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HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells.

Stanton RJ, Prod'homme V, Purbhoo MA, Moore M, Aicheler RJ, Heinzmann M, Bailer SM, Haas J, Antrobus R, Weekes MP, Lehner PJ, Vojtesek B, Miners KL, Man S, Wilkie GS, Davison AJ, Wang EC, Tomasec P, Wilkinson GW - Cell Host Microbe (2014)

Bottom Line: Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations.Among these, the deletion of the UL/b' domain is associated with loss of virulence.An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection & Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK. Electronic address: stantonrj@cf.ac.uk.

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UL135 Decreases NK Cell Cytotoxicity, Degranulation, and IFN-γ Production and Adhesion as Well as T Cell Degranulation and IFN-γ Production(A–E) Cytotoxicity assays were set up with the NKL cell line against HFFF-hCAR (A), IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR (B), or IFN-α-activated T-cell-depleted PBMC against autologous skin fibroblasts (SFs; C). NK cell degranulation assays were performed with IFN-α-activated PBMC against HFFF-hCAR (D) or autologous SF (E).(F) Intracellular IFN-γ stainings were set up with IFN-α-activated PBMC against HFFF-hCAR.(G) Adhesion assays were performed with HFFF-hCAR infected with RAd-UL135 or empty control vector (RAd-Ctrl) and NK cells.(H) T cell degranulation assays were set up with the 7E7 T cell clone against MRC-5 pulsed with antigenic peptide. T cells incubated with PMA-iono or without peptide are shown as controls.(I–J) T cell intracellular IFN-γ staining assays were set up with the D7IE1 (I) and D7pp65 (J) T cell lines against autologous SF infected with RAd-Ctrl or RAd-UL135 and pulsed with peptide.(K–L) NK cell degranulation assays were set up with IFN-α-activated PBMC against allogeneic HFFF-hCAR (K) or autologous SF infected for 48 hr with HCMV strains AD169, Merlin, MerlinΔUL135, MerlinΔUL141, MerlinΔUL135/ΔUL141, or RAd-UL135, RAd-UL141, and RAd-Ctrl (L).(M) T cell degranulation assays were set up with a pp65-specific T cell line against autologous SF either mock infected or infected with Merlin or MerlinΔUL135.(N) Adhesion assays were performed with HFFF infected with the indicated viruses and NK cells. Results from three experiments were normalized and combined.Results are means ± SD of triplicate (A–C, G, H, and N), duplicate (D–F), or quadruplicate (I–M) samples and are representative of three independent experiments (A–M). Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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fig1: UL135 Decreases NK Cell Cytotoxicity, Degranulation, and IFN-γ Production and Adhesion as Well as T Cell Degranulation and IFN-γ Production(A–E) Cytotoxicity assays were set up with the NKL cell line against HFFF-hCAR (A), IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR (B), or IFN-α-activated T-cell-depleted PBMC against autologous skin fibroblasts (SFs; C). NK cell degranulation assays were performed with IFN-α-activated PBMC against HFFF-hCAR (D) or autologous SF (E).(F) Intracellular IFN-γ stainings were set up with IFN-α-activated PBMC against HFFF-hCAR.(G) Adhesion assays were performed with HFFF-hCAR infected with RAd-UL135 or empty control vector (RAd-Ctrl) and NK cells.(H) T cell degranulation assays were set up with the 7E7 T cell clone against MRC-5 pulsed with antigenic peptide. T cells incubated with PMA-iono or without peptide are shown as controls.(I–J) T cell intracellular IFN-γ staining assays were set up with the D7IE1 (I) and D7pp65 (J) T cell lines against autologous SF infected with RAd-Ctrl or RAd-UL135 and pulsed with peptide.(K–L) NK cell degranulation assays were set up with IFN-α-activated PBMC against allogeneic HFFF-hCAR (K) or autologous SF infected for 48 hr with HCMV strains AD169, Merlin, MerlinΔUL135, MerlinΔUL141, MerlinΔUL135/ΔUL141, or RAd-UL135, RAd-UL141, and RAd-Ctrl (L).(M) T cell degranulation assays were set up with a pp65-specific T cell line against autologous SF either mock infected or infected with Merlin or MerlinΔUL135.(N) Adhesion assays were performed with HFFF infected with the indicated viruses and NK cells. Results from three experiments were normalized and combined.Results are means ± SD of triplicate (A–C, G, H, and N), duplicate (D–F), or quadruplicate (I–M) samples and are representative of three independent experiments (A–M). Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Mentions: The levels of protection elicited by pUL135 rivalled those achieved with gpUL141. In a representative chromium release assay, pUL135 reduced specific lysis by 64% at an E:T ratio of 40:1 with the immortalized NKL cell line (Figure 1A). In cytolysis assays, pUL135 also elicited protection against a heterogeneous primary NK cell population, irrespective of whether assays were performed in an allogeneic (Figure 1B) or an autologous setting (Figure 1C). In the autologous assay, pUL135 elicited 49.5% inhibition of NK-cell-mediated cytolysis at an E:T ratio of 100:1. Moreover, using a CD107-mobilization assay, protection was elicited against IFN-α-activated NK cells from 13 different donors. In allogeneic assays, NK cell degranulation of ten bulk cultures was significantly inhibited by pUL135 expression with a mean difference of 36.8% inhibition (Figure 1D). Efficient pUL135-mediated inhibition was also observed in autologous NK degranulation assays with peripheral blood mononuclear cell (PBMC) bulk cultures from three other donors with an average inhibition of 42.2% (Figure 1E). A panel of 57 NK cell clones from two donors were expanded following single-cell sorting (Table 1). In autologous assays, 25 of 57 clones were either unable to kill target cells or not substantially affected by the expression of pUL135. The remaining NK cell clones (56.1%) were all inhibited by pUL135. Although 33% of clones were inhibited by gpUL141, 12.3% were still activated. In contrast, pUL135 was exclusively associated with an inhibitory response in this series of experiments. In addition to direct killing of target cells by cell-mediated cytotoxicity, inflammatory cytokines released by NK cells play a crucial role in suppressing virus replication; IFN-γ expression was suppressed when NK cells were cocultured with targets expressing either pUL135 or gpUL141 in comparison to vector control (Figure 1F). NK activation requires the establishment of cell:cell contact culminating in formation of an IS, and UL135 reduced the efficiency of NK cells adhesion to fibroblast targets (Figure 1G).


