Limits...
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.

Show MeSH

Related in: MedlinePlus

Interactions with ABI1/ABI2 Are Required for UL135 to Protect Against NK Cells(A and B) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), or siRNA against talin-1 and talin-2 (siRNA-TLN1/2). They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(C and D) Cytotoxicity assays were performed with IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR transfected with siRNA-Ctrl (C) or siRNA-ABI1/ABI2 (D) and then infected with either RAd-Ctrl or RAd-UL135.(E) HFFF-hCAR were infected with the indicated adenovirus vectors, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(F) Cells were transfected with control siRNA or siRNA against CYFIP1. They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(G) HFFF-hCAR were infected with RAd expressing ABI1 and ABI2 (RAd-ABI1+ABI2), RAd-UL135, or empty control vector (RAd-Ctrl) in the combinations indicated, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC. Results of are means ± SD of quadruplicate samples. Results are representative of three independent experiments. Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(H and J) HFFF-hCAR were infected with RAd-Ctrl or RAd-UL135. NKL cells were added 48 hr postinfection, allowed to settle, and then fixed and stained with phalloidin. Images show the immune synapse between fibroblasts and NKL cells.(I) Quantitation of NKL:HFFF interactions in (H).(J) 3D reconstruction of the IS is shown in the top panel. Arrows indicate actin fibers in the target that define the inner or outer edge of the synapse.(K and L) Fibroblasts expressing lifeact-Citrine were infected with RAd-Ctrl or RAd-UL135 and incubated with NKL-cell-expressing lifeact-mCherry 48 hr later.(K) Differential imaging of F-actin within a fibroblast (green) and NKL cell (red) at the IS.(L) Actin fibers in RAd-Ctrl infected fibroblast that align with the extent of actin polymerization in the NKL cell are highlighted in white.(M) Intensity profiles of actin along the indicated regions of the IS within the fibroblast (green) or NKL cell (red).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4150922&req=5

fig6: Interactions with ABI1/ABI2 Are Required for UL135 to Protect Against NK Cells(A and B) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), or siRNA against talin-1 and talin-2 (siRNA-TLN1/2). They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(C and D) Cytotoxicity assays were performed with IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR transfected with siRNA-Ctrl (C) or siRNA-ABI1/ABI2 (D) and then infected with either RAd-Ctrl or RAd-UL135.(E) HFFF-hCAR were infected with the indicated adenovirus vectors, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(F) Cells were transfected with control siRNA or siRNA against CYFIP1. They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(G) HFFF-hCAR were infected with RAd expressing ABI1 and ABI2 (RAd-ABI1+ABI2), RAd-UL135, or empty control vector (RAd-Ctrl) in the combinations indicated, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC. Results of are means ± SD of quadruplicate samples. Results are representative of three independent experiments. Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(H and J) HFFF-hCAR were infected with RAd-Ctrl or RAd-UL135. NKL cells were added 48 hr postinfection, allowed to settle, and then fixed and stained with phalloidin. Images show the immune synapse between fibroblasts and NKL cells.(I) Quantitation of NKL:HFFF interactions in (H).(J) 3D reconstruction of the IS is shown in the top panel. Arrows indicate actin fibers in the target that define the inner or outer edge of the synapse.(K and L) Fibroblasts expressing lifeact-Citrine were infected with RAd-Ctrl or RAd-UL135 and incubated with NKL-cell-expressing lifeact-mCherry 48 hr later.(K) Differential imaging of F-actin within a fibroblast (green) and NKL cell (red) at the IS.(L) Actin fibers in RAd-Ctrl infected fibroblast that align with the extent of actin polymerization in the NKL cell are highlighted in white.(M) Intensity profiles of actin along the indicated regions of the IS within the fibroblast (green) or NKL cell (red).

Mentions: The functional significance of the interaction of pUL135 with ABI1/ABI2 and talin in eliciting protection against NK cells was investigated with siRNA. The interaction with ABI1/ABI2, rather than talin, was critical for NK cell evasion whether assessed by CD107a mobilization (Figures 6A and 6B) or cytolysis assays (Figures 6C and 6D). This correlation was supported in experiments utilizing the characterized pUL135 deletion mutants. Only versions of pUL135 that bound ABI1/ABI2 elicited protection against NK cells (Figure 6E). Indeed, pUL135 functioned even more effectively as an NK evasion function when the talin-interacting domain was deleted. pUL135’s direct interaction with ABI1/ABI2 was not sufficient, a second WRC member (CYFIP1) was also required for pUL135 in order to induce protection against NK cell attack (Figure 6F). The recruitment of the entire WRC is thus implicated in pUL135 function. In the absence of pUL135, neither the ablation of ABI1/ABI2 nor CYFIP1 had an impact on NK cell recognition, indicating that pUL135 used ABI1/ABI2 to recruit the WRC and then subverted its activity to suppress NK cells.


