Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Type of study
Language
Publication year range
1.
PLoS Pathog ; 7(5): e1002049, 2011 May.
Article in English | MEDLINE | ID: mdl-21589903

ABSTRACT

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.


Subject(s)
Epitopes, T-Lymphocyte/immunology , HIV-1/immunology , Proteasome Endopeptidase Complex/physiology , T-Lymphocytes, Cytotoxic/immunology , gag Gene Products, Human Immunodeficiency Virus/immunology , Adult , Amino Acid Sequence , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/physiology , Female , HIV Antigens/metabolism , HIV Infections/immunology , HIV Infections/virology , HIV-1/genetics , HIV-1/metabolism , HLA-B7 Antigen/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Molecular Sequence Data , Mutation , Polymorphism, Genetic , Proteasome Endopeptidase Complex/immunology , RNA, Viral/chemistry , RNA, Viral/genetics , Sequence Analysis, DNA , T-Lymphocytes, Cytotoxic/virology , Viral Load , Young Adult , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolism
2.
Nat Struct Mol Biol ; 16(2): 219-25, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19169257

ABSTRACT

The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone exert no effect. However, 26S proteasomes from the yeast alpha3DeltaN open-gate mutant and the rpt2YA and rpt5YA mutants with impaired gating can still be activated (approximately 1.3-fold to 1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S proteasome that facilitate channeling of substrates and their access to active sites. In consequence, polyUb substrates will allosterically stimulate their own degradation.


Subject(s)
Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Crystallography, X-Ray , Humans , Mucin-1/metabolism , Saccharomyces cerevisiae/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...