PMC

PARP is not cleaved in JHLa1 cells. The blot in Fig. 3A was stripped and immunoblotted with anti-PARP antibody. The 85-kDa band consistent with the PARP cleavage product was seen in STS-treated HEK293 (lane 2) and JHL15 (lane 4) but not JHLa1 (lane 6) cells.

ICP10ΔPK and ICP10ΔRR are growth defective in primary hippocampal cultures. Single-step growth assays were done in hippocampal cultures infected with HSV-2, ICP10ΔPK, or ICP10ΔRR (5 PFU/cell). Virus titers were determined at 0 to 48 h postinfection, and results are expressed as mean 10⁵ PFU per milliliter ± SEM.

ICP10 PK and its mutants are present in 30-min-infected primary hippocampal cultures. Extracts from hippocampal cultures mock infected (lane 1) or infected (10 PFU/cell) with HSV-2 neutralized with gD MAb (lane 2), HSV-2 (lane 3), ICP10ΔPK (lane 4), or ICP10ΔRR (lane 5) were immunoblotted with ICP10 antibody. Blots were stripped and reprobed with actin antibody control.

TUNEL-positive cells in virus-infected hippocampal cultures are neurons. Primary hippocampal cultures infected with HSV-2 (10 PFU/cell; 24 h) (A and B) or mock infected (C and D) were stained with NF-160 antibody (A and C) and assayed by TUNEL (B and D). Similar results were obtained with TuJ1 antibody (data not shown). ICP10ΔPK-infected primary hippocampal cultures were assayed by TUNEL (E and H) and stained with TuJ1 (F) or GFAP (I) antibody. TUNEL colocalized with TuJ1 (G) but not GFAP (J) staining.

Cell penetration is required for HSV-2-mediated activation of MEK/MAPK in primary hippocampal cultures. (A) Extracts from cells mock infected with MEM (lane 1) or infected (24 h) with 10 PFU of HSV-2 (lane 2) or HSV-2 neutralized with the IgG fraction from a gD MAb (lane 3) were immunoblotted with antibody specific for P-MAPK1/2 (upper bands) followed by MAPK1/2 (bottom bands). (B) Densitometric scanning of the bands in panel A expressed as P-MAPK/MAPK for both isoforms. (C) Extracts from cells mock infected (lane 1) or infected with HSV-2 neutralized with preimmune (lane 2) or HSV-2 hyperimmune (lane 3) serum were immunoblotted with antibody specific for P-MAPK1/2 (upper bands) followed by MAPK (bottom bands). (D) Densitometric scanning of bands in panel C expressed as P-MAPK/MAPK ratios for both isoforms.

STS-induced DNA fragmentation is inhibited in JHLa1 cells. HEK293 (lanes 2, 3, 6, and 7) and JHLa1 (lanes 4, 5, 8, and 9) cells were treated with 250 nM STS (lanes 3 and 4) or 300 mM d-Mann (lanes 7 and 8) or mock treated with DMSO (control for STS) (lanes 2 and 5) or MEM (control for d-Mann) (lanes 6 and 9). Genomic DNA was extracted at 24 h postinfection and separated on agarose gels as described in Materials and Methods.

MAPK is activated in HSV-2- but not ICP10ΔPK-infected hippocampal cultures. (A) Cells were infected with HSV-2 (lanes 2 and 4) or ICP10ΔPK (lanes 3 and 5) (10 PFU/cell) or mock infected with growth medium (lane 1) and harvested at 0.5 and 24 h postinfection. Proteins were resolved by SDS-PAGE (8.5% acrylamide gels), transferred to nitrocellulose membranes, and immunoblotted with antibody specific for P-MAPK1/2. Blots were stripped and reblotted with antibody to MAPK1/2. (B) Protein levels were quantitated by densitometric scanning, and the results are expressed as P-MAPK/MAPK ratios for both isoforms.

MEK/MAPK and PI3-K/Akt pathways are involved in survival of HSV-2-infected and uninfected hippocampal cultures, respectively. (A) Hippocampal cultures were infected with HSV-2 (10 PFU/cell, 24 h) or mock infected with MEM in the absence or presence (10 to 20 μM) of U0126 and analyzed by TUNEL. Cells in five randomly chosen microscopic fields were counted, the percent positive cells in mock-infected cultures was subtracted, and the results are expressed as mean percent TUNEL-positive (apoptotic) cells ± SEM. *, P < 0.01 versus untreated HSV-2-infected cells by ANOVA. (B) Hippocampal cultures were infected with HSV-2 or mock infected in the absence or presence of LY294002 (10 to 100 mM) and analyzed by TUNEL as in A. *, P < 0.01 versus untreated mock-infected cells by ANOVA.