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)

Interactions with ABI1/ABI2 Are Required for UL135 to Protect Against NK Cells(A and B) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), or siRNA against talin-1 and talin-2 (siRNA-TLN1/2). They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(C and D) Cytotoxicity assays were performed with IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR transfected with siRNA-Ctrl (C) or siRNA-ABI1/ABI2 (D) and then infected with either RAd-Ctrl or RAd-UL135.(E) HFFF-hCAR were infected with the indicated adenovirus vectors, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(F) Cells were transfected with control siRNA or siRNA against CYFIP1. They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(G) HFFF-hCAR were infected with RAd expressing ABI1 and ABI2 (RAd-ABI1+ABI2), RAd-UL135, or empty control vector (RAd-Ctrl) in the combinations indicated, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC. Results of are means ± SD of quadruplicate samples. Results are representative of three independent experiments. Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(H and J) HFFF-hCAR were infected with RAd-Ctrl or RAd-UL135. NKL cells were added 48 hr postinfection, allowed to settle, and then fixed and stained with phalloidin. Images show the immune synapse between fibroblasts and NKL cells.(I) Quantitation of NKL:HFFF interactions in (H).(J) 3D reconstruction of the IS is shown in the top panel. Arrows indicate actin fibers in the target that define the inner or outer edge of the synapse.(K and L) Fibroblasts expressing lifeact-Citrine were infected with RAd-Ctrl or RAd-UL135 and incubated with NKL-cell-expressing lifeact-mCherry 48 hr later.(K) Differential imaging of F-actin within a fibroblast (green) and NKL cell (red) at the IS.(L) Actin fibers in RAd-Ctrl infected fibroblast that align with the extent of actin polymerization in the NKL cell are highlighted in white.(M) Intensity profiles of actin along the indicated regions of the IS within the fibroblast (green) or NKL cell (red).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4150922&req=5

fig6: Interactions with ABI1/ABI2 Are Required for UL135 to Protect Against NK Cells(A and B) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), or siRNA against talin-1 and talin-2 (siRNA-TLN1/2). They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(C and D) Cytotoxicity assays were performed with IFN-α-activated T-cell-depleted PBMC against HFFF-hCAR transfected with siRNA-Ctrl (C) or siRNA-ABI1/ABI2 (D) and then infected with either RAd-Ctrl or RAd-UL135.(E) HFFF-hCAR were infected with the indicated adenovirus vectors, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(F) Cells were transfected with control siRNA or siRNA against CYFIP1. They were infected 24 hr later with RAd-UL135 or RAd-Ctrl. NK degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC.(G) HFFF-hCAR were infected with RAd expressing ABI1 and ABI2 (RAd-ABI1+ABI2), RAd-UL135, or empty control vector (RAd-Ctrl) in the combinations indicated, and NK cell degranulation assays were performed 48 hr postinfection with IFN-α-activated PBMC. Results of are means ± SD of quadruplicate samples. Results are representative of three independent experiments. Two-way ANOVA test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.(H and J) HFFF-hCAR were infected with RAd-Ctrl or RAd-UL135. NKL cells were added 48 hr postinfection, allowed to settle, and then fixed and stained with phalloidin. Images show the immune synapse between fibroblasts and NKL cells.(I) Quantitation of NKL:HFFF interactions in (H).(J) 3D reconstruction of the IS is shown in the top panel. Arrows indicate actin fibers in the target that define the inner or outer edge of the synapse.(K and L) Fibroblasts expressing lifeact-Citrine were infected with RAd-Ctrl or RAd-UL135 and incubated with NKL-cell-expressing lifeact-mCherry 48 hr later.(K) Differential imaging of F-actin within a fibroblast (green) and NKL cell (red) at the IS.(L) Actin fibers in RAd-Ctrl infected fibroblast that align with the extent of actin polymerization in the NKL cell are highlighted in white.(M) Intensity profiles of actin along the indicated regions of the IS within the fibroblast (green) or NKL cell (red).
Mentions: The functional significance of the interaction of pUL135 with ABI1/ABI2 and talin in eliciting protection against NK cells was investigated with siRNA. The interaction with ABI1/ABI2, rather than talin, was critical for NK cell evasion whether assessed by CD107a mobilization (Figures 6A and 6B) or cytolysis assays (Figures 6C and 6D). This correlation was supported in experiments utilizing the characterized pUL135 deletion mutants. Only versions of pUL135 that bound ABI1/ABI2 elicited protection against NK cells (Figure 6E). Indeed, pUL135 functioned even more effectively as an NK evasion function when the talin-interacting domain was deleted. pUL135’s direct interaction with ABI1/ABI2 was not sufficient, a second WRC member (CYFIP1) was also required for pUL135 in order to induce protection against NK cell attack (Figure 6F). The recruitment of the entire WRC is thus implicated in pUL135 function. In the absence of pUL135, neither the ablation of ABI1/ABI2 nor CYFIP1 had an impact on NK cell recognition, indicating that pUL135 used ABI1/ABI2 to recruit the WRC and then subverted its activity to suppress NK cells.

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