Highly stable oligomerization forms of HIV-1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV-1 LTR.
The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV-1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N-terminal, C-terminal and conformation-dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV-LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV-1 LTR promoter.
Tosi
G
G
Department of Clinical and Biological Sciences, School of Medicine, University of Insubria, Varese, Italy.
Meazza
R
R
De Lerma Barbaro
A
A
D'Agostino
A
A
Mazza
S
S
Corradin
G
G
Albini
A
A
Noonan
D M
DM
Ferrini
S
S
Accolla
R S
RS
D000478:Alkylation; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D018604:Epitope Mapping; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015696:Gene Products, tat / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology; D015483:HIV Antibodies / Q000276:immunology / Q000494:pharmacology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D019163:Reducing Agents / Q000494:pharmacology; D012996:Solutions; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D015483:HIV Antibodies; D016056:Immunodominant Epitopes; D019163:Reducing Agents; D012996:Solutions; D054322:tat Gene Products, Human Immunodeficiency Virus