HSV-2 glycoprotein gD targets the CC domain of tetherin and promotes tetherin degradation via lysosomal pathway

Spread of HSV-2 progeny virus can occur both by release of mature infectious virus particles into the extracellular medium and by viral cell-to-cell spread. Having demonstrated that the release of cell-free HSV-2 progeny virions was restricted by tetherin [11], we further asked whether tetherin could affect cell-to-cell spread of HSV-2 by using infectious center assay. Firstly, the plaque formation on HeLa monolayer with or without anti-HSV-2 antibody treatment was tested. The area of plaques was relatively uniform in the presence of anti-HSV-2 antibody, while some small spots existed in the samples without anti-HSV-2 antibody (Fig. 1a and b). The following experiments were all performed under the anti-HSV-2 antibody treatment. Subsequently, the HeLa monolayers pretreated with tetherin or control siRNA were examined. Western blot analysis indicated that siRNA knockdown of tetherin efficiently reduced the expression of tetherin (Fig. 1c). As shown in Fig. 1d and e, compared to control siRNA-pretreated HeLa cells, the plaque size was modestly increased upon tetherin siRNA pretreatment. Accordingly, two polarized epithelial cell types (HaCaT and ARPE-19) [32, 33] were used in the infectious center assay. As shown in Fig. 1f and g, the plaque size was significantly reduced on the pBST2 transfected HaCaT and ARPE-19 monolayers, confirming an interference with the cell-to-cell spread of HSV-2 in the presence of tetherin. Taken together, the reduced size of HSV-2 plaques by tetherin informs that tetherin at least partially inhibits the direct cell-to-cell spread of HSV-2 during plaque formation.

The interaction between HSV gD expressed in the progeny virus producing cells and the neighbouring host cells is essential for cell-to-cell spread. We previously demonstrated that several glycoproteins of HSV-2, including gD, can downregulate the expression of tetherin. To map the tetherin domains required for HSV-2 gD-mediated downregulation, we constructed a panel of deletion mutants of tetherin (Fig. 2a) and performed coimmunoprecipitation assays using cells transiently expressing wild type tetherin or its mutants. The expression of tetherin mutants was confirmed by western blot using an anti-tetherin antibody (Fig. 2b). Subsequently, the precleared cell lysates from the transfected cells were incubated with an anti-flag antibody or an isotype control antibody. The precipitates were analyzed by western blot using the antibody against tetherin. The tetherin mutants delTM, delCT and delGPI but not delCC were specifically coimmunoprecipitated by the flag antibody (Fig. 2c, d, e and f, left panels). Co-IP experiments were also performed by pulling down with the antibody against tetherin followed by western blot with the flag antibody. The antibody against tetherin was able to specifically precipitate the immune complexes that contained gD-flag and delTM, delCT or delGPI (Fig. 2c, d, e and f, right panels), indicating that only the CC domain of tetherin is required for the physical interaction with HSV-2 gD. Taken together, these data suggest that tetherin is down-regulated through a specific interaction between HSV-2 gD and the CC domain of tetherin.