HCMV pUL135 remodels the actin cytoskeleton to impair immune recognition of infected cells.

Stanton RJ, Prod'homme V, Purbhoo MA, Moore M, Aicheler RJ, Heinzmann M, Bailer SM, Haas J, Antrobus R, Weekes MP, Lehner PJ, Vojtesek B, Miners KL, Man S, Wilkie GS, Davison AJ, Wang EC, Tomasec P, Wilkinson GW - Cell Host Microbe (2014)

UL135 Decreases NK Cell Cytotoxicity, Degranulation, and IFN-γ Production and Adhesion as Well as T Cell Degranulation and IFN-γ Production(A–E) Cytotoxicity assays were set up with the NKL cell line against HFFF-hCAR (A), IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR (B), or IFN-α-activated T-cell-depleted PBMC against autologous skin fibroblasts (SFs; C). NK cell degranulation assays were performed with IFN-α-activated PBMC against HFFF-hCAR (D) or autologous SF (E).(F) Intracellular IFN-γ stainings were set up with IFN-α-activated PBMC against HFFF-hCAR.(G) Adhesion assays were performed with HFFF-hCAR infected with RAd-UL135 or empty control vector (RAd-Ctrl) and NK cells.(H) T cell degranulation assays were set up with the 7E7 T cell clone against MRC-5 pulsed with antigenic peptide. T cells incubated with PMA-iono or without peptide are shown as controls.(I–J) T cell intracellular IFN-γ staining assays were set up with the D7IE1 (I) and D7pp65 (J) T cell lines against autologous SF infected with RAd-Ctrl or RAd-UL135 and pulsed with peptide.(K–L) NK cell degranulation assays were set up with IFN-α-activated PBMC against allogeneic HFFF-hCAR (K) or autologous SF infected for 48 hr with HCMV strains AD169, Merlin, MerlinΔUL135, MerlinΔUL141, MerlinΔUL135/ΔUL141, or RAd-UL135, RAd-UL141, and RAd-Ctrl (L).(M) T cell degranulation assays were set up with a pp65-specific T cell line against autologous SF either mock infected or infected with Merlin or MerlinΔUL135.(N) Adhesion assays were performed with HFFF infected with the indicated viruses and NK cells. Results from three experiments were normalized and combined.Results are means ± SD of triplicate (A–C, G, H, and N), duplicate (D–F), or quadruplicate (I–M) samples and are representative of three independent experiments (A–M). Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150922&req=5

