Figure 1.
Identification of the interaction between UL70 and two isoforms of DNAJB6 by yeast two-hybrid screen and coimmunoprecipitation.
(A–B) Yeast strain AH109 was co-transformed with the combination of one BD and one AD plasmid as indicated (A–B). Transformed yeast cells containing both plasmids were first grown on SD-minus Trp/Leu plates (DDO) to maintain the two plasmids, then colonies were replica plated onto SD-minus Trp/Leu/Ade/His plates (QDO) and also subjected to beta-gal activity test (x-gal) by filter lift staining. No interactions were identified between BD-UL70 and control empty vector AD-vector or between the control empty vector BD-vector and AD-DNAJB6 isoform a and b. Positive interactions were identified between BD-UL70 and AD-DNAJB6 isoform a and b, as well as BD-p53 and AD-T, which served as a positive control. All samples in the three panels (DDO, QDO, and X-gal) were prepared from the same experiments at the same time and in parallel. (C–D) Human U251 cells were co-transfected with a combination of two plasmids expressing FLAG- and Myc-tagged proteins, and then harvested at 48 hours posttransfection. The input protein samples (80 µg) (Input) (lanes 1, 4, 7, 10, 13, and 16) and samples (15 µg) that were either immunoprecipitated with anti-Myc (IP (anti-Myc)) (lanes 2, 5, 8, 11, 14, and 17) or anti-FLAG antibodies (IP (anti-FLAG)) (lanes 3, 6, 9, 12, 15, and 18) were separated on SDS-containing polyacrylamide gels, and assayed with Western blot analysis using anti-Myc (anti-Myc) (C) and anti-FLAG antibodies (anti-FLAG) (D), respectively.
Figure 2.
Schematic diagram of DNAJB6a and DNAJB6b and their deletion mutants that interact with UL70 as identified by the two hybrid screen in yeast (YTH) and co-immunoprecipitation (co-IP) in U251 cells.
DNAJB6 (a and b) proteins share the N-terminal 231 amino acids, which include the highly conserved DnaJ homology region (in blue), a Gly/Phe-rich region (in green), and a linker region (in red) but differ at the less conserved C-terminal region (in yellow and pink for DNAJB6a and b, respectively) in which a NLS (in orange) is present in the longer DNAJB6a but is absent in the shorter DNAJB6b [38], [40]. The NLS was mutated and deleted in mutants DNAJB6a-Mut and DNAJB6a-Del, respectively. The interactions that were positive and negative in the two hybrid screen or co-IP were marked as “+” and “−”, respectively.
Table 1.
Plasmid constructs used in the study.
Figure 3.
Co-localization of UL70 and DNAJB6a and DNAJB6b expressed in human cells.
Cells were transfected with the construct containing the sequence of Myc-tagged UL70 alone (A) and in the presence of the constructs containing the sequences of FLAG-tagged DNAJB6 (a and b) (B and C), fixed at 48 hours posttransfection, stained with antibodies, and visualized using a microscope. The images of Myc-tagged UL70 (b, e, i), FLAG-tagged DNAJB6a (f) and DNAJB6b (j), and the nuclei stained with DAPI (a, d, h) were used to generate the composite images (c, g, k). The images show different levels of magnification.
Figure 4.
Role of the nuclear localization signal of DNAJB6 isoform a in the nuclear import of UL70.
U251 cells were transfected with constructs pRK11-FLAG-DNAJB6a, pRK11-FLAG-DNAJB6a-Mut, and pRK11-FLAG-DNAJB6a-Del alone (A) and in the presence of construct plasmid pCMV-Myc-UL70 (B). At 48 hours posttransfection, cells were fixed, stained with antibodies, and visualized using a microscope. The images of Myc-tagged UL70 (k, o), FLAG-tagged DNAJB6a (b), DNAJB6a-Mut (e and l), and DNAJB6a-Del (h and p) and the nuclei stained with DAPI (a, d, g, j, and n) were used to generate the composite images (c, f, i, m, and q).
Figure 5.
Up- and down-regulated expression of DNAJB6 in cells.