The known antagonists of tetherin can lead the degradation of tetherin in late endosomes, lysosomes or proteasomes [34]. To examine whether the gD mediated-downregulation of tetherin undergoes lysosomal or proteasomal degradation pathway, HeLa cells were transfected with pcDNA3.1 or gD expressing plasmid followed by cultivation in the presence of a mixture of lysosome protease inhibitors (LPI; containing leupeptin, pepstatin A, and E64d) or the proteasome protease inhibitor (PPI; containing MG132). The paralleled HeLa cells transfected with gM expressing plasmid were included as the control. At 24 hours post transfection, cells were processed for immunofluorescence staining for subcellular localization of tetherin, gD/gM and a lysosome marker cathepsin D or a proteasome marker 20S proteasome. As shown in Fig. 3a and c, colocalization of gD, tetherin and cathepsin D was seen in gD expressing cells treated with lysosomal protease inhibitors, whereas tetherin and cathepsin D did not apparently colocalize in the pcDNA3.1- or gM-transfected cells. Tetherin colocalized with the 20S proteasome in the presence or absence of HSV-2 gD (Fig. 3b and d), implying that tetherin likely physiologically undergoes proteasomal degradation in the absence of HSV-2 gD. Western blot assay demonstrated that the lysosome protease inhibitors (LPI) rescued tetherin from degradation in the gD-transfected cells (Fig. 3e). To confirm that the gD mediated-downregulation of tetherin undergoes lysosomal degradation, we used a lysosome enrichment kit (Thermo) to isolate and enrich the intact lysosomes from cells transfected with pcDNA3.1 or plasmids expressing gD. The prepared cell extract was ultracentrifuged by density gradient centrifugation. We collected the corresponding band and detected the finally harvested lysosome pellets by western blot. As shown in Fig. 3f, the lysosome marker LAMP1, tetherin and gD all existed in the separated samples from gD-transfected cells. In contrast, only LAMP1 existed in the samples from pcDNA3.1 transfected cells. These data together indicate that the gD mediated-downregulation of tetherin undergoes lysosomal degradation.

Human cervical epithelial cell line HeLa, human keratinocyte cell line HaCaT, African green monkey kidney cell line Vero and embryonic kidney cell line 293 T were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10 % FBS, 100 Units/ml penicillin and 100 Units/ml streptomycin at 37 °C in 5 % CO2. Human retinal pigment epithelial cell line ARPE-19 was grown in DMEM/F-12 medium (50:50) supplemented with 10 % FBS, 100 Units/ml penicillin and 100 Units/ml streptomycin at 37 °C in 5 % CO2. HSV-2 (G strain) was obtained from LGC standards and propagated in Vero cells. Virus stock was stored at −80 °C before used for infection.

The open reading frames (ORF) of human tetherin amplified from pBST2 (Origene) was subcloned into pcDNA3.1(−) to construct the plasmid pWT (expressing full-length human tetherin). A panel of deletion mutants of tetherin were made and subcloned into pcDNA3.1(−), named delCT expressing 20–180 aa of human tetherin, delTM expressing 45–180 aa of human tetherin, delCC expressing human tetherin with the deleption of 100–154 aa and delGPI expressing 1–161 aa of human tetherin, respectively. The delTM without secretory leader peptide is not efficient for tetherin mutant to anchor on the cell surface membrane, we therefore fused the secretory leader peptide MDAMKRGLCCVLLLCGAVFVSPSQE from tissue plasminogen activator to tetherin codons 45–180 as described previously [45]. The glycoprotein gD and gM fused to flag tag were cloned into pcDNA3.1 (+) (named pgD-flag and pgM-flag) as described previously. The construction, cloning and propagation of plasmids were carried out using standard techniques. All constructs were verified by DNA sequencing (Sunny Biotechnology Co. Ltd, Shanghai, China).

Tetherin siRNAs (SI02777054) and control siRNA (1027281) were purchased from Qiagen. HeLa cells were plated onto 6-well culture dishes overnight. Hela monolayers were transfected with tetherin siRNA or control siRNA using HiPerFect Transfection Reagent (301704; Qiagen) according to the manufacturer's instructions.

Infectious center assay was carried out as previously described [46]. In brief, HeLa, HaCaT and ARPE-19 cells plated in 6-well plates at 50 % confluence were exposed to HSV-2 at an MOI of 5 at 37 °C. After 90 min of incubation, cells were washed once with PBS and then treated with 0.1 M citrate buffer (pH 3.0) for 1 min to inactivate extracellular virus particles. The monolayer was then washed twice with PBS to remove the low-pH buffer, and cells were placed in growth medium supplemented with anti-HSV-2 antibody (PAB13979; Abnova) at a dilution of 1:1000 to neutralize extracellular HSV-2. After a total incubation of 5.5 h, the infected cells were detached with trypsin-EDTA, resuspended in growth medium, and ~100 cells were plated onto 50 % confluent monolayers of uninfected HaCaT and ARPE-19 cells transfected with pBST2 or pcDNA3.1, or of uninfected HeLa cells pretreated with tetherin or control siRNA. Cells were maintained in growth medium containing anti-HSV-2 polyclonal antibody. After 2 days, the cells were fixed and stained for HSV-2 antigens, followed by the addition of the peroxidase-conjugated secondary antibody and substrates. Plaques were photographed and the plaque areas were compared.

