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The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains.

Machold RP, Wiertz EJ, Jones TR, Ploegh HL - J. Exp. Med. (1997)

Bottom Line: Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules.Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized.The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Human cytomegalovirus downregulates the expression of human class I major histocompatibility complex (MHC) molecules by accelerating destruction of newly synthesized class I heavy chains. The HCMV genome contains at least two genes, US11 and US2, each of which encode a product sufficient for causing the dislocation of newly synthesized class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol. Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules. Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized. The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.

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Related in: MedlinePlus

The Kb degradation intermediate is a deglycosylated heavy  chain. US11+ cells were infected with vaccinia virus expressing H-2Kb  (lacking cytoplasmic tail), labeled for 10 min with [35S]methionine, and  chased as indicated. Immunoprecipitated Kb heavy chains from each chase  point were either kept on ice (−) or treated with recombinant N-glycanase (+) before resolution by SDS-PAGE (A) or 1D-IEF (B).
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Figure 2: The Kb degradation intermediate is a deglycosylated heavy chain. US11+ cells were infected with vaccinia virus expressing H-2Kb (lacking cytoplasmic tail), labeled for 10 min with [35S]methionine, and chased as indicated. Immunoprecipitated Kb heavy chains from each chase point were either kept on ice (−) or treated with recombinant N-glycanase (+) before resolution by SDS-PAGE (A) or 1D-IEF (B).

Mentions: For immunoprecipitation of mouse class I heavy chains, a rabbit polyclonal antiserum (RafHC) which recognizes non-assembled or unfolded heavy chains was used (12). Cell pellets were each lysed in 1 ml ice-cold lysis mix (0.5% NP-40, 50 mM Tris/HCl, pH 7.4, 5 mM MgCl, 1 mM PMSF, and 10 mM iodoacetamide), and the postnuclear supernatant precleared twice with 10% fixed Staphylococcus aureus before specific immunoprecipitation of murine class I heavy chains with RafHC. To enhance the visualization of degradation intermediates, the RafHC immunoprecipitates were boiled in denaturation buffer (2% SDS, 50 mM Tris/HCl, pH 7.8, 1 mM EDTA, 5 mM DTT), and the murine class I material re-immunoprecipitated with RafHC before gel analysis. The N-glycanase digestions in Fig. 2 were performed according to the manufacturer instructions (Boehringer Mannheim, Germany). SDS-PAGE and one dimensional isoelectric focusing were performed as described (13).


The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains.

Machold RP, Wiertz EJ, Jones TR, Ploegh HL - J. Exp. Med. (1997)

The Kb degradation intermediate is a deglycosylated heavy  chain. US11+ cells were infected with vaccinia virus expressing H-2Kb  (lacking cytoplasmic tail), labeled for 10 min with [35S]methionine, and  chased as indicated. Immunoprecipitated Kb heavy chains from each chase  point were either kept on ice (−) or treated with recombinant N-glycanase (+) before resolution by SDS-PAGE (A) or 1D-IEF (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211711&req=5

Figure 2: The Kb degradation intermediate is a deglycosylated heavy chain. US11+ cells were infected with vaccinia virus expressing H-2Kb (lacking cytoplasmic tail), labeled for 10 min with [35S]methionine, and chased as indicated. Immunoprecipitated Kb heavy chains from each chase point were either kept on ice (−) or treated with recombinant N-glycanase (+) before resolution by SDS-PAGE (A) or 1D-IEF (B).
Mentions: For immunoprecipitation of mouse class I heavy chains, a rabbit polyclonal antiserum (RafHC) which recognizes non-assembled or unfolded heavy chains was used (12). Cell pellets were each lysed in 1 ml ice-cold lysis mix (0.5% NP-40, 50 mM Tris/HCl, pH 7.4, 5 mM MgCl, 1 mM PMSF, and 10 mM iodoacetamide), and the postnuclear supernatant precleared twice with 10% fixed Staphylococcus aureus before specific immunoprecipitation of murine class I heavy chains with RafHC. To enhance the visualization of degradation intermediates, the RafHC immunoprecipitates were boiled in denaturation buffer (2% SDS, 50 mM Tris/HCl, pH 7.8, 1 mM EDTA, 5 mM DTT), and the murine class I material re-immunoprecipitated with RafHC before gel analysis. The N-glycanase digestions in Fig. 2 were performed according to the manufacturer instructions (Boehringer Mannheim, Germany). SDS-PAGE and one dimensional isoelectric focusing were performed as described (13).

Bottom Line: Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules.Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized.The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
Human cytomegalovirus downregulates the expression of human class I major histocompatibility complex (MHC) molecules by accelerating destruction of newly synthesized class I heavy chains. The HCMV genome contains at least two genes, US11 and US2, each of which encode a product sufficient for causing the dislocation of newly synthesized class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol. Based on a comparison of their abilities to degrade the murine class I molecules H-2Kb, Kd, Db, Dd, and Ld, the US11 and US2 gene products have non-identical specificities for class I molecules. Specifically, in human astrocytoma cells (U373-MG) transfected with the US11 gene, the Kb, Db, Dd, and Ld molecules expressed via recombinant vaccinia virus are rapidly degraded, whereas in US2-transfected cells, only Db and Dd are significantly destabilized. The diversity in HCMV-encoded functions that interfere with class I-restricted presentation likely evolved in response to the polymorphism of the MHC.

Show MeSH
Related in: MedlinePlus