ABSTRACT
A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4(S27N) had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4(S27N) enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.
Subject(s)
CD4 Antigens/metabolism , Gene Expression Regulation, Viral , Gene Products, tat/genetics , HIV Infections/metabolism , HIV-1/pathogenicity , rab4 GTP-Binding Proteins/metabolism , Antigens, CD/metabolism , Apoptosis , Base Sequence , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Chloramphenicol O-Acetyltransferase/metabolism , Dependovirus/genetics , Disease Susceptibility , Exons/genetics , Flow Cytometry , Gene Products, tat/pharmacology , Genes, Dominant , HIV Core Protein p24/metabolism , HIV Infections/virology , HIV Long Terminal Repeat/genetics , HeLa Cells , Humans , Introns/genetics , Jurkat Cells , Lysosomes , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Nucleic Acid , Transfection , rab4 GTP-Binding Proteins/genetics , tat Gene Products, Human Immunodeficiency VirusABSTRACT
OBJECTIVE: Systemic lupus erythematosus (SLE) patients produce autoantibodies to HRES-1/p28, a human endogenous retrovirus-encoded nuclear protein. To identify cross-reactive viral antigens capable of triggering autoreactivity, HRES-1/p28 epitopes were mapped by SLE antibodies. METHODS: Forty-four peptides overlapping HRES-1/p28 and 13 viral peptides were synthesized on cellulose membrane and tested for recognition by antibodies from 16 HRES-1 Western blot seropositive SLE patients. Transfusion-transmitted virus (TTV) was detected by gene amplification in sera of 211 SLE patients, 78 healthy SLE family members, 199 unrelated healthy donors, and 91 rheumatoid arthritis (RA) patients. RESULTS: HRES-1/p28 residues 41-55, 121-135, and 156-170 were recognized by 12/16 (75.0%), 11/16 (68.8%), and 9/16 lupus sera (56.25%) and considered immunodominant. HRES-1/p28 residues 121-135 harbor cross-reactive epitope with retroviral peptides and the 70 K U1snRNP lupus autoantigen. HRES-1/p28 residues 41-55 and 156-170 exhibited the highest prevalence of cross-reactivity with TTV peptide ORF2a (14/16, 87%). Prevalence of TTV DNA was increased in lupus patients (120/211) with respect to healthy (66/199; P < 0.0001) or RA controls (23/91; P < 0.0001). TTV prevalence in healthy lupus relatives (40/78) was decreased with respect to lupus patients (80/121; P = 0.0184) and increased with respect to unrelated healthy donors (66/199; P = 0.0026). HRES-1/p28 Western blot reactivity was observed in 12/23 TTV PCR-negative donors and 43/58 TTV PCR-positive donors (P < 0.0281). CONCLUSIONS: Increased prevalence of TTV and molecular mimicry with HRES-1/p28 may contribute to generation of antinuclear antibodies and pathogenesis of SLE.
Subject(s)
Antigens, Nuclear/immunology , Autoantigens/immunology , Circoviridae Infections/immunology , Immunodominant Epitopes/immunology , Lupus Erythematosus, Systemic/immunology , Retroviridae Proteins/immunology , Torque teno virus/immunology , Adult , Amino Acid Sequence , Antibodies/immunology , Antibody Affinity/immunology , Antigens, Nuclear/genetics , Autoantigens/genetics , Circoviridae Infections/epidemiology , Cross Reactions/genetics , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immunodominant Epitopes/genetics , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/virology , Male , Middle Aged , Molecular Sequence Data , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Prevalence , Retroviridae Proteins/genetics , Sequence Homology, Amino Acid , Torque teno virus/genetics , Viral Proteins/genetics , Viral Proteins/immunology , Viremia/diagnosis , Viremia/epidemiologyABSTRACT
Homozygous deletion of three nucleotides coding for Ser-171 (S171) of TAL-H (human transaldolase) has been identified in a female patient with liver cirrhosis. Accumulation of sedoheptulose 7-phosphate raised the possibility of TAL (transaldolase) deficiency in this patient. In the present study, we show that the mutant TAL-H gene was effectively transcribed into mRNA, whereas no expression of the TALDeltaS171 protein or enzyme activity was detected in TALDeltaS171 fibroblasts or lymphoblasts. Unlike wild-type TAL-H-GST fusion protein (where GST stands for glutathione S-transferase), TALDeltaS171-GST was solubilized only in the presence of detergents, suggesting that deletion of Ser-171 caused conformational changes. Recombinant TALDeltaS171 had no enzymic activity. TALDeltaS171 was effectively translated in vitro using rabbit reticulocyte lysates, indicating that the absence of TAL-H protein in TALDeltaS171 fibroblasts and lymphoblasts may be attributed primarily to rapid degradation. Treatment with cell-permeable proteasome inhibitors led to the accumulation of TALDeltaS171 in whole cell lysates and cytosolic extracts of patient lymphoblasts, suggesting that deletion of Ser-171 led to rapid degradation by the proteasome. Although the TALDeltaS171 protein became readily detectable in proteasome inhibitor-treated cells, it displayed no appreciable enzymic activity. The results suggest that deletion of Ser-171 leads to inactivation and proteasome-mediated degradation of TAL-H. Since TAL-H is a regulator of apoptosis signal processing, complete deficiency of TAL-H may be relevant for the pathogenesis of liver cirrhosis.