fig1: UL135 Decreases NK Cell Cytotoxicity, Degranulation, and IFN-γ Production and Adhesion as Well as T Cell Degranulation and IFN-γ Production(A–E) Cytotoxicity assays were set up with the NKL cell line against HFFF-hCAR (A), IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR (B), or IFN-α-activated T-cell-depleted PBMC against autologous skin fibroblasts (SFs; C). NK cell degranulation assays were performed with IFN-α-activated PBMC against HFFF-hCAR (D) or autologous SF (E).(F) Intracellular IFN-γ stainings were set up with IFN-α-activated PBMC against HFFF-hCAR.(G) Adhesion assays were performed with HFFF-hCAR infected with RAd-UL135 or empty control vector (RAd-Ctrl) and NK cells.(H) T cell degranulation assays were set up with the 7E7 T cell clone against MRC-5 pulsed with antigenic peptide. T cells incubated with PMA-iono or without peptide are shown as controls.(I–J) T cell intracellular IFN-γ staining assays were set up with the D7IE1 (I) and D7pp65 (J) T cell lines against autologous SF infected with RAd-Ctrl or RAd-UL135 and pulsed with peptide.(K–L) NK cell degranulation assays were set up with IFN-α-activated PBMC against allogeneic HFFF-hCAR (K) or autologous SF infected for 48 hr with HCMV strains AD169, Merlin, MerlinΔUL135, MerlinΔUL141, MerlinΔUL135/ΔUL141, or RAd-UL135, RAd-UL141, and RAd-Ctrl (L).(M) T cell degranulation assays were set up with a pp65-specific T cell line against autologous SF either mock infected or infected with Merlin or MerlinΔUL135.(N) Adhesion assays were performed with HFFF infected with the indicated viruses and NK cells. Results from three experiments were normalized and combined.Results are means ± SD of triplicate (A–C, G, H, and N), duplicate (D–F), or quadruplicate (I–M) samples and are representative of three independent experiments (A–M). Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Mentions: The levels of protection elicited by pUL135 rivalled those achieved with gpUL141. In a representative chromium release assay, pUL135 reduced specific lysis by 64% at an E:T ratio of 40:1 with the immortalized NKL cell line (Figure 1A). In cytolysis assays, pUL135 also elicited protection against a heterogeneous primary NK cell population, irrespective of whether assays were performed in an allogeneic (Figure 1B) or an autologous setting (Figure 1C). In the autologous assay, pUL135 elicited 49.5% inhibition of NK-cell-mediated cytolysis at an E:T ratio of 100:1. Moreover, using a CD107-mobilization assay, protection was elicited against IFN-α-activated NK cells from 13 different donors. In allogeneic assays, NK cell degranulation of ten bulk cultures was significantly inhibited by pUL135 expression with a mean difference of 36.8% inhibition (Figure 1D). Efficient pUL135-mediated inhibition was also observed in autologous NK degranulation assays with peripheral blood mononuclear cell (PBMC) bulk cultures from three other donors with an average inhibition of 42.2% (Figure 1E). A panel of 57 NK cell clones from two donors were expanded following single-cell sorting (Table 1). In autologous assays, 25 of 57 clones were either unable to kill target cells or not substantially affected by the expression of pUL135. The remaining NK cell clones (56.1%) were all inhibited by pUL135. Although 33% of clones were inhibited by gpUL141, 12.3% were still activated. In contrast, pUL135 was exclusively associated with an inhibitory response in this series of experiments. In addition to direct killing of target cells by cell-mediated cytotoxicity, inflammatory cytokines released by NK cells play a crucial role in suppressing virus replication; IFN-γ expression was suppressed when NK cells were cocultured with targets expressing either pUL135 or gpUL141 in comparison to vector control (Figure 1F). NK activation requires the establishment of cell:cell contact culminating in formation of an IS, and UL135 reduced the efficiency of NK cells adhesion to fibroblast targets (Figure 1G).

Bottom Line: Without immune pressure, laboratory-adapted HCMV strains have undergone genetic alterations.Among these, the deletion of the UL/b' domain is associated with loss of virulence.An independent interaction between pUL135 and talin disrupted cell contacts with the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection & Immunity, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XN, UK. Electronic address: stantonrj@cf.ac.uk.

Show MeSH
Related in: MedlinePlus