Novel Host Protein TBC1D16, a GTPase Activating Protein of Rab5C, Inhibits Prototype Foamy Virus Replication

Abstract

Prototype foamy virus (PFV) is a member of the oldest family of retroviruses and maintains lifelong latent infection in the host. The lifelong latent infection of PFV may be maintained by the restriction factors of viral replication in the host. However, the mechanisms involved in PFV latent infection are poorly understood. Here, we found that TBC1D16, a TBC domain-containing protein, is significantly down-regulated after PFV infection. Tre2/Bub2/Cdc16 (TBC) domain-containing proteins function as Rab GTPase-activating proteins (GAPs) and are participates in the progression of some diseases and many signaling pathways. However, whether TBC proteins are involved in PFV replication has not been determined. Here, we found that TBC1D16 is a novel antiviral protein that targets Rab5C to suppress PFV replication. Overexpression TBC1D16 inhibited the transcription and expression of Tas and Gag, and silencing TBC1D16 enhanced the PFV replication. Moreover, the highly conserved amino acid residues R494 and Q531 in the TBC domain of TBC1D16 were essential for inhibiting PFV replication. We also found that TBC1D16 promoted the production of PFV-induced IFN-β and the transcription of downstream genes. These results suggest that TBC1D16 might be the first identified TBC proteins that inhibited PFV replication and the mechanism by which TBC1D16 inhibited PFV replication could provide new insights for PFV latency.

Keywords: Rab5C; TBC1D16; prototype foamy virus (PFV); type I IFN; viral replication.

Figure 1

The expression of TBC1D16 is down-regulated in PFV-infected cells. (A) Volcano map of differentially expressed genes in the host after PFV infection. (B) Heat map of 20 selected genes with the most significant differences in expression. A scale from 3.41 to -14.54 represents folds of differential expression. (C) Expression of ten differentially expressed genes encoding functional proteins in PFV-infected HEK293T cells compared with mock-infected cells (*p < 0.05, **p < 0.01, ***p < 0.001 and ns, no significance).

Figure 2

TBC1D16 inhibits the replication of PFV. (A) After infection with PFV (MOI = 0.5) for 48 h, the mRNA expression of TBC1D16 in HEK293T and HT1080 cells was detected by qPCR (***p < 0.001). (B) The relative viral load in the presence or absence of overexpressed TBC1D16 was analyzed in PFV indicator cell line. RL-TK (1.5 μg) was transfected as an internal control. (C, D) pCMV-Flag-TBC1D16 or pCMV-Flag (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, the cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA or protein expression of PFV Tas and Gag was detected by qPCR (**p < 0.01 and ***p < 0.001). (E) pCMV-Flag-TBC1D16 or pCMV-Flag (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, the cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of PFV Tas and Gag was detected by western blotting. (F, G) The specific shRNA and shControl (as a negative control) were transfected into HEK293T and HT1080 cells for 24 h to knock down the expression of TBC1D16 and then were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of PFV Tas and Gag was detected by qPCR (***p < 0.001). (H) The specific shRNA and shControl (as a negative control) were transfected into HEK293T and HT1080 cells for 24 h to knock down the expression of TBC1D16 and then were infected with PFV (MOI = 0.5) for 48 h. The protein expression of PFV Tas and Gag was detected by western blotting.

Figure 3

TBC1D16 affects the Tas-dependent transactivation of the PFV LTR and IP promoters. (A) Structural diagram of PFV LTR and IP promoter. (B) HEK293T cells seeded in 24-well plates were contransfected with pCMV-Flag-TBC1D16 (400 ng, and pCMV-Flag as an empty control), pRL-TK (3 ng), and pGL3-PFV-LTR-luc (30 ng) or pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter. These cotransfected plasmids were combined with pTK-Tas (30 ng for pGL3-PFV-LTR-luc or 20 ng for pGL3-PFV-IP-luc). A luciferase reporter assay was used to detect LTR and IP promoter activity (Renilla luciferase as an internal control) (**p < 0.01, ***p < 0.001). (C) Luciferase assays were performed similarly as in (B). But these cotransfected plasmids were combined without pTK-Tas (ns, no significance). (D) The specific shRNA of TBC1D16 and shControl (as a negative control), pRL-TK (3 ng), pTK-Tas (30 ng for pGL3-PFV-LTR-luc or 20 ng for pGL3-PFV-IP-luc) and pGL3-PFV-LTR-luc (30 ng) or pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter were transfected into HEK293T cells for 48 h. Luciferase reporter assay was used to detect the LTR and IP promoter activity (Renilla luciferase as an internal control) (***p < 0.001). (E, F) pCMV-Flag-TBC1D16 (3 μg) and pGL3-PFV-LTR-luc (3 μg) or pGL3-PFV-IP-luc (3 μg) were cotransfected into HEK293T cells for 48 h, and ChIP assay was used to detect the enrichment of TBC1D16 on the PFV LTR and IP promoter. ChIP-qPCR data were normalized by the percent input method (%input with IgG as control). The data are presented as the means ± SD (*p < 0.05 and **p < 0.01). (G) pCMV-Flag-TBC1D16 (400 ng and pCMV-Flag as an empty control), pRL-TK (3 ng), pTK-Tas (30 ng) and truncated pGL3-PFV-LTR-luc (30 ng) firefly luciferase reporter were cotransfected into HEK293T cells for 48 h. Luciferase reporter assay was used to detect the truncated LTR promoter activity (Renilla luciferase as an internal control) (***p < 0.001 and ns, no significance).

