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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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Related in: MedlinePlus

Expression of UL135(A) HFFF-hCAR were infected with RAd-UL135 or an empty control vector (RAd-Ctrl) for 48 hr, and HFFF were mock infected (M) or infected with HCMV strain Merlin or MerlinΔUL135 for the indicated times. Proteins were separated by SDS-PAGE, and western blot was performed for UL135 (V5 antibody) or actin.(B) HFFF-hCAR were infected with RAd-UL135 or RAd-Ctrl for 48 hr, and samples were stained for UL135 (V5 antibody) and giantin (golgi marker). HFFF were infected with the indicated strains of HCMV for 48 hr and stained for UL135 (V5 antibody) or IE1/IE2 (MerlinΔUL135) and giantin.(C) HFFF were infected with the indicated strains of HCMV and stained for either surface or intracellular UL135 expression 48 hr later.(D and E) Brightfield images of HFFF-hCAR infected with RAd-UL135 or RAd-Ctrl (D) and HFFF (E) mock infected or infected with HCMV strain Merlin or MerlinΔUL135 for 48 hr.
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fig2: Expression of UL135(A) HFFF-hCAR were infected with RAd-UL135 or an empty control vector (RAd-Ctrl) for 48 hr, and HFFF were mock infected (M) or infected with HCMV strain Merlin or MerlinΔUL135 for the indicated times. Proteins were separated by SDS-PAGE, and western blot was performed for UL135 (V5 antibody) or actin.(B) HFFF-hCAR were infected with RAd-UL135 or RAd-Ctrl for 48 hr, and samples were stained for UL135 (V5 antibody) and giantin (golgi marker). HFFF were infected with the indicated strains of HCMV for 48 hr and stained for UL135 (V5 antibody) or IE1/IE2 (MerlinΔUL135) and giantin.(C) HFFF were infected with the indicated strains of HCMV and stained for either surface or intracellular UL135 expression 48 hr later.(D and E) Brightfield images of HFFF-hCAR infected with RAd-UL135 or RAd-Ctrl (D) and HFFF (E) mock infected or infected with HCMV strain Merlin or MerlinΔUL135 for 48 hr.

Mentions: UL135 exhibits a high degree of sequence conservation in characterized HCMV strains and clinical isolates. An ortholog is present in chimpanzee cytomegalovirus (CMV) but not CMV species of the lower primates (Umashankar et al., 2011). pUL135 is exceptionally proline-rich (60 of 308 amino acids [aa]), contributing to predictions that it contains 22 potential SH3 binding sites and is 80% “structurally disordered.” pUL135 was expressed at slightly higher levels from RAd-UL135 in comparison to HCMV (Figure S2A) and was synthesized as two species with molecular masses of 38kDa and 40kDa in comparison to a predicted size of 33 kDa. During productive HCMV infection, pUL135 was expressed during early phase (24 hr), but, unusually for an HCMV gene, levels declined through the late phase (Figure 2A). pUL135 is membrane associated (Umashankar et al., 2011) and predicted to contain an N-terminal transmembrane domain and two N- and 42 O-linked glycosylation sites. However, it was not sensitive to PNGaseF or O-glycosidase, indicating these glycosylation sites were not used (Figure S2B). In order to optimize detection in the context of HCMV infection, a sequence providing a C-terminal V5 tag was inserted into UL135. When expressed in isolation from RAd-UL135,or in the context of HCMV infection, pUL135 colocalized with markers of the Golgi apparatus and at the plasma membrane (Figure 2B). Golgi localization was more prominent when UL135 was expressed in isolation, most likely because of the expression of other HCMV genes and reorganization of the Golgi apparatus, during infection. A monoclonal antibody raised to pUL135 was able to detect the protein on cells only once they had been permeabilized (Figure 2C). The data imply that pUL135 is anchored in the Golgi and plasma membranes via its N-terminal hydrophobic domain with the main body of the protein exposed to the cytosol, which is consistent with previous data (Umashankar et al., 2011). This topological orientation in the plasma membrane would make it difficult for pUL135 to act directly as an inhibitory ligand.


