Plasmids

PTEN-L complementary DNA (cDNA) was amplified from total RNA obtained from SeV-stimulated HEK293T cells. PTEN-L was cloned into pEF by using Afl II and Mlu I, the pCDNA3.1 vector by using EcoR I and Xho I, and pHAGE by using Xho I and BamH I. PTEN-L (ATG/ATA) and C297S-L were constructed with a Site-Directed Mutagenesis Kit (TOYOBO), with which the canonical ATG start codon of PTEN was changed to ATA, and the CTG codon was changed to ATG.

Lentiviral packaging and infection

A lentiviral system was utilized to obtain stably transfected cells. PTEN, PTEN-L (ATG/ATA and CTG/ATG) and C297S-L were cloned separately into the lentiviral vector pHAGE. Then, the lentiviral backbone (8μg), psPAX2 (6μg) and pMD2.G (3μg) were transiently transfected into HEK293T cells plated on 100-mm dishes. After 48h, supernatants were collected and used to infect Pten^−/− MEF or PC-3 with polybrene (8μg/ml). Cells were infected twice to obtain higher transduction efficiency, and puromycin (0.5μg/ml) was used to screen positive cells.

Western blotting

Cells were washed once with PBS and lysed in RIPA buffer (50mM Tris, pH 7.6, 1% NP-40, 150mM NaCl, 0.1% SDS). Protein samples were mixed with 5 × SDS loading buffer and boiled for 5min. Samples were resolved by SDS–PAGE, transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK), blocked with TBS containing 0.1% Tween-20 and 5% skim milk and probed with antibodies to PTEN, p-IRF3 (Ser396), IκBα, p-IκBα (CST), Flag (Sigma, St Louis, MO, USA), p65 (Abcam, Cambridge, MA, USA), IRF3, 6 × His tag, LMNB1, β-tubulin, GAPDH and β-actin (Proteintech, Chicago, IL, USA). The antibody to p-IRF3 (Ser97) was obtained in our previous study.¹¹

RNA isolation and quantitative RT-PCR

Total RNA was isolated from Pten^−/− MEF and PC-3 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), per the manufacturer’s instructions. mRNA was reverse transcribed into cDNA by M-MLV Reverse Transcriptase (Promega). qRT-PCR was performed with an ABI 7300 Real TIME PCR System. GAPDH was used to normalize the input samples via the ΔΔCt method. Primer sequences were as reported in our previous work.¹¹

VSV plaque assays

The indicated stably transfected cells were plated one day before infection. After infection with VSV-GFP for 2 h, cells were washed three times with PBS, and fresh medium was added. After 24h, supernatants were collected and diluted to infect VERO cells that were plated on 24-well plates at 90% confluence. After 2h, supernatants were removed, and methylcellulose was added to plates. Three days later, cells were fixed with formaldehyde (4%) for 20min and stained with crystal violet (0.2%) for 2h. Plaques were counted to obtain the viral titer (pfu/ml).

ELISA

Cells were infected with SeV for 8h. Cytokines were detected by using a human IFN-β ELISA kit (R&D, Minneapolis, MN, USA), human IL-6 ELISA kit (Dakewe Biotech, Shenzhen, China), mouse IFN-β ELISA kit (Biolegend, San Diego, CA, USA), and mouse IL-6 and CXCL1 ELISA kits (Neobioscience Technology, Shenzhen, China).

PTEN-L assays in cell cultures

Briefly, 10μg of indicated expression plasmids were transfected into HEK293T cells, which were plated on 100mm-dishes. After 48h, supernatants were harvested, centrifuged and filtered through a 0.45-micron filter. Pten^−/− MEF cells were plated on 12-well plate for 24h. The culture medium of the 12-well plate was removed, and supernatants collected from HEK293T were added to Pten^−/− MEF cultures. After 24h, supernatants were removed, and VSV-GFP was used to infect the treated cells. Supernatants were also incubated with Pierce Ni-NTA Magnetic Agarose Beads (Thermo Fisher) and then used to treat Pten^−/− MEF, as described above. Purified recombinant PTEN-L protein was added into the culture medium of Pten^−/− MEFs for 24h, and then VSV-GFP was used to infect the treated Pten^−/− MEF.

