A MONOCLONAL-ANTIBODY DEFINES A NOVEL HIV TYPE-1 TAT DOMAIN INVOLVED IN TRANS-CELLULAR TRANSACTIVATION

In the present study, a CAT assay, a beta-galactosidase assay, and imm unofluorescence analysis have been used to study the cellular uptake o f the HIV-1 Tat protein, An anti-Tat MAb binding to an epitope compris ing both the basic domain and the RGD sequence inhibits trans-activati on by exogenous Tat, Two different full-length recombinant Tat protein s mere 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 an ti-Tat MAb was taken up by COS and HeLa cells, This indicates that the re are conformational differences between the two Tat proteins and tha t a correct folding of the epitope recognized by the anti-Tat MAb is r equired 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 s hown in vitro and also inhibition of trans-cellular trans-activation b y fetal calf serum in tissue culture was demonstrated. The specific in hibition of the cellular uptake of Tat by an anti-Tat monoclonal antib ody and the blocking of uptake by serum components implies specific bi nding of Tat to the cell membrane.