DR5 is a restriction factor for human herpesviruses

Abstract

Restriction factors are dominant proteins that target different essential steps of the viral life cycle; thus, these proteins provide an early line of defense against viruses. Here, we found that the internalization of DR5, an important receptor of the extrinsic apoptotic pathway, initiates apoptosis to inhibit Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication. An evolutionary analysis of the DR5 sequence demonstrated that three amino acids underwent positive selection in primates. Notably, one of these positive selection sites, A62, is essential for the antiviral function of DR5 and is important for the binding of DR5 to its ligand, TNF-related apoptosis-inducing ligand. Moreover, DR5 exhibits broad antiviral activity against and inhibits various herpesviruses, including Epstein-Barr virus, herpes simplex virus type 1, and herpes simplex virus type 2. As a countermeasure, the KSHV K5 protein interacts with DR5 and promotes DR5 degradation through the lysosomal and proteasomal degradation pathways; lysine 245 of DR5 is essential for K5-induced DR5 degradation. These findings demonstrate that DR5 is a restriction factor for human herpesviruses.

Keywords: DR5; Herpesvirus; KSHV; restriction factor.

Fig. 1.

DR5 inhibits KSHV replication. (A–C) Overexpression of DR5 inhibits KSHV replication. (A) DR5 expression was evaluated via qPCR and immunoblotting. (B) Extracellular viral loads after DNase treatment were quantified via qPCR. (C) Viral gene expression was analyzed via qPCR. (D–F) Knockdown of DR5 promotes KSHV lytic replication. (D) si-NC or si-DR5 was transfected into iSLK.RGB cells. After 24 h, the cells were collected, and the expression of DR5 was detected via qPCR and immunoblotting. (E) iSLK.RGB cells expressing DR5 siRNA were treated with Dox. After 48 h, the viral loads in the supernatants were quantified via qPCR. (F) Viral gene expression was detected via qPCR. Representative results from three biological replicates are presented. The error bars indicate the SDs. The data were analyzed with Student’s multiple t tests (ns, no significant difference; *P < 0.05; **P < 0.01; ****P < 0.0001).

Fig. 2.

DR5 inhibits KSHV replication through the induction of apoptosis. (A and B) DR5-expressing cells, DR5-knockdown cells, and control cells were treated with Dox for 24 h and 48 h. Then, caspase-Glo 3/7 reagents were added to the culture medium, and the luminescence of each sample was measured with a luminometer according to the manufacturer’s instructions. (C) DR5-expressing cells and control cells were treated with DMSO or inhibitors of caspase-3/8 and caspase-3, after which the viral loads in the supernatants were detected via qPCR. (D) DR5-expressing cells and control cells were treated with Dox for 24 h to induce KSHV reactivation. Then, the cells were incubated with DMSO, chloroquine, or bafilomycin A1 for another 10 h, and the viral loads in the supernatants were determined by qPCR. (E–G) Viral loads in the supernatants, the percentage of GFP-expressing HEK293T cells infected with viral supernatants and viral gene expression were detected as described above. (H) DR5-expressing cells, DR5-L311/312A mutant-expressing cells, and control cells were treated with Dox for 24 h and 48 h. Then, caspase 3/7 activity was measured with a luminometer according to the manufacturer’s instructions. Representative results from three biological replicates are presented. The error bars indicate the SDs. The data were analyzed with Student’s t tests (ns, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 3.

DR5 is an ISG. (A) Analysis of the STAT motif and ISRE motif upstream of the DR5 TSS. The red squares indicate the STAT motif, and the green squares indicate the ISRE motif. TSS indicates the transcription start site, and CDS indicates the coding sequence. (B–D) THP-1 cells were treated with IFNα 2/IFN β/IFN for different durations, and the expression of DR5 was determined via qPCR. (E–G) THP-1 cells were treated with IFNα 2/IFN β/IFN for different durations, and the expression of DR5 was determined via western blotting. Representative results from three biological replicates are presented. The error bars indicate the SDs. The data were analyzed with Student’s t tests (***P < 0.001, ****P < 0.0001).

Fig. 4.

The A62D mutation of DR5 attenuates the inhibitory effect of DR5 on KSHV replication due to the reduced ability of DR5 to bind the ligand TRAIL. (A–C) The A62D mutation of DR5 attenuates the inhibitory effect of DR5 on KSHV replication. (A) DR5 expression was evaluated by immunoblotting to validate the successful establishment of iSLK.RGB cell lines with stable expression of DR5 and DR5 mutants. (B) DNase-treated extracellular viral loads were quantified via qPCR. (C) Viral gene expression was analyzed via qPCR. (D and E) The reduced inhibitory effect of the A62D mutant of DR5 on KSHV replication occurred due to a decrease in the ability of DR5 to bind the ligand TRAIL. (D) The ligand TRAIL (1 µg/mL) was coated on a 96-well plate, followed by the incubation of various concentrations of wild-type His-DR5 or His-DR5-A62 mutant with the TRAIL for 2 h at room temperature. Subsequently, anti-His-mouse antibody was added and incubated for 1.5 h, followed by incubation with anti-HRP-mouse antibody for 1 h. A substrate solution was then added to each well and incubated for 20 min at room temperature. The reaction was stopped by adding a stop solution, and the optical density of each well was measured using a microplate reader set to 450 nm. (E) Wild-type DR5-, DR5-A62D-, DR5-A221L-, and DR5-R257H-expressing cells and control cells were constructed from HEK293 cells. These cells were subsequently infected with 30 copies of KSHV DNA for 8 h, after which caspase 3/7 activity was measured with a luminometer according to the manufacturer’s instructions. Representative results from three biological replicates are presented. The error bars indicate the SDs. The data were analyzed with Student’s multiple t tests (ns, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 5.