Purification of recombinant PTEN and PTEN-L

pRSFDuet-1-PTEN-L^ΔN21-His or pRSFDuet-1-PTEN-His was transformed into E. coli BL21 grown in Luria broth (LB) medium containing kanamycin at 37°C. When the OD_600nm value of cell cultures was between 0.6 and 1, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2mM, and cell cultures were induced at 20°C for 18h. Bacterial cells were harvested by centrifugation and lysed by using a high-pressure homogenizer. His-tagged fusion proteins were purified from cell lysates by affinity chromatography using Ni-NTA His-Bind Resin (Novagen, Madison, WI, USA) according to the manufacturer's instructions.

Mouse experiments

Conditional Pten-knockout mice were obtained in our previous work.¹¹ Six-week-old female mice were used in the experiment. The indicated purified PTEN or PTEN-L protein (5mg/kg) was injected into mice though the tail vein for 3 days. Then, 1 × 10⁸ VSV were intraperitoneally injected into mice. All animal experiments were performed in accordance with the Regulations of Hubei Province Laboratory Animal Management and approved by Animal Experiment Ethics Committee in Wuhan University, China.

PTEN-L promotes production of proinflammatory factors

In innate immune responses, both antiviral type I IFN and inflammatory cytokines are commonly induced. However, our previous study has demonstrated that canonical PTEN mainly contributes to the stimulation of type I IFN production but not proinflammatory cytokines.¹¹ Given that PTEN-L, compared with PTEN, contains a long N-terminal extension, it may acquire additional functions. Therefore, we further investigated whether PTEN-L might promote the proinflammatory cytokine response. We used PTEN-knockout MEFs (Pten^−/− MEF) and human PC-3 cells that were reconstituted with different variants of PTEN-L, as described above. Expression of proinflammatory cytokines CXCL1 and IL-6 was detected after challenge with SeV. PTEN-L expression markedly enhanced the mRNA levels of the proinflammatory cytokines in both types of cells infected with SeV, whereas PTEN did not (Figures 2a and b). Furthermore, the levels of secreted IL-6 and CXCL1 in the culture supernatants were significantly higher than those in PTEN-knockout MEFs expressing PTEN or PTEN-L dead control (Figure 2c) and PC-3 (Figure 2d). Collectively, these data suggested that PTEN-L stimulates production of proinflammatory cytokines and type I IFN. This finding is in clear contrast to the notion that PTEN that activates only IFN responses.

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Figure 2

PTEN-L stimulates proinflammatory factors. (a and b) Quantitative RT-PCR was conducted to measure the relative expression levels of CXCL1 and IL-6. Stably transfected MEFs or transiently transfected PC-3 cells were uninfected (UI for short) or infected with SeV (50 HA Units/ml) for 8h. Cells were lysed in TRIzol to isolate RNA, which was reverse transcribed to cDNA for further qPCR analysis. (c and d) Analysis of proinflammatory cytokines from the cell culture supernatants, as assessed by ELISA. The data represent the average of three independent experiments and were analyzed by two-tailed unpaired t test. Graphs indicate mean±s.d. (n=3) derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

PTEN-L enhances cellular antiviral responses

Given that PTEN-L is involved in the type I IFN and inflammatory responses, we next assessed whether PTEN-L affected antiviral activity. We infected indicated types of PC-3 cells with IFN-sensitive VSV-GFP and detected virus propagation. In this commonly used system, the intensity of green fluorescence correlates with the amount of viral replication. As shown in Figure 3a, expression of PTEN-L (ATG/ATA or CTG/ATG) and PTEN remarkably decreased the fluorescence compared with expression of C297S-L or empty vector control. Accordingly, VSV titers and the level of VSV-encoded glycoprotein G (VSV-G) were lower in the PTEN- or PTEN-L-expressing cells (Figure 3b). One band smaller than PTEN-L but larger than PTEN was also observed, which might represent another isoform of PTEN (PTEN-M by size)²⁵ or a degradation product of PTEN-L. Similar results were observed in MEFs (Figures 3c and d). We also detected the levels of secreted IFN-β and IL-6 in the culture supernatants (Figure 3e), and the results were consistent with those after SeV infection, as shown in Figure 2. Altogether, the results showed that PTEN-L substantially promotes antiviral activity in infected cells.