ICP10 PK protects neuronally differentiated PC12 cells from death due to NGF withdrawal. PC12 cells were differentiated by growth (12 to 14 days) in serum-free medium supplemented with 100 ng of NGF per ml. They were transfected with pJW17 (expresses ICP10 PK) or pJHL15 (p139) or mock transfected with FuGene and examined for cell survival by the MTS reduction assay using the Cell Titer 96 Aqueous One solution cell proliferation assay. Results are expressed as percent survival relative to 0 h post-NGF withdrawal ± SEM. *, P < 0.05 versus control; +, P < 0.05 versus pJHL15-transfected cells, by ANOVA.

ICP10 PK inhibits STS- and d-Mann-induced apoptosis in constitutively expressing cells. Parental HEK293 cells and HEK293 cells stably transfected with ICP10 (JHLa1) or the PK-negative ICP10 mutant p139 (JHL15) were treated (24 h) with 250 nM STS or 300 mM d-Mann and assayed for apoptosis by TUNEL. (A) TUNEL-positive and -negative cells were counted in five randomly chosen microscopic fields, and the mean percent apoptotic cells ± SEM was calculated as described in Materials and Methods. *, P < 0.01 versus HEK293; +, P < 0.01 versus JHL15 by ANOVA. (B and C) Apoptotic nuclei were seen for STS-treated HEK293 (B) and JHL15 (C) cells as determined by TUNEL with FITC-conjugated dUTP. (D) Apoptotic nuclei were not seen for STS-treated JHLa1 cells examined by TUNEL with FITC-conjugated dUTP.

ICP10 PK has antiapoptotic activity and inhibits caspase-3 activation in virus-infected hippocampal cultures. (A) Primary hippocampal cultures were infected (24 h) with HSV-2, HSV-2(R), ICP10ΔPK, or ICP10ΔRR (10 PFU/cell) and analyzed by TUNEL. The results are expressed as the mean percent apoptotic cells ± SEM estimated by counting cells in five randomly chosen microscopic fields and subtracting the percent positive cells in mock-infected cultures, as described in Materials and Methods. *, P < 0.01 versus HSV-2 and HSV-2(R) or ICP10ΔRR; +, P = 0.046 versus HSV-2 or HSV-2(R) by ANOVA. (B) Hoechst staining of representative nuclei from TUNEL-positive ICP10ΔPK-infected cells. (C) Hoechst staining of representative nuclei from TUNEL-negative HSV-2-infected cells. (D) Primary hippocampal cultures infected with HSV-2, HSV-2(R), ICP10ΔPK, or ICP10ΔRR as in A were stained with antibody to the activated caspase-3. The results are expressed as the mean percent staining cells ± SEM estimated by counting cells in five randomly chosen microscopic fields as in A. *, P < 0.05 versus HSV-2, HSV-2(R), or ICP10ΔRR by ANOVA.

MAPK activation in HSV-2-infected hippocampal cultures is MEK dependent and does not require de novo viral protein synthesis. (A) Extracts of cells mock infected (lanes 1 and 2) or infected with HSV-2 (lanes 3 and 4) or UV-inactivated HSV-2 (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence of 20 μM U0126 (lanes 2, 4, and 6) were obtained at 30 min postinfection and immunoblotted with antibody specific for P-MAPK1/2, followed by MAPK1/2 as in Fig. . (B) The bands in panel A were analyzed by densitometric scanning, and the results are expressed as P-MAPK/MAPK ratios for both isoforms.

Caspase-3 activation is inhibited in JHLa1 cells. (A) Extracts of HEK293 (lanes 1 and 2), JHL15 (lanes 3 and 4) and JHLa1 (lanes 5 and 6) cells, mock treated (with DMSO) (lanes 1, 3, and 5) or treated (24 h) with 250 nM of STS (lanes 2, 4, and 6) were resolved by SDS-PAGE (7% acrylamide gels), transferred to nitrocellulose membranes, and immunoblotted with antibody specific for the inactive pro-caspase-3. (B) Duplicates of the cultures shown in A were stained with antibody specific for the large fragment of activated caspase-3, and cells in five randomly chosen microscopic fields were counted. Results are expressed as mean percent positive cells ± SEM. *, P < 0.01 versus HEK293; +, P < 0.01 versus JHL15 by ANOVA. The cultures did not stain with normal rabbit serum, and the proportion of untreated staining cells was minimal (<5%).