Article Figures & Data
Figures
- FIG 1
Schematic representation of HHV-6B U26. (A) Schematic representation of the full-length HHV-6B U26 ORF in the HHV-6 genome. The HHV-6B genome comprises three major internal repeat elements (R1 to R3), the origin of replication (oriLyt), and the DR (direct repeat) termini (DRL and DRR). (B) Schematic of pU26, which was predicted to have 8 transmembrane domains by the SOSUI website (http://harrier.nagahama-i-bio.ac.jp/sosui/). (C) Mock- and HHV-6B-infected cells were lysed and subjected to immunoprecipitation (IP) with anti-U26 antibody followed by Western blotting with the same antibody.
- FIG 2
U26 is required for HHV-6B propagation. (A) Growth kinetics of HHV-6B U26. MT4 cells were infected with HHV-6B and harvested at 0 h, 8 h, 16 h, 48 h, and 72 h postinfection. The viral genome copy number in each sample was quantitated by real-time PCR. (B) U26 knockdown efficacy was confirmed by RT-qPCR. MT4 cells were transduced U26 shRNA-expressing lentivirus (shU26) or control lentivirus (shControl), infected with HHV-6B, and harvested at 24 h, 48 h, and 72 h postinfection. Total RNA was extracted from each sample, and U26 expression was measured by RT-qPCR. (C) U26 knockdown had minor effects on the HHV-6B entry process. U26 knockdown cells and control cells were infected with HHV-6B for 2 h at 37°C and then washed with 0.25% trypsin-EDTA. Total DNA was extracted from the infected cells, and real-time PCR was used to measure incoming virus genomes. (D) U26 affected HHV-6B propagation. HHV-6B infected U26 knockdown cells and control cells for 24 h, 48 h, and 72 h. Real-time PCR was used to detect HHV-6 genome copies in culture medium. (E and F) U26 affected the expression of viral proteins. HHV-6B infected MT4 cells, which were transduced with U26 shRNA-expressing lentivirus or control lentivirus and harvested at 24 h, 48 h, and 72h postinfection. Cell lysates were resolved by SDS-PAGE, followed by Western blotting with anti-IE1 (E) and anti-gB (F) antibodies.
- FIG 3
A fraction of U26 is located in mitochondria. (A) Chart showing the possibility of different proteins localizing to mitochondria, as predicted by a mitochondrial prediction website (https://ihg.gsf.de/ihg/mitoprot.html). (B) Confocal microscopy of U26 subcellular localization. HeLa cells expressing HA-U26 were stained with a mitochondrial marker (anti-Tom20) followed by Alexa Fluor 555 goat anti-rabbit secondary Ab (red). These cells were also stained with anti-HA followed by fluorescein isothiocyanate (FITC) 488 goat anti-mouse secondary Ab (green). The nuclei were stained with DAPI (blue). (C) The degree of colocation between U26 and mitochondria was demonstrated by fluorescence intensity profile analysis across the arrowed line (red) in panel B. a.u., arbitrary units. (D) Cytosolic/mitochondrial fractions isolated from U26-Flag-expressing HEK293T cells (left) and HHV-6B-infected cells (right) were immunoblotted with individual antibodies. Anti-Tom20 and antiactin were used as the mitochondrial and cytosolic markers, respectively.
- FIG 4
U26 downregulates MAVS expression. (A) U26 decreased the expression of exogenous MAVS without affecting STING expression. HEK293T cells were transfected with the plasmid for either HA-tagged MAVS or Myc-tagged STING expression alone or together with Flag-tagged U26. Cell lysates were resolved by SDS-PAGE, followed by immunoprecipitation with anti-Flag, anti-HA, and anti-Myc. (B) MAVS affected virus propagation. (Left) MT4 cells were lentivirally transduced with MAVS gRNA or control gRNA and selected with puromycin (0.5 μg/ml). MAVS knockout (KO) efficacy was confirmed by Western blotting. (Middle) MAVS knockout cells and control cells were infected with HHV-6B and harvested at 8 h, 16 h, 24 h, 48 h, and 72 h postinfection. The cell lysates were detected by Western blotting with anti-IE1 and antiactin. (Right) Real-time PCR was used to detect HHV-6 genome copies in infected cells and culture medium. (C) U26-mediated MAVS reduction was dependent on the proteasome pathway rather than lysosome pathways. HEK293T cells expressing Flag-tagged MAVS and HA-tagged U26 were treated with MG132 (left) or chloroquine (right) for 4 h. (D) U26 accelerated the ubiquitination of MAVS. HEK293T cells were transfected with plasmids encoding Flag-tagged MAVS together with HA-tagged K48 ubiquitin or HA-tagged K63 ubiquitin. The cells were prepared for immunoprecipitation assays with anti-Flag antibody followed by immunoblot analysis with the indicated antibodies. (E) Detection of endogenous MAVS protein. Cell lysates of U26-expressing HEK293T or control cells were immunoblotted with anti-MAVS. (F, left) Control shRNA- or U26 shRNA-expressing MT4 cells were infected with HHV-6B for 24 h, and endogenous MAVS was detected with anti-MAVS by immunoblot analysis. (Right) U26 knockdown efficacy was confirmed by RT-qPCR.
- FIG 5
U26 inhibits the RLR/MAVS/IRF3 signal pathway. (A) U26 affected the production of antiviral factors. (Left) HEK293T cells were transfected with plasmids encoding Flag-tagged MAVS and/or U26 and incubated for 24 h. C, control. (Right) Control shRNA- or U26 shRNA-expressing MT4 cells were infected with HHV-6B and harvested at 24 h postinfection. Antiviral factor (IFN-β and IL-6) mRNA expression levels were analyzed by RT-qPCR at the transfection (left) and infection (right) levels. (B) Schematic diagram of the RLR/MAVS signaling pathway. (C) U26 had a minor effect on the NF-κB signaling pathway. HEK293T cells were transfected with Flag-tagged MAVS and HA-tagged U26 and then immunoblotted with the indicated antibodies. (D and E) U26 inhibited MAVS-induced TBK1 and IRF3 phosphorylation. (D) Cell lysates of HEK293T cells expressing Flag-tagged MAVS alone or together with U26 were subjected to immunoblotting with the indicated antibodies. (E) Control shRNA- or U26 shRNA-expressing MT4 cells were infected with HHV-6B. (Left) At 24 h postinfection, IRF3 phosphorylation in these cells was detected by immunoblot analysis. (Right) U26 knockdown efficacy was confirmed by RT-qPCR.
- FIG 6
U26 affects mitochondrial membrane potential. (Top) Target cells were transfected with Flag-tagged U26 and incubated for 24 h. (Bottom) Control shRNA- or U26 shRNA-expressing MT4 cells were infected with HHV-6B and harvested at 24 h postinfection. Cells were incubated with TMRE at room temperature for 5 min in the dark, and ΔΨm was then detected using a flow cytometer.
