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Herpes simplex virus type 2 UL14 gene product has heat shock protein (HSP)-like functions

¹ Laboratory of Virology, Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan
² Department of Environmental Biology, College of Bioscience and Biotechnology, Chubu University, Matsumoto-cho 1200, Kasugai 487-8501, Japan

View larger version (71K): [in a new window]   Fig. 1. The intracellular localization of UL14 protein in 14/Vero cells (A-K) and 14/HEp-2 cells (L) detected by immunofluorescence (A, mock). UL14 protein at 37°C (B) and after heat shock at 43°C for 30 minutes (C). UL14 protein (D) colocalizes with nucleolin (E) after heat shock (F, merge). UL14 protein (G) co-stained with propidium iodide (H) after heat shock (I, merge). UL14 protein localization after 5 hours treatment with ATP-depletion cocktail (J) and 5 hours of 1 M sorbitol treatment (K). UL14 protein localization in M-phase cells after heat shock, co-stained with propidium iodide (L, merged image) shows localization at the centrosomes. Bars, 10 µm.

View larger version (151K): [in a new window]   Fig. 2. UL14 protein colocalizes with cellular protein at 37°C (D-F) and after heat shock (A-C,G-I). 14/Vero cells were detected for UL14 protein (A) and PML (B) after heat shock (C, merge). 14/HEp-2 cells were detected for UL14 protein (D,G) and NuMA (E,H) at 37°C (D-F), and after heat shock (G-I); F and I are the merged images. Arrowheads indicate centrosomal localization of NuMA in M-phase cells.

View larger version (17K): [in a new window]   Fig. 3. (A) Fractionation of 14/HEp-2 cells during continuous heat shock at 43°C. Cells were heat-shocked for up to 180 minutes, and fractionated into insoluble (pellet, P) and soluble (supernatant, S) fractions at 0, 5, 15, 60, 120 and 180 minutes with PBS containing 1% Triton X-100. The samples were separated by SDS-PAGE, subjected to western blotting and finally detected with anti-UL14 polyclonal antibody. The total amount of the protein decreased after 60 minutes of heat shock, but the amount of UL14 protein in the insoluble fractions was sustained. (B) The nucleolar localization of UL14 protein in 14/Vero cells during heat shock at 43°C and recovery at 37°C. Cells were heat-shocked at 43°C for 120 minutes and left to recover at 37°C for up to 23 hours. Cells were fixed at several time points for immunofluorecence. Bright nucleolar staining of UL14 protein was counted and the population was plotted against time. Nearly all of the cells exhibited nucleolar localization of UL14 protein after 10 minutes of heat shock. The protein gradually delocalized from the nucleolus and, subsequently, from the nucleus during recovery at 37°C.

View larger version (45K): [in a new window]   Fig. 4. (A) Immunofluorescence of 14/Vero cells after heat shock. (A-C) Heat-shocked cells were detected for Hsp70 (A) and UL14 protein (B), showing that the two proteins colocalize in the nucleus and nucleolus (C, merge). (D-F) 14/Vero cells were microinjected with anti-Hsp70 polyclonal antibody and treated with heat shock. The cells were detected for the injected antibody (D) and UL14 protein (E; F, merge). UL14 protein still translocated into the nucleus and nucleolus in microinjected cells, indicating that its localization did not depend on Hsp70 function. (B) The comparison of nucleolar localization of UL14 protein and Hsp70 in Vero and 14/Vero cells. Vero and 14/Vero cells were heat-shocked at 43°C for 120 minutes and recovered at 37°C for up to 16 hours. Cells were fixed at several time points for immunofluorecence. Bright nucleolar staining of Hsp70 and/or UL14 protein was counted and the population was plotted against time. In both cells, Hsp70 rapidly translocated to the nucleoli after heat shock and started to delocalize to the cytoplasm during recovery. The rate of delocalization was faster in 14/Vero cells than in Vero cells. In 14/Vero cells, UL14 protein remained in the nucleoli for a longer time than Hsp70. (C) Hsc70/Hsp70 expression in heat-shocked Vero and 14/Vero cells. Cells were continuously heat-shocked at 43°C for up to 60 minutes and samples were collected for immunoblotting at the intervals shown. Anti-Hsp70 Ab was used at a dilution of 1/5000. To show that nearly equal amounts of protein were loaded, bands corresponding to actin were obtained from staining identical SDS-PAGE gels with CBB.

View larger version (35K): [in a new window]   Fig. 5. (A) Homological amino acid sequences conserved in HSV UL14 and Hsp70 family. The red letters are complete matches, and the blue letters are amino acids with similar properties. (B) The comparison of the ability of UL14 wild type and mutant proteins to translocate VP26-FLAG in coexpressing cells. Cells were cotransfected with pFLAG-UL35 and the plasmids indicated (pcDNA3-) and, 24 hours later, fixed and stained for the two proteins. Predominant nuclear localization of VP26-FLAG was counted in at least 150 double-stained cells. The bars show the percentage of coexpressing cells with nuclear localization of VP26-FLAG. (C-E) Double-staining of cells transfected with pcDNA3-UL14R (60,64) A and pFLAG-UL35. It is shown that the UL14 mutant protein (C) colocalizes with VP26-FLAG (D) in the cytoplasm but no nuclear staining of VP26-FLAG is observed (E, merge). Images in C-E are representative of VP26 cytoplasmic localization in the assay shown in Fig. 5B.

View larger version (15K): [in a new window]   Fig. 6. The presence of UL14 protein increases luciferase activity in a dose-dependent manner when cells yield low luciferase activity. HEp-2 cells were cotransfected with pGL3-p (0.25 µg) and pcDNA3-UL14 (1, 0 µg; 2, 0.50 µg; 3, 1.0 µg) and an empty vector pcDNA3, to a total of 1.25 µg DNA per assay. The results are shown as the percentage of relative luciferase activity. The increase in the expression of UL14 protein correlates with the rise in luciferase activity (up to 14-fold). Each assay was done in triplicate, and each point represents the average±s.d. of three experiments. The same samples were subjected to western blotting and detected for luciferase showing that the level of protein is almost constant contrary to its rise in activity. (B) Luciferase activity is increased in cell lines expressing UL14 protein without a change in the level of luciferase protein. HEp-2, 14/HEp-2, Vero and 14/Vero cells were each transfected with 0.5 µg of luciferase-expressing plasmid pGL3-p and luciferase activity was measured 24 hours later. In each cell line, constitutive expression of UL14 protein increased luciferase activity. Luciferase activity was measured as in A. Western blots show that the amount of expressed luciferase is almost unchanged. (C) UL14 protein existence substitutes for the loss in luciferase activity induced by transfection with antisense oligomer to cellular Hsp70 mRNA. HEp-2 and 14/HEp-2 cells were cotransfected with: (1) 0.5 µM of either sense or antisense (a.s.) oligomers and 0.1 µg pGL3-p; (2) 0.5 µM of antisense oligomer, 0.1 µg pGL3-p and 0.25 µg pCDNA3-UL14, - UL14R(60,64)A, or -UL14D(51-90). 14HEp-2 cells were cotransfected with 0.5 µM of either sense or antisense oligomers and 0.1 µg pGL3-p. Luciferase activity was assayed as above and relative activity was compared. The results show that the drop in luciferase activity on transfection of antisense oligomers is less in 14/HEp-2 cells (35%) than in HEp-2 cells (55%). In addition, luciferase coexpressed with UL14 mutant proteins showed lower activity, suggesting that UL14 wt protein compensates for the loss of cellular chaperone activity. Each assay was done in triplicate, and each point represents the average±s.d. of three experiments.