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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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Fate of murine class I MHC heavy chains in US11+ or US2+  cells. Control (nontransfected), US11+, or US2+ cells were infected with  vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail; A), Kd (B),  Db (C), Dd (D), or Ld (E), labeled with [35S]methionine for 10 min and  chased as indicated. Immunoprecipitated murine class I molecules were  resolved on a 12.5% SDS–polyacrylamide gel and visualized by fluorography. The position of the breakdown intermediate is indicated with an asterisk.
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Figure 1: Fate of murine class I MHC heavy chains in US11+ or US2+ cells. Control (nontransfected), US11+, or US2+ cells were infected with vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail; A), Kd (B), Db (C), Dd (D), or Ld (E), labeled with [35S]methionine for 10 min and chased as indicated. Immunoprecipitated murine class I molecules were resolved on a 12.5% SDS–polyacrylamide gel and visualized by fluorography. The position of the breakdown intermediate is indicated with an asterisk.

Mentions: To probe the specificity of the HCMV gene products US11 and US2 for class I MHC molecules, we infected parental U373-MG cells, and transfectants expressing either US11 or US2, with a panel of recombinant vaccinia virus encoding different mouse class I heavy chains, and then performed a pulse chase experiment (10-min labeling, 0-min and 20-min chase points) in the presence of the proteasome inhibitor Cbz-LLL (Fig. 1). At each chase point, aliquots of cells were pelleted and frozen at −80°C before lysis and immunoprecipitation with a rabbit anti-free heavy chain antiserum (RafHC). Immunoprecipitates were denatured by boiling in 2% SDS, and re-immunoprecipitated before analysis by SDS-PAGE. In the case of H-2Kb (lacking cytoplasmic tail) vaccinia virus-infected cells (Fig. 1 A), class I heavy chains synthesized in the 10min pulse are stable throughout the 20-min chase in control cells and in US2+ cells. However, in US11+ cells, the fully intact Kb heavy chains observed immediately upon completion of labeling (Fig. 1 A, 0-min timepoint) are rapidly converted into a faster migrating species. If the pulse chase is performed in the absence of Cbz-LLL, then the Kb heavy chains in US11+ cells are fully degraded by 20 min, and no intermediates accumulate (data not shown).


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)

Fate of murine class I MHC heavy chains in US11+ or US2+  cells. Control (nontransfected), US11+, or US2+ cells were infected with  vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail; A), Kd (B),  Db (C), Dd (D), or Ld (E), labeled with [35S]methionine for 10 min and  chased as indicated. Immunoprecipitated murine class I molecules were  resolved on a 12.5% SDS–polyacrylamide gel and visualized by fluorography. The position of the breakdown intermediate is indicated with an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Fate of murine class I MHC heavy chains in US11+ or US2+ cells. Control (nontransfected), US11+, or US2+ cells were infected with vaccinia virus expressing H-2Kb (lacking the cytoplasmic tail; A), Kd (B), Db (C), Dd (D), or Ld (E), labeled with [35S]methionine for 10 min and chased as indicated. Immunoprecipitated murine class I molecules were resolved on a 12.5% SDS–polyacrylamide gel and visualized by fluorography. The position of the breakdown intermediate is indicated with an asterisk.
Mentions: To probe the specificity of the HCMV gene products US11 and US2 for class I MHC molecules, we infected parental U373-MG cells, and transfectants expressing either US11 or US2, with a panel of recombinant vaccinia virus encoding different mouse class I heavy chains, and then performed a pulse chase experiment (10-min labeling, 0-min and 20-min chase points) in the presence of the proteasome inhibitor Cbz-LLL (Fig. 1). At each chase point, aliquots of cells were pelleted and frozen at −80°C before lysis and immunoprecipitation with a rabbit anti-free heavy chain antiserum (RafHC). Immunoprecipitates were denatured by boiling in 2% SDS, and re-immunoprecipitated before analysis by SDS-PAGE. In the case of H-2Kb (lacking cytoplasmic tail) vaccinia virus-infected cells (Fig. 1 A), class I heavy chains synthesized in the 10min pulse are stable throughout the 20-min chase in control cells and in US2+ cells. However, in US11+ cells, the fully intact Kb heavy chains observed immediately upon completion of labeling (Fig. 1 A, 0-min timepoint) are rapidly converted into a faster migrating species. If the pulse chase is performed in the absence of Cbz-LLL, then the Kb heavy chains in US11+ cells are fully degraded by 20 min, and no intermediates accumulate (data not shown).

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