Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

Abstract

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8(+) T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.

Figure 1

GrM is expressed in HCMV-specific CD8⁺ T cells. (a) FACS plots show the expression of CD8- and HCMV-specific pp65 and IE1 tetramers on CD3⁺ T cells (upper row) or the expression of CD27 and CD45RA (bottom row) on total CD3⁺CD8⁺ T cells (left) or HCMV-specific CD3⁺CD8⁺ T cells (middle, right). (b) FACS histogram visualizes the relative intracellular GrM protein levels in naive CD3⁺CD8⁺ T cells (filled gray) and HCMV-specific CD3⁺CD8⁺ T cells (lines). (c) PBMCs were isolated from a renal transplant patient pre- and post-transplantation. Graph depicts kinetically the absolute numbers of IE1-specific CD3⁺CD8⁺ cells (line) and HCMV viral load (filled gray). FACS plots show the expression of CD8 and IE1 tetramers on CD3⁺ T cells (upper row), and CD27 and CD45RA on total or IE1-specific CD3⁺CD8⁺ T cells (middle rows). Histograms (bottom row) visualize the relative intracellular GrM protein levels in naive CD3⁺CD8⁺ T cells (filled gray) and IE1-specific CD3⁺CD8⁺ T cells (line)

Figure 2

GrM cleaves host cell protein hnRNP K. (a) HeLa cell lysates were incubated with GrM (1 μM) or GrM-SA (1 μM) for 1 h at 37°C and then subjected to 2D-DIGE. A representative protein spot that is identified by mass spectrometry as a cleavage fragment of hnRNP K is depicted. (b) Jurkat lysates (5 μg) were incubated with increasing concentrations of GrM or GrM-SA (300 nM) for 4 h at 37°C and immunoblotted for hnRNP K. (c) Purified His-hnRNP K (1.5 μg) was incubated with increasing concentrations of GrM or GrM-SA (300 nM) for 16 h at 37°C and subjected to SDS-PAGE. Proteins were stained with InstantBlue. (d) Schematic overview of GrM cleavage sites within hnRNP K (NLS, nuclear localization signal; KH, K homology domain; KI, K interactive region; KNS, K nuclear shuttling signal; RGG, arginine–glycine–glycine). (e) HeLa cells were transfected with His-tagged hnRNP K wild-type (wt) or mutant expression plasmids and lysed 2 days post-transfection. Expression of recombinant proteins was determined by subjecting untreated lysates to immunoblotting using anti-6 × His antibody (bottom panel). Lysates were incubated with 300 nM GrM or left untreated for 4 h at 37°C and immunoblotted using anti-6 × His antibody (upper panel) (NS, nonspecific). (f) HFFs were incubated with GrM (2.8 μM) or GrM-SA (2.8 μM) in the presence or absence of SLO (2 μg/ml) for 16 h at 37°C. Lysates were immunoblotted for hnRNP K. (g) HFFs were incubated with GrM (1 μM), GrM-SA (1 μM), GrB (100 nM), or left untreated in the presence or absence of SLO (1 μg/ml) with or without zVAD-fmk (100 μM) for 16 h at 37°C. Cell viability was assessed by staining cells with annexin-V-FLUOS (fluorescein) and PI followed by flow cytometry analysis. Annexin-V and PI double-negative cells were considered to be viable. Bars represent the mean±S.D. of three independent experiments. (h) HFFs were incubated with GrM (1 μM), GrM-SA (1 μM), GrB (100 nM), or left untreated in the presence or absence of SLO (1 μg/ml) with or without zVAD-fmk (100 μM) for 16 h at 37°C. Cells were lysed and immunoblotted using antibodies against active caspase-3 (D175). (i) HFFs were challenged with increasing effector:target (E:T) ratios of LAK cells for 4 h at 37°C. Lysates were subjected to immunoblotting using hnRNP K antibodies. Data depicted are representative of at least two independent experiments

Figure 3

GrM cleaves hnRNP K more efficiently in the presence of RNA. (a) Jurkat cell lysates were pretreated with RNase (100 μg/ml) or left untreated for 30 min at 37°C, followed by incubations with increasing concentrations of GrM or GrM-SA (300 nM) for 4 h at 37°C and immunoblotted for hnRNP K or α-tubulin (upper panel). Band intensities were quantified and depicted in graphs (lower panel). (b) Purified His-hnRNP K (1.5 μg) was pretreated with RNA (100 ng) or left untreated, followed by incubations with increasing concentrations of GrM or GrM-SA (300 nM) for 16 h at 37°C. Samples were subjected to SDS-PAGE and gels were stained with InstantBlue (upper panel). Band intensities were quantified and depicted in graphs (lower panel). Data depicted are representative of at least two independent experiments