Co-immunoprecipitation assay was carried out using Pierce Crosslink Immunoprecipitation Kit (26147; Thermo scientific) according to the manufacturer's instructions. For co-immunoprecipitations of gD-flag and tetherin mutants, mouse monoclonal antibody against FLAG (F1804; Sigma) and rabbit polyclonal antibody to tetherin (11721; NIH AIDS Research and Reference Reagent Program) were used for pull-down experiments. These two antibodies were also used for western blot analysis of the immunoprecipitates and the lysates.

Prepared cell lysates were resolved by 12 % SDS-PAGE and transferred to 0.45 μm polyvinylidene difluoride membranes (Millipore). Nonspecific binding was blocked using 5 % non-fat milk in PBS overnight at 4 °C. The membrane was incubated with primary antibody against tetherin (rabbit polyclonal antibody; 11721; NIH AIDS Research and Reference Reagent Program) at a dilution of 1:5000, FLAG (mouse monoclonal antibody; F1804; Sigma) at a dilution of 1:3000, and β-actin (mouse monoclonal antibody; sc-81178; Santa Cruz) at a dilution of 1:500, for 1 h at 37 °C. The membrane was washed five times with 0.1 % Tween 20/PBS, followed by incubation for 1 h with HRP conjugated goat anti–rabbit secondary antibody (1:10,000; BA1054, Boster) or HRP conjugated goat anti–mouse secondary antibody (1:10,000; BA1050, Boster). After five washes with 0.1 % Tween-20/PBS, the bands were visualized by exposure to FluorChem HD2 Imaging System (Alpha Innotech) after the addition of chemiluminescent substrate (SuperSignal® West Dura Extended Duration Substrate; 34075; Thermo Scientific Pierce).

To detect the accumulation of tetherin in lysosomes or proteasomes, transfected cells were cultured in complete medium in the presence of a mixture of lysosome protease inhibitors (containing leupeptin, pepstatin A and E64d; Sigma) for 16 h or a proteasome inhibitor (MG132; Sigma) for 12 h as previously described [16]. Transfected cells on 35 mm glass bottom culture dishes were washed twice with phosphate-buffered saline (PBS), followed by fixation with 4 % (w/v) cold paraformaldehyde for 30 min at room temperature. Cells were permeabilized with PBST (PBS-0.2 % (v/v) Triton X-100) for 10 min at room temperature and then blocked with PBS-2 % (w/v) BSA for 1 h at room temperature. Cells were incubated for 1 h at 37 °C with mouse monoclonal antibody against FLAG (F1804; Sigma) at a dilution of 1:200 and followed by incubation for 1 h at 37 °C with Cy3-conjugated goat anti-mouse secondary antibody (Beyotime, China) at a dilution of 1:200 in PBS-2 % (w/v) BSA. Thereafter, cells were incubated for 1 h at 37 °C with mouse monoclonal antibody against tetherin (H00000684-M15, Abnova) at a dilution of 1:200 and rabbit polyclonal antibody against cathepsin D (sc-10725; Santa Cruz) (or 20S proteasome α7/α8 (sc-67344; Santa Cruz)) at a dilution of 1:100 in PBS-2 % (w/v) BSA, followed by incubation for 1 h at 37 °C with Cy5-conjugated goat anti-mouse secondary antibody (Beyotime, China), FITC-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at a dilution of 1:200 in PBS-2 % (w/v) BSA. Cells were washed three times after each incubation with PBS and then twice with distilled water. Nuclei were dyed by Hoechst 33258 (Invitrogen). Stained cells were analyzed using confocal microscopy (PerkinElmer UltraView VoX).

All experiments were repeated for at least three times. Data are presented as mean ± SD unless otherwise specified. The difference of mean value was analyzed by a paired Student’s t-test. P < 0.05 was considered statistically significant.