A monoclonal antibody defines a novel HIV type 1 Tat domain involved in trans-cellular trans-activation.
In the present study, a CAT assay, a beta-galactosidase assay, and immunofluorescence analysis have been used to study the cellular uptake of the HIV-1 Tat protein. An anti-Tat MAb binding to an epitope comprising both the basic domain and the RGD sequence inhibits trans-activation by exogenous Tat. Two different full-length recombinant Tat proteins were used in these studies. The inhibitory MAb, however, recognized only one of the recombinant Tat proteins. Immunofluorescence analysis demonstrated that only the Tat protein recognized by the inhibitory anti-Tat MAb was taken up by COS and HeLa cells. This indicates that there are conformational differences between the two Tat proteins and that a correct folding of the epitope recognized by the anti-Tat MAb is required for cellular uptake. The recombinant Tat taken up by the cells was distributed between the nucleoli, the nucleoplasm, and along the nuclear membrane. Interactions between Tat and serum components were shown in vitro and also inhibition of trans-cellular trans-activation by fetal calf serum in tissue culture was demonstrated. The specific inhibition of the cellular uptake of Tat by an anti-Tat monoclonal antibody and the blocking of uptake by serum components implies specific binding of Tat to the cell membrane.
Valvatne
H
H
Laboratory for Biotechnology, University of Bergen, Norway.
Szilvay
A M
AM
Helland
D E
DE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000821:virology*; D002522:Chlorocebus aethiops; D015696:Gene Products, tat / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D015483:HIV Antibodies / Q000276:immunology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015696:Gene Products, tat; D015483:HIV Antibodies; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D001616:beta-Galactosidase