed at 48 h postinfection, and equal amounts were immunoblotted with rabbit anti-VZV IE63 antibody. Another aliquot of each lysate was incubated with mouse anti-ASF1a monoclonal antibody MPH7, and the immunoprecipitates were immunoblotted with rabbit anti-VZV IE63 antibody. The faint bands in the ROka (lane 6) and ROka63-AccI (lane 8) cells at 26 kDa are due to the light chain of the MPH7 antibody. IP, immunoprecipitation. (B) ASF1 interacts very weakly with IE63-5M in ROka63-5M-infected MeWo cells. MeWo cells infected with VZV ROka or ROka63-5M were lysed at 48 h postinfection. An aliquot of each lysate was incubated with mouse anti-VZV IE63 (9A12) monoclonal antibody, and immune complexes were recovered on protein G-Sepharose beads and eluted. A portion of cell lysate (input), eluted immune complexes, and a portion of supernatant remaining after immunoprecipitation (unbound lysate) were subjected to immunoblotting using rabbit anti-VZV IE63 antibody and rabbit anti-ASF1 antibody. (C) COS cells were transiently transfected with a plasmid expressing myc-tagged, full-length ASF1a (p408) and plasmid pCI63, pmaxGFP, pcDNA63-5M, or pcDNA63-10M. At 48 h posttransfection, the cells were lysed and equal amounts of clarified lysates were immunoblotted with mouse anti-myc antibody (to detect ASF1a) and rabbit anti-VZV 63 antibody. Another aliquot of each lysate was incubated with mouse anti-myc antibody, and the immune complexes were immunoblotted with rabbit anti-VZV IE63 or mouse anti-myc antibodies. (D) Map showing ASF1 binding site and other domains of VZV IE63. “NLS” are nuclear localization signals, and “phosphorylation” is the region which contains phosphorylation sites mutated in ROka63-5M.

Subcellular localization of ASF1 and VZV IE63. (A and B) HeLa cells were cotransfected with plasmid p408 expressing myc-tagged ASF1a and either plasmid pCI-HA63 expressing HA-tagged, full-length VZV IE63 (A) or pCI-HA63KpnI, which expresses the HA-tagged amino-terminal 208 amino acids of VZV IE63 (B). At 48 h posttransfection, the cells were fixed, permeabilized, and stained with mouse anti-myc monoclonal antibody and rat anti-HA monoclonal antibody, followed by Alexa Fluor 488 (green)-conjugated goat anti-rat IgG antibody, Alexa Fluor 594 (red)-conjugated goat anti-mouse IgG₁ antibody, and DAPI. The arrows in panels A and B indicate cells expressing only ASF1a or IE63. (C) Cultured guinea pig enteric neurons were uninfected (top row) or infected with either cell-free (middle row) or cell-associated (bottom row) VZV, and at 3 days postinfection, the neurons were fixed, permeabilized, and stained with mouse anti-IE63 monoclonal antibody 9D12, rabbit anti-ASF1a antibody, and guinea pig anti-neuron specific ubiquitin carboxyl-terminal hydrolase L1 antibody, followed by goat anti-rabbit Alexa Fluor 488 (green), goat anti-mouse Alexa Fluor 594 (red), and biotin labeled anti-guinea pig antibody, with avidin-conjugated Alexa 680 (purple) and bisbenzimide used to stain DNA (blue).