HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex

Abstract

Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

Keywords: DYRK2; EDD; HIV-1; Protein Degradation; TERT; Telomerase; Telomeres; Ubiquitination; VPRBP; Vpr.

FIGURE 1.

Vpr inhibits telomerase activity by TERT down-regulation. A, down-regulation of TERT protein expression by Vpr. HeLa cells stably expressing FLAG-TERT (HeLa-TERT cells) were transiently transfected with each construct (Vpr, Vif, Vpu, and Nef). Twenty-four hours later, cells were harvested for immunoblot analysis of FLAG-TERT. The level of TERT protein was normalized to TUBA4A (tubulin) and quantified using ImageJ. B, no effects of Vpr on TERT transcription. HeLa, HeLa-TERT, and 293T cells were transiently transfected with a FLAG-tagged Vpr construct and harvested for gene expression analysis using semiquantitative RT-PCR assays. GAPDH and HPRT were used as internal controls. C, down-regulation of TERT expression by Vpr in a dose-dependent manner. A Vpr construct was transfected into HeLa-TERT cells at different doses. Twenty-four hours later, cells were harvested for immunoblot analysis. D, time-dependent effect of Vpr on down-regulation of TERT protein expression. HeLa-TERT cells were transfected with 2 μg of Vpr and collected for immunoblot analysis at different times. Vpr expression in the cells was quantified by immunoblotting (FLAG). TUBA4A was used as an internal control. E, suppression of telomerase activity by Vpr expression. HeLa cells were transiently transfected with a Vpr expression plasmid. Twenty-four hours later, cells were collected to quantify telomerase activity using TRAP assays. The telomeric repeat sequence (TTAGGG) increment indicates telomerase activity. I.C., internal control.

FIGURE 2.

Vpr induces TERT ubiquitination. A, increased TERT ubiquitination by Vpr in vivo. HeLa cells were transiently transfected with three plasmids (HA-UBC, FLAG-TERT, and FLAG-Vpr) for in vivo ubiquitination assays. Thirty-six hours later, cell lysates were immunoprecipitated (IP) with anti-HA antibody (UBC), and TERT ubiquitination ((Ub)n-TERT) in the lysates was analyzed by immunoblotting (IB; FLAG). Although TERT expression was down-regulated by Vpr (Input panels, lanes 2 and 3), TERT ubiquitination was increased (IP: HA panels, lanes 2 and 3). Blots were quantified using ImageJ. H.C., heavy chain. B, increased TERT ubiquitination by Vpr in vitro. Recombinant TERT and DYRK2 proteins (in the absence and presence of Vpr protein) were incubated with EDVP E3 ligase components, UBE1, UBE2D2, and ATP and analyzed by immunoblotting. C and D, CUL4A-independent Vpr-induced telomerase inhibition. C, deletion of CUL4A using CUL4A siRNA transfection. HeLa cells were transiently transfected with control siRNA (siControl) and six different CUL4A siRNAs (siCUL4A-1–6) and analyzed by immunoblotting. S.E., short exposure; L.E., long exposure. D, Vpr-induced telomerase inhibition in a CUL4A-independent manner. HeLa cells were transiently transfected with siRNAs (control versus CUL4A) and Vpr expression plasmids. Twenty-four hours later, cells were analyzed for endogenous telomerase activity using qPCR-based TRAP assays. RU, relative units. Error bars indicate S.D. (n = 3).

FIGURE 3.

VPRBP mediates Vpr-induced TERT degradation. A and B, Vpr-induced TERT down-regulation via VPRBP. HeLa-TERT cells were transiently transfected with plasmids encoding wild-type Vpr or LTNP mutants (Q65R, F72L, and R77Q). Thirty-six hours after transfection, cells were harvested for immunoblotting (IB; A) and qPCR-based TRAP assays (B). RU, relative units. C, decreased binding of the Vpr LTNP mutant Q65R to TERT. HeLa cells were transiently transfected with the FLAG-TERT, HA-Vpr, or HA-Vpr Q65R plasmid. Cell lysates were then analyzed for interaction between TERT and Vpr (wild-type or Q65R) using reciprocal immunoprecipitation (IP) and immunoblotting. D, lack of binding of Vpr Q65R to VPRBP. HeLa cells were transfected with VPRBP and Vpr (wild-type or Q65R mutant). Each sample was then subjected to immunoprecipitation and immunoblot analysis. H.C., heavy chain. E, VPRBP mediation of Vpr-induced TERT destabilization. HeLa-TERT cells were cotransfected with control or VPRBP siRNA (si-VPRBP) with Vpr expression plasmid. Cells were analyzed by immunoblotting for TERT protein levels. S.E., short exposure; L.E., long exposure.

FIGURE 4.

Vpr promotes TERT-VPRBP association. A, Vpr increases TERT-VPRBP interaction in vitro. TERT-VPRBP interaction was quantified using in vitro binding assays. In vitro translated TERT and Vpr (wild-type or Q65R mutant) were incubated with maltose-binding protein (MBP)-purified VPRBP protein and analyzed using maltose-binding protein pulldown assays. IB, immunoblot. B, Vpr promotion of TERT-VPRBP interaction in vivo. HeLa-TERT cells were transfected with Vpr (wild-type or Q65R mutant) and analyzed for TERT-VPRBP interaction by immunoprecipitation (IP; FLAG-TERT) and immunoblotting (HA-VPRBP). H.C., heavy chain. C, association of Vpr with the endogenous EDVP E3 ligase complex. HeLa cells transfected with EDVP components were analyzed for Vpr-EDVP interaction by immunoprecipitation and immunoblotting.

FIGURE 5.

Vpr inhibits endogenous telomerase activity. A, Vpr inhibition of endogenous telomerase activity in immortalized T lymphocytes. Jurkat and SupT1 cells were stably transduced with HA-tagged Vpr (wild-type or Q65R mutant) retrovirus or control HA-tagged EGFP retrovirus. Four days after selection with hygromycin, cells were harvested for TRAP assays. Autoradiography was quantified using ImageJ. I.C., internal control. B and C, inhibition of telomerase activity by HIV infection. H9 cells (parental) and chronically HIV-infected H9 cells were analyzed for telomerase activity (qPCR-based TRAP assay) (B). RU, relative units. Human CD4⁺CD3⁺ T cells were isolated from PBMCs and infected with HIV. At each time point, CD4⁺CD3⁺ T cells were collected and analyzed for telomerase activity using qPCR-based TRAP assays (C). RNase-treated samples were included as negative controls.

FIGURE 6.

Illustration of the mechanism of Vpr-induced telomerase deregulation. DYRK2-induced phosphorylation of TERT induces TERT-EDVP association (arrow i) in the presence of Vpr. Vpr promotes TERT-VPRBP binding, which facilitates DYRK2-associated EDVP E3 ligase-mediated ubiquitination of TERT (arrow ii). Polyubiquitinated ((Ub)n) TERT is then targeted by proteasome-mediated degradation, which results in loss of telomerase activity. The decreased telomerase activity elicits telomere crisis during successive cell division and subsequent cell senescence (canonical function of telomerase). Alternatively, telomerase inhibition deregulates signaling pathways, stem cell activity, and gene expression (non-canonical function of telomerase).