Prototype foamy virus downregulates RelB expression to facilitate viral replication

Abstract

Foamy viruses (FVs) are classified in the subfamily Spumaretrovirinae and bridge the gap between Orthoretrovirinae and Hepadnaviridae. FVs have strong cytopathic effects against cells cultured in vitro. However, they establish lifelong latent infections without evident pathology in the host. The roles of cellular factors in FV replication are poorly understood. To better understand this area, we determined the transcriptomes of HT1080 cells infected with prototype foamy virus (PFV) to measure the effect of PFV infection on the expression of cellular genes. We found that the level of RelB mRNA, a member of the nuclear factor-κB (NF-κB) protein family, was significantly decreased as a result of PFV infection, and this was further confirmed with real-time PCR. Interestingly, overexpression of RelB reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. This inhibitory effect of RelB results from diminished transactivation of the viral long terminal repeat (LTR) promoter and an internal promoter (IP) by viral Tas protein. Together, these data demonstrate that PFV infection downregulates the viral inhibitory host factor RelB, which otherwise restricts viral gene expression.

Keywords: IP; LTR; RelB; prototype foamy virus; transcription.

Fig. 1

Downregulation of RelB by PFV infection. (A) After PFV infection of HT1080 cells for 24 h, RNA was extracted for transcriptome sequencing. (B) After HT1080 cells were infected with PFV for 24 h, the extracted RNA was reverse‐transcribed into cDNA, and the gene‐specific real‐time primers were designed for PCR to detect change of RelB mRNA levels. The results were the average of three independent experiments, and data were analyzed using GraphPad Prism software (paired t‐test, compared with mock, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars indicate standard deviations.

Fig. 2

Overexpression of RelB inhibits PFV replication. A and B HEK293T cells (A) and HT1080 cells (B) were transfected with pcPFV (0.5 μg) and the empty vector or HA‐RelB (0.5 μg). Forty‐eight hours post‐transfection, supernatants (500 μL) or transfected cells (1/10 of total) were cocultured with PFVL indicator cell line for another 48 h and luciferase activity was measured then (compared with pcPFV, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The rest of cells were lysed and then immunoblotted with Gag, Bet, HA, and tubulin antibodies. (C) HT1080‐Control and HT1080 stably expressing RelB cells were infected with 200 μL of PFV whose titers had been determined using PFVL indicator cells (D). At 48 h postinfection, supernatants (500 μL) or infected HT1080 cell lines (1/10 of total) were cocultured with PFVL cells (compared with pQCXIP + PFV, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The infected HT1080 cell lines were also lysed and then immunoblotted with Gag, Bet, RelB, and tubulin antibodies. One‐way ANOVA was used to perform statistical test. All transfections were performed in triplicate. The results shown represent the averages of the results of three independent experiments. Error bars indicate standard deviations.

Fig. 3

Knockdown of endogenous RelB promotes PFV replication. (A) Equal amount of cells (HEK293T, HeLa, and HT1080) were collected and lysed for western blotting analysis. (B) HeLa cells were infected with shRNA viral particles and negative control. Forty‐eight hours postinfection, cells were collected for western blotting analysis. (C–E) HeLa‐shControl and HeLa‐shRelB cells were transfected with pcPFV infectious clone (1.5 μg). Forty‐eight hours post‐transfection, C supernatants (500 μL) or D cells (1/10 of total) were cocultured with PFVL cells (compared with shControl + pcPFV, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (E) The rest of cells were collected for western blotting analysis. All transfections were performed in triplicate. One‐way ANOVA was used to perform statistical test. All transfections were performed in triplicate. The results shown represent the averages of the results of three independent experiments. Error bars indicate standard deviations.

Fig. 4

RelB downregulates the activation of PFV LTR and IP by Tas. (A and B) Levels of integrated proviral DNA were measured in semiquantitative PCR (A) and real‐time PCR (B) as described in Materials and Methods (two‐way ANOVA was used to perform statistical test). (C) HEK293T cells were transfected with LTR‐luc (0.025 μg), IP‐luc (0.01 μg), or CMV‐luc (0.025 μg) and combined with 3.1‐Tas (0.1 μg) and HA‐RelB (0.3 μg). To normalize transfection efficiency, pCMV‐β‐gal (0.025 μg) was cotransfected. Total DNA amounts were adjusted to 1 μg with an empty vector. At 48 h post‐transfection, luciferase activities were measured and corrected by β‐gal catalytic activities (one‐way ANOVA was used to perform statistical test. Compared with 3.1‐Tas, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The amount of Tas protein and RelB protein was detected by western blotting. (D) The same experimental process also repeated in HeLa cells. All transfections were performed in triplicate. The results shown represent the averages of the results of three independent experiments. Error bars indicate standard deviations.

Fig. 5

The RelB interacts with Tas. (A) HA‐RelB (4 μg) was transiently transfected into HEK293T cells together with the empty vector (control) or Flag‐Tas (4 μg, respectively). Co‐IP was performed with an anti‐Flag antibody. Samples from both cell lysates and immunoprecipitates were analyzed by western blotting using HA and Flag antibodies. (B) Flag‐Tas (4 μg) was transiently transfected into HEK293T cells together with the empty vector (control) or HA‐RelB (4 μg, respectively). Co‐IP was performed with an anti‐HA antibody. Samples from both cell lysates and immunoprecipitates were analyzed by western blotting using HA and Flag antibodies. (C) Flag‐RelB (4 μg) was transiently transfected into 293T cells together with the empty vector (control), Tat wild‐type (4 μg), or truncation mutant Tat 1‐37 (4 μg). Co‐IP was performed with an anti‐GFP antibody. Samples from both cell lysates and immunoprecipitates were analyzed by western blotting using Flag and GFP antibodies. (D) HeLa cells were transfected with Flag‐Tas (0.5 μg) or HA‐RelB (0.5 μg) or both. An indirect IFA was used to localize RelB (with rabbit anti‐HA antibody and FITC‐conjugated goat anti‐rabbit secondary antibody) and Tas (with mouse anti‐Flag antibody and TRITC‐conjugated goat anti‐mouse secondary antibody). Scale bar = 10 μm. (E) The percentage of RelB associated with Tas was quantified by counting the number of RelB‐positive cells, which are positive for Tas is shown. Between 30 and 35 images were analyzed. All transfections were performed in triplicate. The results shown represent the averages of the results of three independent experiments. Error bars indicate standard deviations.