The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1

Abstract

Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stimulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P. Codogno, and A. Esclatine, Autophagy 4:1-8, 2008) by a mechanism that requires de novo viral protein expression. Exploration of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Although this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2α by PKR has been described as a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.

Fig 1

Autophagosome formation at early and late time points after HCMV infection. MRC5 cells were transfected with GFP-LC3 for 48 h and then mock-infected or infected with HCMV or with UV-inactivated HCMV (UV-HCMV) for 4, 6, 8, and 24 h. Cells were fixed and immunostained for pp65 viral protein (red). (A) Representative images; bar, 10 μm. (B and C) Autophagy was quantified in pp65-positive cells by counting the number of GFP-LC3 puncta per cell. The results are the means of three independent experiments. Between 50 and 100 cells were analyzed per assay. *, P < 0.05; **, P < 0.01 (t test).

Fig 2

(A) Supernatant of HCMV-infected cells is able to stimulate autophagy. MRC5 cells were treated during 4 h with supernatant of HCMV-infected cells clarified of viral particles and cellular debris by ultracentrifugation (see Materials and Methods) or infected with UV-inactivated HCMV, followed by immunoblotting with anti-LC3, anti-p62, or anti-actin antibodies. Lysosomal inhibitors E64 and pepstatin A (pepst) were added under all conditions, to block the lysosomal degradation of LC3-II and p62. (B) Accumulation of LC3 during HCMV infection is independent of the autophagic level. MRC5 cells were infected with HCMV at an MOI of 1 during 6, 24, or 48 h and treated or not with E64, followed by immunoblotting with anti-LC3 and anti-actin antibodies. Note that the level of LC3-II in infected cells does not change in the presence of lysosomal inhibitor.

Fig 3

Inhibition of autophagic flux requires expression of viral genes. (A) Representative images of MRC5 cells transfected with mRFP-GFP-LC3 plasmid and infected with HCMV or UV-inactivated HCMV. Red and yellow dots indicate GFP⁻ RFP⁺ and GFP⁺ RFP⁺ puncta, respectively. (B) Autolysosomes were quantified by counting GFP⁻ RFP⁺ puncta per cell. The results are the means of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05 (t test). Amino acid-starved cells (EBSS) were used as a positive control (C) Immunoblot analysis of p62 levels in cells infected with HCMV or UV-inactivated HCMV for the indicated times. Lysosomal inhibitors E64/pepstatin A were added to all conditions, to block the lysosomal degradation. Actin immunoblotting was used as a loading control. (D) Effect of HCMV infection on long-lived protein degradation. [¹⁴C]valine-radiolabeled MRC5 cells were mock infected or infected with HCMV or UV-inactivated HCMV for 8 or 24 h, and proteolysis was measured as described in Materials and Methods. Cells were treated with 200 nM wortmannin (Plus WN) or not (No WN). The results are the means of three independent experiments. *, P < 0.05.

Fig 4

Bcl-2 is not responsible for autophagy inhibition in HCMV-infected cells. (A) Bcl-2 protein and mRNA expression. Immunoblot analysis of Bcl-2 protein in mock-infected cells and in cells infected with HCMV at MOIs of 1 and 3 from 24 to 72 h. Bcl-2 mRNA level was analyzed in control cells, in cells in starvation medium (EBSS), and in cells infected with HCMV at an MOI of 1 from 6 to 72 h or with UV-inactivated HCMV for 24 h. The results are the means of three independent experiments. (B) Coimmunoprecipitation of Beclin 1 and Bcl-2 in MRC5 cells. MRC5 cells were mock infected, infected with HCMV (MOI = 1) for 2 days, or grown in rich medium (RM) for 4 h prior to coimmunoprecipitation. Rich medium is known to inhibit autophagy. Cell lysates were immunoprecipitated with preimmune goat serum or a goat polyclonal anti-Beclin 1 antibody followed by immunoblotting with the indicated antibodies. (C and D) Accumulation of hyperphosphorylated Bcl-2 during HCMV infection in MRC5 fibroblasts. (C) Immunoblot analysis of the phosphorylated Bcl-2 (Ser70). MRC5 cells were mock infected or infected with HCMV at an MOI of 1 from 12 to 72 h. Actin immunoblotting was used as a loading control. (D) MRC5 cells were mock infected or infected with HCMV at an MOI of 1 from 18 to 72 h and at an MOI of 3 from 18 to 48 h. An increased staining of phosphorylated Bcl-2 (juxtanuclear green staining) was observed in the HCMV-infected cells compared to mock-infected cells (nuclear red staining shows HCMV IE antigens).