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Figure 3

PTEN-L enhances cellular antiviral responses. (a and c) PTEN-null cells reconstituted with empty vector (EV), PTEN, PTEN-L^atg/ata (ATG/ATA), PTEN-L^ctg/atg (CTG/ATG) and the phosphatase dead mutant C297S-L were exposed to VSV-GFP (MOI=0.01) infection for 36h. The replication of VSV-GFP was assessed by microscopy. (b and d) Supernatants were subjected to VSV plaque assays. Cells were lysed and then subjected to SDS–PAGE. VSV replication was analyzed by anti-VSV-G and β-actin immunoblotting. PTEN expression was analyzed by anti-PTEN immunoblotting. (e) Pten^−/− MEF supernatants were also collected for ELISA. Graphs represent mean±s.d. (n=3) derived from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Secreted PTEN-L mediates type I IFN responses and antiviral immunity

PTEN-L has been identified as a secreted and cell-penetrating protein;²⁰ thus, PTEN-L protein might potentially be developed into an antiviral agent for direct application. We overexpressed His-tagged PTEN, PTEN-L, phosphatase-dead mutant C297S-L and PTEN-L^ΔN21&R6 (ΔN21&R6 for short) in HEK293T cells. The PTEN-L ΔN21&R6 mutant contained a deletion of both the secretion and penetration signal and was incapable of secreting and entering cells. Cell culture supernatants that might have contained the secreted PTEN-L were then collected and used to treat PTEN-knockout MEFs. As shown in Figure 4a, the PTEN-knockout MEFs treated with secreted PTEN-L but not the phosphatase-dead mutant displayed less virus propagation. Treatment with supernatants from PTEN- and ΔN21&R6-expressing cell cultures did not exert antiviral effects on PTEN-knockout MEFs (Figure 4a), but this lack of activity might have resulted from the inability to secrete PTEN and ΔN21&R6. To demonstrate that overexpressed PTEN-L was secreted from HEK293T cells and penetrated Pten^−/− MEF, the supernatants were incubated with Ni-NTA magnetic agarose beads that bound to the 6x-histidine-tagged PTEN-L. PTEN-L and C297S-L were secreted from HEK293T and penetrated Pten^−/−MEFs but not PTEN and ΔN21&R6 (Figure 4b). Thus, phosphatase activity is important for the role of PTEN-L in antiviral activity. In agreement with fluorescence assay results, the virus titer was decreased (Figure 4c), and type I IFN and CXCL1 mRNA levels were increased in the cells treated with PTEN-L compared with control cells (Figure 4d).

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Figure 4

Secreted PTEN-L mediates type I IFN responses and antiviral immunity. (a) Pten^−/− MEFs were treated with the supernatants of HEK293T cell cultures transfected with the following PTEN variants: PTEN-L^atg/ata (ATG/ATA), C297S-L, PTEN and PTEN-L^ΔN21&R6 (ΔN21&R6) or empty vector (EV). Then, cells were infected with VSV-GFP. VSV-GFP replication was observed by microscopy. (b) HEK293T supernatants were collected. Cells were lysed and then subjected to SDS–PAGE. Collected supernatants were incubated with Ni-NTA magnetic agarose beads to pull down His-tagged protein. Treated MEFs were lysed and subjected to SDS–PAGE analysis of secreting and cell-penetrating of PTEN-L. (c) Pten^−/− MEF supernatants were subjected to VSV plaque assays. (d) Pten^−/− MEF cells were lysed in TRIzol to isolate RNA, which was reverse transcribed to cDNA. Quantitative RT-PCR was conducted to measure the relative expression levels of IFN-B1 and CXCL1. The data represent the average of three independent experiments and were analyzed by two-tailed unpaired t test. Graphs show the mean±s.d. (n=3) derived from three independent experiments. *P<0.05, ***P<0.001, ****P<0.0001.