KSHV K5 interacts with DR5 and degrades DR5. (A–C) KSHV K5 interacts with DR5. (A) A stable SLK cell line expressing the FLAG-K5 gene was established via lentiviral transduction. Cell lysates were immunoprecipitated with FLAG antibody-conjugated agarose beads and analyzed via western blotting with an anti-DR5 antibody. (B) Cell lysates from SLK cells expressing FLAG-K5 were immunoprecipitated with DR5 antibody and protein A/G-conjugated agarose beads. Subsequently, the samples were analyzed by western blotting with an anti-FLAG antibody. (C) HeLa cells were transfected with FLAG-DR5, HA-K5, or FLAG-DR5 and HA-K5 and then stained with anti-HA and anti-FLAG antibodies and Alexa Fluor 488 and Alexa Fluor 555 secondary antibodies. Colocalization was viewed by a DM6000B fluorescence microscope. (D–G) KSHV K5 degrades DR5. (D) SLK cells expressing K5 or transfected with the empty control were stained with anti-DR5-PE or anti-IgG1-PE and analyzed by flow cytometry. (E) HEK293T cells were transfected with HA-K5 or FLAG-DR5 or FLAG-DR5 and HA-K5, and co-IP was performed with FLAG antibody-conjugated agarose beads. Ubiquitination was detected with an anti-Ub antibody. (F and G) DR5 expression in wild-type RGB cells and mutant RGB cells in which K5 was deleted. A recombinant K5 deletion virus was constructed and introduced into iSLK cells, after which the cells were treated with Dox for 8, 24, 36, 48, or 72 h. The expression of DR5 was measured via western blotting.

Fig. 6.

DR5 has broad antiviral activity against EBV, HSV-1, and HSV-2. (A) DR5 inhibits EBV infection. HEK293 cells expressing wild-type DR5 and the DR5-A62D, DR5-A221L, and DR5-R257H mutants and control cells were infected with the EBV B95.8 strain for 24 h. The extracellular viral loads were subsequently quantified via qPCR after DNase treatment. To ensure DNA extraction quality, pGL3-luc plasmid DNA was introduced during viral DNA extraction. Relative DNA copy numbers were determined via qPCR using primers for EBV-Bam-W and pGL3. The control values were normalized to 1. (B) DR5 inhibits HSV-1 infection. Cell lines were infected with HSV-1 for 24 h. The viral loads in the supernatants were subsequently quantified via qPCR via primers for HSV-1 UL30 and pGL3. The control values were normalized to 1. (C) DR5 inhibits HSV-2 infection. Cell lines were infected with HSV-2 for 24 h. The viral loads in the supernatants were quantified via qPCR using primers for HSV-2 gD and pGL3. The control values were normalized to 1. Representative results from three biological replicates are presented. The error bars indicate the SDs. The data were analyzed with Student’s multiple t tests (ns, no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (D) EBV BDLF3 Mediates DR5 Degradation. HEK293T cells were transfected with FLAG-DR5 and HA-Vector, or FLAG-DR5 and HA-BDLF3 for 36 h, followed by cell lysis. The levels of DR5 and BDLF3 were assessed via western blot using anti-HA and anti-FLAG antibodies. (E) HSV-1 Induces DR5 Degradation. HEK293T cells were transfected with FLAG-DR5 and HA-Vector, or FLAG-DR5 and HSV-1 HA-UL56 for 36 h, then underwent cell lysis. The expression of DR5 and HSV-1 UL56 was analyzed by western blot using anti-HA and anti-FLAG antibodies. (F) HSV-2 promotes DR5 degradation. HEK293T cells were transfected with FLAG-DR5 and HA-Vector, or FLAG-DR5 and HSV-2 HA-UL56 for 36 h, then lysed. The levels of DR5 and HSV-2 UL56 were determined via western blot using anti-HA and anti-FLAG antibodies. (G) Schematic depicting the role of DR5 in regulating KSHV lytic replication. The host protein DR5 acts as a restriction factor, suppressing KSHV lytic replication through the induction of lysosomal-dependent apoptosis. To antagonize its inhibitory impact on KSHV lytic replication, the K5 protein interacts with DR5, promoting DR5 degradation.