Fig 5

Activation of the JNK pathway during HCMV infection. (A) HCMV activates JNK phosphorylation. MRC5 cells were mock infected or infected with HCMV at MOIs of 1 and 3 from 6 to 72 h. Total cell lysates were prepared, and samples were subjected to immunoblot analysis of JNK and phosphorylated JNK (P-JNK). (B) Immunoblot analysis of the phosphorylated c-Jun during HCMV infection. MRC5 cells were mock infected or infected with HCMV at MOIs of 1 and 3 from 24 to 96 h. (C) Cells were infected with HCMV during 48 h and treated with SP600125, a selective JNK inhibitor, during different time frames as indicated in the diagram (conditions 1 to 4). SP600125 blocks Bcl-2 overexpression when added during the first 24 h of infection (lane 2) and phosphorylation of Bcl-2 when added from 24 until 48 h p.i. (lane 3). SP600125 blocks phosphorylation of c-Jun when added during 24 or 48 h (lanes 3 and 4) (D) RelB mRNA analysis by real-time PCR. MRC5 cells were mock infected or infected with HCMV at an MOI of 1 for 6 to 72 h, infected with UV-inactivated HCMV for 24 h, or grown in starvation medium (EBSS) for 4 h. The results are the means of three independent experiments. (E) Confocal analysis of the expression of RelB in HCMV-infected cells. MRC5 cells were infected with HCMV at an MOI of 1 for 48 h. Whereas RelB presents a marked nuclear staining in HCMV-infected cells, it has a diffuse (nuclear and cytoplasmic) staining in mock-infected cells. The cells were observed under a confocal laser scanning microscope (Zeiss LSM 510) and analyzed with Imaris software for three-dimensional reconstruction.

Fig 6

The viral protein TRS1 interacts with Beclin 1 and inhibits autophagic flux. (A) Coimmunoprecipitation of Beclin 1 and TRS1 in HCMV-infected MRC5 cells. MRC5 cells were mock infected or infected with HCMV at an MOI of 1 for 48 h. Cell lysates were immunoprecipitated with preimmune goat serum or a goat polyclonal anti-Beclin 1 antibody followed by immunoblotting with an anti-TRS1 antibody. (B) Immunoblot analysis of LC3 in HeLa cells transfected with TRS1 and cultured in starvation medium. Actin immunoblotting was used as a loading control. (C) Representative images of HeLa cells cotransfected with mRFP-GFP-LC3 and TRS1 plasmids for 24 h and cultured in starvation medium during the last 4 h of infection. Red and yellow dots indicate GFP⁻ RFP⁺ and GFP⁺ RFP⁺ puncta, respectively. Bar, 10 μm. (D) Quantitation of autophagy as measured by the number of LC3 puncta per cell, total puncta per cell (GFP⁺ RFP⁺ and GFP⁻ RFP⁺ puncta), GFP⁺ RFP⁺ puncta per cell, and GFP⁻ RFP⁺ puncta per cell. Only TRS1-expressing cells were quantified as described in Materials and Methods. The results are the means of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05; **, P < 0.01 (t test). (E) Immunoblot analysis of p62 protein in HeLa cells transfected with TRS1 plasmids and cultured in starvation medium. Actin immunoblotting was used as a loading control.

Fig 7

The N-terminal region of TRS1 is required for inhibition of autophagy and Beclin 1 binding. (A) Schematic representation of TRS1 showing position of the region required for PKR binding (amino acids 679 to 738) and the dsRNA-binding domain (amino acids 74 to 248). Different TRS1 constructs were used: C-terminal deleted fragment [TRS1 (1-738)], PKR-binding deleted fragment [TRS1 (1-679)], and N-terminal deleted fragments [TRS1 (45-795) and (93-795)]. (B) Representative images of GFP-LC3 staining in HeLa cells cotransfected for 48 h with GFP-LC3 and indicated TRS1 constructs. Four hours before fixation, cells were grown in starvation medium to induce autophagy. TRS1-transfected cells were visualized by an anti-His antibody, and GFP-LC3-positive cells with GFP-LC3 dots were quantified (C). The results are the means of three independent experiments. Between 50 and 100 cells were analyzed per assay. *, P < 0.05 (t test). (D) Coimmunoprecipitation of endogenous Beclin 1 and indicated His-tagged mutant TRS1 in HeLa cells transfected with the indicated plasmids.

Fig 8

TRS1 inhibits autophagy independently of PKR. Murine PKR^−/− and PKR^+/+ cells were cotransfected for 48 h with GFP-LC3 and His-TRS1 or FLAG-ICP34.5 constructs. Four hours before fixation, cells were grown in starvation medium to induce autophagy. TRS1-transfected cells were visualized by an anti-His antibody, and ICP34.5-transfected cells were visualized by an anti-FLAG antibody. (A) Representative images of GFP-LC3 in PKR^−/− and PKR^+/+ cells. Inserts show the viral protein staining. (B) GFP-LC3 positive cells with GFP-LC3 dots were quantified. The results are the means of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05; **, P < 0.01 (t test).

Fig 9

TRS1 is required for inhibition of autophagy in fibroblasts. (A) Expression of TRS1 and IEA in MRC5 cells infected with HCMV wild-type and ΔTRS1 mutant virus. (B) MRC5 cells were infected with HCMV wild-type or ΔTRS1 mutant virus at an MOI of 1 during 48 or 72 h and transfected for 48 h with GFP-LC3. When indicated, cells were grown in starvation medium (EBSS) 4 h before fixation to induce autophagy. Autophagy was quantified in IEA-positive cells by counting the number of GFP-LC3 puncta per cell. The results are the mean of three independent experiments. Twenty cells were analyzed per assay. *, P < 0.05; ns, not significant (t test). (C) Immunoblot analysis of Bcl-2 protein in cells infected with wild-type HCMV or ΔTRS1 mutant virus at an MOI of 1, from 24 to 72 h. (D) Accumulation of hyperphosphorylated Bcl-2 during ΔTRS1 mutant virus infection in MRC5 fibroblasts at 24, 48, and 72 h p.i. Nuclear red staining corresponds to viral IE antigens.