Figure 4

The TBC domain of TBC1D16 is important for TBC1D16 to inhibit PFV transcription and replication. (A) Schematic diagram of full-length TBC1D16, indicating the C-terminal TBC domain and Ser rich (SR) domain,and truncation mutants used in subsequent analyses. (B, C) pCMV-Flag-TBC1D16 truncated plasmids (pCMV-Flag as an empty control) were transfected into HEK293T (B) and HT1080 (C) cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of PFV Tas and Gag was detected by qPCR (***p < 0.001 and ns, no significance). (D) pCMV-Flag-TBC1D16 truncated plasmids (pCMV-Flag as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of PFV Tas and Gag was detected by western blotting. The expression of TBC1D16 truncated plasmids were detected by anti-Flag antibody in HEK293T cells. (E) pCMV-Flag-TBC1D16 truncated plasmids (400 ng and pCMV-Flag as an empty control), pRL-TK (3 ng), pTK-Tas (30 ng) and pGL3-PFV-LTR-luc (30 ng) firefly luciferase reporter plasmid were cotransfected into HEK293T cells for 48 h. Luciferase reporter assay was used to detect LTR promoter activity (Renilla luciferase was used as an internal control) (***p < 0.001 and ns, no significance). (F) pCMV-Flag-TBC1D16 truncated plasmids (400 ng and pCMV-Flag as an empty control), pRL-TK (3 ng), pTK-Tas (20 ng) and pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter plasmid were cotransfected into HEK293T cells for 48 h. Luciferase reporter assay was used to detect IP promoter activity (Renilla luciferase as an internal control) (***p < 0.001 and ns, no significance).

Figure 5

Highly conserved amino-acid residues R494 and Q531 in the TBC domain are important for TBC1D16 to inhibit PFV replication. (A) Schematic diagram of the key locus in the TBC domain of TBC1D16 indicating three signature motifs, which were specific locus mutants used in subsequent analyses. (B, C) pCMV-Flag-TBC1D16 specific locus mutant plasmids (pCMV-Flag as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of PFV Tas and Gag was detected by qPCR (***p < 0.001 and ns no significance). (D) pCMV-Flag-TBC1D16 specific locus mutant plasmids (pCMV-Flag as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of PFV Tas and Gag was detected by western blot. (E, F) pCMV-Flag-TBC1D16 specific locus mutant plasmids (400 ng and pCMV-Flag as an empty control), pRL-TK (3 ng), pTK-Tas (30 ng for pGL3-PFV-LTR-luc or 20 ng for pGL3-PFV-IP-luc) and pGL3-PFV-LTR-luc (30 ng) or pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter plasmid were cotransfected into HEK293T cells for 48 h. Luciferase reporter assay was used to detect LTR and IP promoter activity (Renilla luciferase as an internal control) (***p < 0.001).

Figure 6

Rab5C, rather than Rab4A, inhibits the replication of PFV. (A, B) Gene expression of Rab5C and Rab4A after PFV infection. After infection with PFV for 48 h, the mRNA expression of Rab5C and Rab4A in HEK293T and HT1080 cells was detected by qPCR (***p < 0.001 and ns, no significance). (C) The relative viral load in the presence or absence of overexpressed Rab5C or Rab4A was analyzed in PFV indicator cell line. RL-TK (1.5 μg) was transfected as an internal control. (D, E) pCMV-HA-Rab5C or pCMV-HA-Rab4A and pCMV-HA (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of PFV Tas and Gag was detected by qPCR (***p < 0.001). (F) pCMV-HA-Rab5C and pCMV-HA (as an empty control) were transfected into HEK293T and HT1080 cells for 24h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of Tas and Gag were detected by western blotting. (G, H) The specific shRNA of Rab5C or shControl (as a negative control) was transfected into HEK293T and HT1080 cells for 24 h to knockdown the expression of Rab5C and then the cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of PFV Tas and Gag was detected by qPCR (**p < 0.01 and ***p < 0.001). (I) The specific shRNA of Rab5C or shControl (as a negative control) was transfected into HEK293T and HT1080 cells for 24 h to knock down the expression of Rab5C, and then the cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of Tas and Gag were detected by western blotting.

