The U95 protein of human herpesvirus 6B interacts with human GRIM-19: silencing of U95 expression reduces viral load and abrogates loss of mitochondrial membrane potential

Abstract

To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.

FIG. 1.

Coimmunoprecipitation of HHV-6B U95 and human GRIM-19 proteins in MT-4 cells. MT-4 cells were transfected with the pCMV-HA-U95 and/or pCMV-Myc-GRIM-19 construct. Mock controls were mixed lysates from cells transfected with plasmids expressing either the HA or Myc tag only. (A) Immunoprecipitation (IP) of cell lysates with anti-HA polyclonal antibody was performed, except for the Myc-GRIM-19 lysate, which acted as the positive control for Western blotting with anti-Myc. (B) Immunoprecipitation (IP) of cell lysates with anti-Myc monoclonal antibody was performed, except for the HA-U95 lysate, which acted as the positive control for Western blotting with anti-HA. The precipitates were fractionated by Tris-glycine-SDS-polyacrylamide gel electrophoresis (12%) and subjected to Western blot analysis using anti-HA antibody or anti-Myc antibody to detect HA-U95 or Myc-GRIM-19, respectively.

FIG. 2.

Intracellular colocalization of HHV-6B U95 with GRIM-19. MT-4 cells were cotransfected with pCMV-Myc-GRIM-19 and pCMV-HA-U95 constructs, fixed, immunolabeled, and examined by immunofluorescence microscopy. Detection of Myc-GRIM-19 with mouse anti-Myc antibody (A) and of HA-U95 with rabbit anti-HA antibody (B) was performed. The secondary antibodies were FITC-tagged anti-mouse antibody and rhodamine phalloidin-tagged anti-rabbit antibody, respectively. (C) Colocalization was observed by superimposing panels A and B. To serve as negative controls, MT-4 cells were cotransfected with empty pCMV-Myc and pCMV-HA vectors, fixed, immunolabeled, and examined by immunofluorescence microscopy. Reactions with mouse anti-Myc antibody (D) and rabbit anti-HA antibody (E) are shown. The secondary antibodies were FITC-tagged anti-mouse antibody and rhodamine phalloidin-tagged anti-rabbit antibody, respectively. (F) Superimposed image of panels D and E.

FIG. 3.

Colocalization of HHV-6B U95 with endogenous GRIM-19 in MT-4 cells. MT-4 cells were transfected with the pCMV-HA-U95 construct, immobilized onto glass slides by use of a Cytospin centrifuge, fixed, immunolabeled, and examined by immunofluorescence microscopy. (A) Detection of HA-U95 with rabbit anti-HA antibody staining and counterlabeling with FITC-tagged anti-rabbit antibody. (B and E) Detection of endogenous cellular GRIM-19 by mouse monoclonal antibody staining and counterlabeling with rhodamine phalloidin-tagged anti-mouse antibody. (C) Colocalization is displayed by superimposing panels A and B. (D) Mitotracker Green FM dye staining of mitochondria as long, tubular organelles extending from the nucleus. (F) Colocalization is displayed by superimposing panels D and E.

FIG. 4.

Real-time RT-PCR analyses of relative U95 mRNA expression levels in HHV-6B-infected MT-4 cells following U95 shRNA treatment compared with those in controls. HHV-6B-infected MT-4 cells were transfected with the U95 shRNA construct or with the control scrambled shRNA construct after infection for 1 h. Real-time RT-PCR was performed 1 and 4 h following shRNA transfection. Samples were normalized against the G3PDH housekeeping gene and with untransfected but HHV-6B-infected MT-4 cells at each respective time point as the reference sample (i.e., change of onefold for U95 transcription). Statistical analyses were then performed, with P values of <0.05 denoting significant differences.

FIG. 5.

Transmission electron micrographs of HHV-6B-infected cells without and with U95 shRNA treatment. (A) After 5 h of infection of MT-4 cells with HHV-6B, typical features of mitochondrial swelling (SW), with severe disruption of mitochondrial structures, showing myelin-like figures (MY) and a loss of cristae, were observed. (B) After 1 h of HHV-6B infection, MT-4 cells were transfected with U95 shRNA. At 4 h post-RNAi, swelling of the mitochondria was significantly reduced, and numerous cristae (CR) could be observed clearly. Magnification, ×50,000. At the time of infection of MT-4 cells with HHV-6B (C) and at 1 h postinfection (D), little or no disruption of mitochondrial structures was observed. At 4 h (E) and 7 h (F) postinfection, there were increasingly severe structural abnormalities of mitochondria, including myelin-like figures (MY) and a loss of cristae. (G) At 7 h postinfection, uninfected MT-4 cells generally displayed normal mitochondrial structures. Bars, 1 μm.

FIG. 6.

Assessment of mitochondrial membrane potential by MitoProbe DiOC₂(3) assay. MT-4 cells were analyzed by flow cytometry using a 488-nm excitation wavelength, with 530/30-nm-band-pass and 650-nm-long-pass detection filters. Cells were resuspended in PBS, and the distribution of fluorescent signals (logarithmic scale) of 20,000 cells was observed. The x axes of the left and right panels depict the intensities of green and red fluorescence, respectively. Each y axis indicates the number of cells, while each area under the curve is equivalent to 20,000 cell events. (A) Unstained MT-4 cells served as a control to exclude autofluorescence. (B) DiOC₂(3)-stained uninfected MT-4 cells. (C) DiOC₂(3)-stained HHV-6B-infected MT-4 cells showing a reduction in red fluorescence signals. (D) DiOC₂(3)-stained HHV-6B-infected MT-4 cells subjected to U95 shRNA treatment at 1 h postinfection. (E) DiOC₂(3)-stained HHV-6B-infected MT-4 cells subjected to scrambled shRNA treatment at 1 h postinfection, indicating decreased red fluorescence signals. (F) Effect of HHV-6B RNAi treatment on mitochondrial membrane potential in HHV-6B-infected MT-4 cells. The bar chart illustrates the percentages of cells in different treatment and control groups emitting red, green, or both fluorescence signals by flow cytometry.