Extracellular application of PTEN-L protein mediates antiviral responses in cell cultures and in vivo

To demonstrate that the antiviral effect was caused by secreted PTEN-L instead of other antiviral molecules, supernatants of indicated transfected HEK293T cells were incubated with Ni-NTA magnetic agarose beads to deplete the His-tagged PTEN-L and then used to treat Pten^−/− MEFs. As shown in Figure 5a, after depletion of PTEN-L, the inhibition of virus propagation by the PTEN-L supernatant was no longer evident. However, regardless of incubation with beads, the supernatants of empty vector and PTEN did not exhibit any antiviral effects. The results of VSV titers and the levels of VSV-G were consistent with the fluorescence results (Figure 5b). We also prepared recombinant PTEN-L protein (PTEN-L-His) from an E. coli expression system. The purified PTEN-L-His or PBS control was added into the culture medium of Pten^−/− MEFs. As shown in Figure 5c, the PTEN-knockout MEFs treated with PTEN-L-His exhibited less virus propagation. Moreover, VSV titers and VSV-G levels were lower in PTEN-L-His-treated cells than in cells treated with PBS (Figure 5c). Altogether, these results indicated that PTEN-L inhibits virus replication when it is applied extracellularly in cell cultures.

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Figure 5

Extracellular application of PTEN-L protein mediates antiviral responses in cell cultures and in vivo. (a) The supernatants of indicated transfected HEK293T cells were incubated with or without Ni-NTA magnetic agarose beads and used to treat Pten^−/− MEF. VSV-GFP (MOI=0.01) was used to infect the treated Pten^−/− MEF, and VSV-GFP replication was observed by microscopy. (b) Supernatants were subjected to VSV plaque assays. Cells were lysed and then subjected to SDS-PAGE. VSV replication was analyzed by anti-VSV-G and β-actin immunoblotting. (c) Pten^−/− MEF cells were treated with PBS or 100nM purified proteins from pRSFDuet-1-PTEN-L^ΔN21-His (PTEN-L-His) and infected by VSV-GFP (MOI=0.01). Supernatants were subjected to VSV plaque assays. Cells were lysed and then subjected to SDS-PAGE. VSV replication was analyzed by anti-VSV-G and β-actin immunoblotting. PTEN-L-His penetration into Pten^−/− MEF cells was confirmed by anti-PTEN immunoblotting. (d) Recombinant purified PTEN-His and PTEN-L-His used for mice experiments were analyzed by SDS-PAGE and Coomassie BB staining. PTEN mutant mice were injected with purified PTEN or PTEN-L protein (5mg/kg) for three days and then infected intraperitoneally with VSV at 1 × 10⁸ pfu per mouse (n5; 6 weeks old). Quantitative RT-PCR analysis of mRNA encoding VSV-G protein in the spleen in the indicated treated mice after infected with VSV for 24h. (e) ELISA analysis of IFN-β and IL-6 in serum from PTEN- or PTEN-L-treated mice was also performed. The data represent average of three independent experiments and were analyzed by two-tailed unpaired t test. Graphs indicate mean±s.d. (n=3) derived from three independent experiments. **P<0.01, ****P<0.0001.

We also observed the antiviral effect of recombinant PTEN-L protein in vivo by using a mouse model. Purified PTEN-L-His or PTEN-His was injected into PTEN-mutant mice though the tail vein. VSV was then used to infect mice. In accordance with the observations in the cell culture model, we found that VSV titers in mice injected with PTEN-L-His were noticeably lower than those of mice injected with PTEN-His (Figure 5d). Moreover, we also assessed IFN-β and IL-6 serum levels in mice injected with indicated proteins after VSV challenge. In agreement with results from the cell culture model, injection of the long isoform PTEN-L caused PTEN-mutant mice to acquire higher antiviral innate immune responses (Figure 5e). Altogether, treatment of cells or animals with PTEN-L protein promoted antiviral responses and suppressed viral infection.

We thank Dr Hong Wu for providing Pten^−/− MEFs and Dr Hongliang Li for Pten^flox/flox mice and Dr Mingzhou Chen for providing VSV as a gift. This work was supported by the National Nature Science Foundation of China (grant 81620108020), the China ‘973’ Basic Research Program (#2013CB911101) and Hubei Provincial Science & Technology Innovation Team grant (#2015CFA009).