Figure 7

QESTIGAAF (amino-acid residues 50-58) is important for Rab5C to inhibit PFV replication. (A) Schematic diagram of the effector region (amino-acid residues 50-58) of Rab5C. (B–D) QESTIGAAF (amino-acid residues 50-58) is important for Rab5C to inhibit PFV replication. (B, C) pCMV-HA-Rab5C or pCMV-HA-Rab5CΔ(50-58) (400 ng and pCMV-HA as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. In another group, pCMV-HA-Rab5C or pCMV-HA-Rab5CΔ(50-58) were cotransfected with specific shRNA of Rab5C into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of Tas and Gag were detected by qPCR (***p < 0.001 and ns, no significance). (D) pCMV-HA-Rab5C or pCMV-HA-Rab5CΔ(50-58) (400 ng and pCMV-HA as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. In another group, pCMV-HA-Rab5C or pCMV-HA-Rab5CΔ(50-58) were cotransfected with specific shRNA of Rab5C into HEK293T cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of Tas and Gag was detected by Western blotting. (E) QESTIGAAF (amino-acid residues 50-58) is important for the interaction between Rab5C and TBC1D16. pCMV-Flag-TBC1D16 (3 μg) and pCMV-HA-Rab5C (3 μg) or pCMV-HA-Rab5CΔ(50-58) (3 μg) were cotransfected into HEK293T cells for 48 h, then Co-immunoprecipitation and immunoblot analysis were used to detect the interaction between TBC1D16 and Rab5C or Rab5CΔ(50-58).

Figure 8

The interaction between TBC1D16 and Rab5C is essential to inhibit PFV replication. (A) The interaction of Rab5C and TBC1D16. pCMV-Flag-TBC1D16 specific locus mutant plasmids (3 μg) and pCMV-HA-Rab5C (3 μg) were cotransfected into HEK293T cells for 48 h. Co-immunoprecipitation and immunoblot analysis were used to detect the interaction between TBC1D16 with different locus mutants and Rab5C in HEK293T cells. (B, C) pCMV-Flag-TBC1D16 or pCMV-HA-Rab5C and pCMV-Flag (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. In another group, pCMV-HA-Rab5C and specific shRNA of TBC1D16 or pCMV-Flag-TBC1D16 and specific shRNA of Rab5C were cotransfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA expression of Tas and Gag was detected by qPCR (***p < 0.001 and ns, no significance). (D) pCMV-Flag-TBC1D16 or pCMV-HA-Rab5C and pCMV-Flag (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. In another group, pCMV-HA-Rab5C and specific shRNA of TBC1D16 or pCMV-Flag-TBC1D16 and specific shRNA of Rab5C were cotransfected into HEK293T and HT1080 cells for 24 h. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The protein expression of Tas and Gag was detected by western blot analysis. (E) pCMV-Flag-TBC1D16 (400 ng), pRL-TK (3 ng), pTK-Tas (30 ng for pGL3-PFV-LTR-luc or 20 ng for pGL3-PFV-IP-luc) and pGL3-PFV-LTR-luc (30 ng) or pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter plasmid were cotransfected with specific shRNA of Rab5C into HEK293T cells for 48 h. Luciferase reporter assay was used to detect the LTR and IP promoter activity (Renilla luciferase as an internal control) (***p < 0.001 and ns, no significance). (F, G) pCMV-Flag-TBC1D16 (424-635) and pCMV-Flag (as an empty control) were transfected into HEK293T and HT1080 cells for 24 h. These cotransfected plasmids were combined with or without specific shRNA of Rab5C. After transfection, cells were infected with PFV (MOI = 0.5) for 48 h. The mRNA or protein expression of PFV Tas and Gag was detected by qPCR and western blotting (***p < 0.001 and ns, no significance). (H) pCMV-Flag-TBC1D16 (424-635) (400 ng), pRL-TK (3 ng), pTK-Tas (30 ng for pGL3-PFV-LTR-luc or 20 ng for pGL3-PFV-IP-luc) and pGL3-PFV-LTR-luc (30 ng) or pGL3-PFV-IP-luc (20 ng) firefly luciferase reporter were cotransfected with specific shRNA of Rab5C into HEK293T cells for 48 h. Luciferase reporter assay was used to detect the LTR and IP promoter activity (Renilla luciferase as an internal control) (***p < 0.001 and ns, no significance).