Figure 4

HnRNP K is essential for HCMV replication. (a) HFFs were transduced with hnRNP K-specific or control shRNA and lysed at different time points. Lysates were subjected to immunoblotting using antibodies against hnRNP K or α-tubulin (loading control). (b) HnRNP K-deficient and control HFFs were infected with HCMV and viral load in supernatants at different time points was assessed by quantitative PCR. (c) Cell viability of uninfected or HCMV-infected transduced HFFs was determined by WST-1 assay and relative cell viability of uninfected control shRNA-treated cells at 0 h.p.i. was set to 1. (d) HFFs were seeded at different cell densities and infected with HCMV at a multiplicity of 1.0 plaque-forming unit per cell. Viral load in supernatants at 96 h.p.i. was assessed by quantitative PCR and a linear regression curve was plotted. Data depicted represent the mean±S.D. of triplicates

Figure 5

HnRNP K regulates IE2 protein expression. (a) HFFs were transduced with hnRNP K-specific or control shRNA, infected with HCMV, and lysed at different time points. Lysates were subjected to immunoblotting using antibodies against IE1/2, pp71, pp65, pp28, hnRNP K, and α-tubulin. (b) HeLa cells were co-transfected with pGL3-SV40 (50 ng), pRL-CMV (10 ng), complemented with pCGN-71 vector (1 μg) or pSG5-His-hnRNP K, and pcDNA3.1⁺ vector (1 μg total DNA). Cells were lysed at 48 h post-transfection, and luciferase activity was assessed. Relative luciferase activity is represented as fold CMV promoter (MIEP) induction. Lysates used in the luciferase reporter assay were subjected to immunoblotting using an anti-pp71 or anti-6 × His antibody. (c) HCMV-infected hnRNP K-deficient and control HFFs were lysed at 8 h.p.i. and mRNA was isolated and subjected to RT-PCR and quantitative PCR using IE2 and GAPDH specific primers and probes. Relative IE2 mRNA levels were normalized for GAPDH mRNA levels and the mean from control cells was set at 1. (d) HCMV-infected transduced HFFs were lysed at different time points and immunoblotted for the C terminus of IE2 or α-tubulin. (e) HCMV-infected hnRNP K-deficient and control HFFs were treated with MG132 (250 nM) or left untreated at 48 h.p.i. and lysed at 72 h.p.i. Lysates were subjected to immunoblotting using antibodies against IE2, hnRNP K, α-tubulin, and ubiquitin. Data depicted are representative of triplicate experiments. (f) HCMV-infected HFFs were lysed at 72 h.p.i. and incubated with His-hnRNP K or His-β-tubulin as a negative control for 16 h at 4°C. Immunoprecipitation of His-tagged proteins was followed by isolation of mRNA, RT-PCR, and quantitative PCR using specific primers and probes against UL123 (IE1), UL122 (IE2), UL54, and GAPDH. The mean mRNA levels from His-β-tubulin control samples for each mRNA were set to 1 and all samples were normalized for GAPDH mRNA levels. Bars represent the mean±S.D. of triplicates. (Inset) Samples after immunoprecipitation were subjected to immunoblotting using anti-6 × His antibody

Figure 6

HnRNP K localization and GrM-mediated decrease of IE2 protein expression. (a) Immunofluoresence images of uninfected (upper panel) and HCMV-infected (lower panel) HFFs at 72 h.p.i. Nuclei are stained blue and hnRNP K is visualized in green. Bars, 50 μm. (b) HCMV-infected fibroblasts were treated with GrM-SA (500 nM) or GrM (500 nM) in the presence of SLO (1 μg/ml) at 2 h.p.i. and cells were lysed at indicated time points. Lysates were subjected to immunoblotting using antibodies against IE2 or β-tubulin. Data depicted are representative of at least two independent experiments performed in triplicates. (c) HCMV-infected fibroblasts were incubated with GrM (500 nM) or GrM-SA (500 nM) in the presence of SLO (1 μg/ml) at 2 h.p.i. HCMV viral load in supernatants at 72 h.p.i. was assessed by quantitative PCR. Bars represent the mean±S.D. of triplicates. (d) HCMV-infected fibroblasts were incubated with GrM (500 nM) or GrM-SA (500 nM) in the presence of SLO (1 μg/ml) at 2 h.p.i. Relative cell viability at 24 h.p.i. was assessed by WST-1 assay. Bars represent the mean±S.D. of triplicates