Western blot analysis of the levels of DNAJB6a (lanes 1–4) and b (lanes 5–8) and Myc-tagged UL70 in the parental U251 cells (U251) (lanes 1 and 5), the DNAJB6a-expressing U251-6a (U251-6a) (lane 4) and DNAJB6b-expressing cells (U251-6b) (lane 8), or U251 cells that were transfected with anti-DNAJB6a siRNA (6a-siRNA) (lane 3), anti-DNAJB6b siRNA (6b-siRNA) (lane 7), or control siRNA (C-siRNA) (lanes 2 and 6). The expression of cellular actin was used as the internal control. Cells were transfected with pCMV-Myc-UL70 in the absence and presence of siRNAs. Forty-eight hours after transfection, cells were infected with HCMV at MOI of 1. Protein samples were prepared at 48–72 hours postinfection. The membranes were reacted with antibodies, stained using a Western chemiluminescent substrate kit (GE Healthcare), and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [54], [55]. A dilution series of the samples was analyzed and the results were compared in order to accurately determine the protein levels. Quantitation was performed in the linear range of protein detection [54], [55].
Figure 6.
Effect of up- and down-regulation of the expression of DNAJB6 on the cellular distribution of UL70.
Immunofluorescence microscopy of the cellular localization of UL70 in the parental U251 cells (U251), cells overexpressing DNAJB6a (U251-6a) and b (U251-6b), or the cells treated with anti-DNAJB6a (6a-siRNA) and anti-DNAJB6b siRNA (6b-siRNA). Cells were infected with HCMV (MOI = 5) at 48 hours posttransfection, fixed at 48–72 hours postinfection, stained with antibodies, and visualized. The images of Myc-tagged UL70 (green) (a, d, g, j, m) and the nuclei stained with DAPI (blue) (b, e, h, k, n) were used to generate the composite images (c, f, i, l, o). The images show different levels of magnification.
Figure 7.
Effect of up- and down-regulation of the expression of DNAJB6 on the distribution of UL70 in nuclear and cytoplasmic fractions.
Different cells (e.g. parental U251 cells, U251-6a and U251-6b cells, and 6a- and 6b-siRNA treated cells) were transfected with pCMV-Myc-UL70 in the absence and presence of siRNAs. At 48 hours posttransfection, cells were infected with HCMV (MOI = 1). At 48–72 hours postinfection, cells were harvested and separated into nuclear and cytoplasmic fractions. Equivalent amounts of each fraction were analyzed by immunoblotting with anti-Myc. The purity of the nuclear and cytoplasmic fractions was assayed by immunoblotting with anti-histone H1 and anti-Actin, respectively. The membranes were reacted with antibodies, stained using a Western chemiluminescent substrate kit (GE Healthcare), and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [54], [55]. A dilution series of the samples was analyzed and the results were compared in order to accurately determine the protein levels. Quantitation was performed in the linear range of protein detection [54], [55]. The experiments were in duplicate and repeated three times. The standard deviation is indicated by the error bar (lower panel).
Figure 8.
Effect of up- and down-regulation of the expression of DNAJB6 on HCMV gene expression, and viral genomic synthesis and production.
(A) Western blot analysis of the levels of HCMV IE1, UL44, and UL99 in the parental U251 cells (U251) (lane 1), cells overexpressing DNAJB6a (U251-6a) (lane 4) and b (U251-6b) (lane 6), or U251 cells that were transfected with either anti-DNAJB6a (6a-siRNA) (lane 3) and DNAJB6b siRNA (6b-siRNA) (lane 5) or control siRNA (C-siRNA) (lane 2). The expression of cellular actin was used as the internal control. At 48 hours posttransfection, cells were infected with HCMV (MOI = 1). Protein samples were prepared at 48–72 hours postinfection. (B) To assay the level of intracellular viral DNA, cells were harvested at 72 hours postinfection. To assay the level of viral production, total infection cultures were collected at 5 days postinfection and viral titers were determined. The values of the relative levels of HCMV DNA and titers, which are the means from triplicate experiments, represent the ratios of the levels of viral DNA or titers in different cells to those in the parental U251 cells (U251), respectively. The analyses were repeated three times and the standard deviation is indicated by the error bar.