Figure 2.
Figure 2. EB1 and CLIP-170 are required for early HSV-1 infection. (A) NHDFs were treated with control or independent EB1 siRNAs (#1 or #2) and then infected with HSV-1–GFP-Us11 at MOI 5 for 16 h. Representative fluorescence (GFP-Us11) and phase images are shown. (B) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then infected at MOI 5 for 16 h with either of two different HSV-1 strains: wild-type strain F or strain Patton (Patton (GFP-Us11)). Levels of infectious virus in freeze-thawed culture lysates are presented on a log scale of plaque forming units (pfu) per milliliter. Error bars represent standard deviation. (C) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then infected at MOI 5 for 16 h with HSV-1 (Patton (GFP-Us11)). Whole cell extracts were analyzed by WB with the indicated antibodies. ICP0, ICP4, and ICP22 are IE proteins, whereas anti–HSV-1 antibody detects several viral structural proteins made later in infection. A sample of control siRNA–treated NHDFs was treated with 100 µg/ml cycloheximide (CHX) for 4 h to induce the onset of apoptosis as a control for detection of PARP cleavage. (D) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then fixed and stained for Tyr-tubulin (blue), EB1 (green), or CLIP-170 (red). Representative confocal planes are shown. White boxes highlight MT end staining with EB1, or MT ends lacking EB1 or CLIP-170 in EB1-depleted cells. Yellow arrowheads highlight examples of CLIP-170 patches localized along the MT lattice. (E) NHDFs or NHDFs expressing dominant-negative EB1 (EB1-DN) were fixed and stained for Tyr-tubulin (blue), EB1 (green), or CLIP-170 (red). Representative confocal planes are shown. White boxes highlight MT ends with EB1 and CLIP-170 in NHDFs, or MT ends lacking CLIP-170 in EB1-DN cells. Yellow arrowheads highlight examples of CLIP-170 patches localized along the MT lattice under each condition. Exposure settings required to image endogenous EB1 saturate images in EB1-DN–expressing cells, which also accumulates at MT plus ends (Video 1 A). (F) NHDF or NHDF-EB1-DN were mock infected or infected at MOI 5 for 5 h and then whole cell lysates were analyzed by WB using the indicated antibodies. Migration of endogenous EB1 and EB1-DN are highlighted to the right. FL, full length, CL, cleaved forms of PARP.

EB1 and CLIP-170 are required for early HSV-1 infection. (A) NHDFs were treated with control or independent EB1 siRNAs (#1 or #2) and then infected with HSV-1–GFP-Us11 at MOI 5 for 16 h. Representative fluorescence (GFP-Us11) and phase images are shown. (B) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then infected at MOI 5 for 16 h with either of two different HSV-1 strains: wild-type strain F or strain Patton (Patton (GFP-Us11)). Levels of infectious virus in freeze-thawed culture lysates are presented on a log scale of plaque forming units (pfu) per milliliter. Error bars represent standard deviation. (C) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then infected at MOI 5 for 16 h with HSV-1 (Patton (GFP-Us11)). Whole cell extracts were analyzed by WB with the indicated antibodies. ICP0, ICP4, and ICP22 are IE proteins, whereas anti–HSV-1 antibody detects several viral structural proteins made later in infection. A sample of control siRNA–treated NHDFs was treated with 100 µg/ml cycloheximide (CHX) for 4 h to induce the onset of apoptosis as a control for detection of PARP cleavage. (D) NHDFs were treated with control, EB1, or CLIP-170 siRNAs and then fixed and stained for Tyr-tubulin (blue), EB1 (green), or CLIP-170 (red). Representative confocal planes are shown. White boxes highlight MT end staining with EB1, or MT ends lacking EB1 or CLIP-170 in EB1-depleted cells. Yellow arrowheads highlight examples of CLIP-170 patches localized along the MT lattice. (E) NHDFs or NHDFs expressing dominant-negative EB1 (EB1-DN) were fixed and stained for Tyr-tubulin (blue), EB1 (green), or CLIP-170 (red). Representative confocal planes are shown. White boxes highlight MT ends with EB1 and CLIP-170 in NHDFs, or MT ends lacking CLIP-170 in EB1-DN cells. Yellow arrowheads highlight examples of CLIP-170 patches localized along the MT lattice under each condition. Exposure settings required to image endogenous EB1 saturate images in EB1-DN–expressing cells, which also accumulates at MT plus ends (Video 1 A). (F) NHDF or NHDF-EB1-DN were mock infected or infected at MOI 5 for 5 h and then whole cell lysates were analyzed by WB using the indicated antibodies. Migration of endogenous EB1 and EB1-DN are highlighted to the right. FL, full length, CL, cleaved forms of PARP.

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