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)

Expression of UL135(A) HFFF-hCAR were infected with RAd-UL135 or an empty control vector (RAd-Ctrl) for 48 hr, and HFFF were mock infected (M) or infected with HCMV strain Merlin or MerlinΔUL135 for the indicated times. Proteins were separated by SDS-PAGE, and western blot was performed for UL135 (V5 antibody) or actin.(B) HFFF-hCAR were infected with RAd-UL135 or RAd-Ctrl for 48 hr, and samples were stained for UL135 (V5 antibody) and giantin (golgi marker). HFFF were infected with the indicated strains of HCMV for 48 hr and stained for UL135 (V5 antibody) or IE1/IE2 (MerlinΔUL135) and giantin.(C) HFFF were infected with the indicated strains of HCMV and stained for either surface or intracellular UL135 expression 48 hr later.(D and E) Brightfield images of HFFF-hCAR infected with RAd-UL135 or RAd-Ctrl (D) and HFFF (E) mock infected or infected with HCMV strain Merlin or MerlinΔUL135 for 48 hr.
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Related In: Results  -  Collection

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fig2: Expression of UL135(A) HFFF-hCAR were infected with RAd-UL135 or an empty control vector (RAd-Ctrl) for 48 hr, and HFFF were mock infected (M) or infected with HCMV strain Merlin or MerlinΔUL135 for the indicated times. Proteins were separated by SDS-PAGE, and western blot was performed for UL135 (V5 antibody) or actin.(B) HFFF-hCAR were infected with RAd-UL135 or RAd-Ctrl for 48 hr, and samples were stained for UL135 (V5 antibody) and giantin (golgi marker). HFFF were infected with the indicated strains of HCMV for 48 hr and stained for UL135 (V5 antibody) or IE1/IE2 (MerlinΔUL135) and giantin.(C) HFFF were infected with the indicated strains of HCMV and stained for either surface or intracellular UL135 expression 48 hr later.(D and E) Brightfield images of HFFF-hCAR infected with RAd-UL135 or RAd-Ctrl (D) and HFFF (E) mock infected or infected with HCMV strain Merlin or MerlinΔUL135 for 48 hr.
Mentions: UL135 exhibits a high degree of sequence conservation in characterized HCMV strains and clinical isolates. An ortholog is present in chimpanzee cytomegalovirus (CMV) but not CMV species of the lower primates (Umashankar et al., 2011). pUL135 is exceptionally proline-rich (60 of 308 amino acids [aa]), contributing to predictions that it contains 22 potential SH3 binding sites and is 80% “structurally disordered.” pUL135 was expressed at slightly higher levels from RAd-UL135 in comparison to HCMV (Figure S2A) and was synthesized as two species with molecular masses of 38kDa and 40kDa in comparison to a predicted size of 33 kDa. During productive HCMV infection, pUL135 was expressed during early phase (24 hr), but, unusually for an HCMV gene, levels declined through the late phase (Figure 2A). pUL135 is membrane associated (Umashankar et al., 2011) and predicted to contain an N-terminal transmembrane domain and two N- and 42 O-linked glycosylation sites. However, it was not sensitive to PNGaseF or O-glycosidase, indicating these glycosylation sites were not used (Figure S2B). In order to optimize detection in the context of HCMV infection, a sequence providing a C-terminal V5 tag was inserted into UL135. When expressed in isolation from RAd-UL135,or in the context of HCMV infection, pUL135 colocalized with markers of the Golgi apparatus and at the plasma membrane (Figure 2B). Golgi localization was more prominent when UL135 was expressed in isolation, most likely because of the expression of other HCMV genes and reorganization of the Golgi apparatus, during infection. A monoclonal antibody raised to pUL135 was able to detect the protein on cells only once they had been permeabilized (Figure 2C). The data imply that pUL135 is anchored in the Golgi and plasma membranes via its N-terminal hydrophobic domain with the main body of the protein exposed to the cytosol, which is consistent with previous data (Umashankar et al., 2011). This topological orientation in the plasma membrane would make it difficult for pUL135 to act directly as an inhibitory ligand.

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