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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 Interacts Independently with the WRC and Talin and Inhibits Cytoskeletal Remodelling(A–E) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), siRNA against talin-1 and talin-2 (siRNA-TLN1/2), or siRNA against CYFIP1. They were infected with RAd-UL135 or RAd-Ctrl 24 hr later. Assays were performed 48 hr postinfection.(A and B) Cells were fixed and stained for actin (phalloidin).(C and D) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and representative of three independent experiments. Two-way ANOVA test. ∗∗∗p < 0.001.(E) Cells were lysed, and UL135 was immunoprecipitated with its V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(F–J) HFFF-hCAR were infected with the indicated adenovirus vectors, and assays were performed at 48 hr postinfection.(F) UL135 was immunoprecipitated with the V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(G) Cells were fixed and stained for actin (phalloidin).(H) Cells were fixed and stained for UL135 (V5 antibody) and ABI1. Accumulation of ABI1 at sites of actin protrusions are indicated with arrows.(I) Cells were stained for UL135 (V5 antibody) and talin. Accumulation of talin at sites resembling focal adhesions are indicated with arrows.(J) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and are representative of three independent experiments. One-way ANOVA test. ∗∗∗p < 0.001.
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fig5: UL135 Interacts Independently with the WRC and Talin and Inhibits Cytoskeletal Remodelling(A–E) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), siRNA against talin-1 and talin-2 (siRNA-TLN1/2), or siRNA against CYFIP1. They were infected with RAd-UL135 or RAd-Ctrl 24 hr later. Assays were performed 48 hr postinfection.(A and B) Cells were fixed and stained for actin (phalloidin).(C and D) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and representative of three independent experiments. Two-way ANOVA test. ∗∗∗p < 0.001.(E) Cells were lysed, and UL135 was immunoprecipitated with its V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(F–J) HFFF-hCAR were infected with the indicated adenovirus vectors, and assays were performed at 48 hr postinfection.(F) UL135 was immunoprecipitated with the V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(G) Cells were fixed and stained for actin (phalloidin).(H) Cells were fixed and stained for UL135 (V5 antibody) and ABI1. Accumulation of ABI1 at sites of actin protrusions are indicated with arrows.(I) Cells were stained for UL135 (V5 antibody) and talin. Accumulation of talin at sites resembling focal adhesions are indicated with arrows.(J) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and are representative of three independent experiments. One-way ANOVA test. ∗∗∗p < 0.001.

Mentions: pUL135 consistently induced cell rounding, loss of contact adhesions, and disassembly of stress fibers. RNA interference was used to investigate the roles played by the various pUL135-interacting cellular proteins (Figure S5A). Ablation of both ABI1 and ABI2 (ABI1/ABI2) was associated with a very slight reduction in F-actin levels in control cells, consistent with a requirement for ABI1/ABI2 in the WRC (Figures 5A andS5B). In contrast, pUL135 expression induced a very dramatic loss of F-actin. Moreover, following ABI1/ABI2 knockdown, pUL135 lost the capacity to depolymerize F-actin fibers throughout the center of the cell. This implies that, rather than directly inhibiting ABI1/ABI2, pUL135 is commandeering the WRC to actively promote F-actin depolymerization. To determine whether the entire WRC was involved in pUL135-mediated actin remodelling, the WRC member CYFIP1 was targeted with small interfering RNA (siRNA). Although knockdown had no obvious effect on actin, CYFIP1 was required for pUL135-mediated depolymerization of actin (Figures 5B and S5C). Therefore, ABI1/ABI2 was not the only WRC component necessary for pUL135-mediated actin remodelling.


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 Interacts Independently with the WRC and Talin and Inhibits Cytoskeletal Remodelling(A–E) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), siRNA against talin-1 and talin-2 (siRNA-TLN1/2), or siRNA against CYFIP1. They were infected with RAd-UL135 or RAd-Ctrl 24 hr later. Assays were performed 48 hr postinfection.(A and B) Cells were fixed and stained for actin (phalloidin).(C and D) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and representative of three independent experiments. Two-way ANOVA test. ∗∗∗p < 0.001.(E) Cells were lysed, and UL135 was immunoprecipitated with its V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(F–J) HFFF-hCAR were infected with the indicated adenovirus vectors, and assays were performed at 48 hr postinfection.(F) UL135 was immunoprecipitated with the V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(G) Cells were fixed and stained for actin (phalloidin).(H) Cells were fixed and stained for UL135 (V5 antibody) and ABI1. Accumulation of ABI1 at sites of actin protrusions are indicated with arrows.(I) Cells were stained for UL135 (V5 antibody) and talin. Accumulation of talin at sites resembling focal adhesions are indicated with arrows.(J) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and are representative of three independent experiments. One-way ANOVA test. ∗∗∗p < 0.001.
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fig5: UL135 Interacts Independently with the WRC and Talin and Inhibits Cytoskeletal Remodelling(A–E) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), siRNA against talin-1 and talin-2 (siRNA-TLN1/2), or siRNA against CYFIP1. They were infected with RAd-UL135 or RAd-Ctrl 24 hr later. Assays were performed 48 hr postinfection.(A and B) Cells were fixed and stained for actin (phalloidin).(C and D) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and representative of three independent experiments. Two-way ANOVA test. ∗∗∗p < 0.001.(E) Cells were lysed, and UL135 was immunoprecipitated with its V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(F–J) HFFF-hCAR were infected with the indicated adenovirus vectors, and assays were performed at 48 hr postinfection.(F) UL135 was immunoprecipitated with the V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.(G) Cells were fixed and stained for actin (phalloidin).(H) Cells were fixed and stained for UL135 (V5 antibody) and ABI1. Accumulation of ABI1 at sites of actin protrusions are indicated with arrows.(I) Cells were stained for UL135 (V5 antibody) and talin. Accumulation of talin at sites resembling focal adhesions are indicated with arrows.(J) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and are representative of three independent experiments. One-way ANOVA test. ∗∗∗p < 0.001.
Mentions: pUL135 consistently induced cell rounding, loss of contact adhesions, and disassembly of stress fibers. RNA interference was used to investigate the roles played by the various pUL135-interacting cellular proteins (Figure S5A). Ablation of both ABI1 and ABI2 (ABI1/ABI2) was associated with a very slight reduction in F-actin levels in control cells, consistent with a requirement for ABI1/ABI2 in the WRC (Figures 5A andS5B). In contrast, pUL135 expression induced a very dramatic loss of F-actin. Moreover, following ABI1/ABI2 knockdown, pUL135 lost the capacity to depolymerize F-actin fibers throughout the center of the cell. This implies that, rather than directly inhibiting ABI1/ABI2, pUL135 is commandeering the WRC to actively promote F-actin depolymerization. To determine whether the entire WRC was involved in pUL135-mediated actin remodelling, the WRC member CYFIP1 was targeted with small interfering RNA (siRNA). Although knockdown had no obvious effect on actin, CYFIP1 was required for pUL135-mediated depolymerization of actin (Figures 5B and S5C). Therefore, ABI1/ABI2 was not the only WRC component necessary for pUL135-mediated actin remodelling.

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