CLONING AND CHARACTERIZATION OF A NOVEL CELLULAR PROTEIN, TDP-43, THAT BINDS TO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 TAR DNA-SEQUENCE MOTIFS

Human immunodeficiency virus type 1 (HIV-1) gene expression is modulat ed by both viral and cellular factors. A regulatory element in the HIV -1 long terminal repeat known as TAR, which extends from nucleotides - 18 to +80, is critical for the activation of gene expression by the tr ansactivator protein, Tat. RNA transcribed from TAR forms a stable ste m-loop structure which serves as the binding site for both Tat and cel lular factors. Although TAR RNA is critical for Tat activation, the ro le that TAR DNA plays in regulating HIV-1 gene expression is not clear . Several studies have demonstrated that TAR DNA can bind cellular pro teins, such as UBP-1/LBP-1, which repress HIV-1 gene expression and ot her factors which are involved in the generation of short, nonprocessi ve transcripts. In an attempt to characterize additional cellular fact ors that bind to TAR DNA, a lambda gt11 expression cloning strategy in volving the use of a portion of TAR DNA extending from -18 to +28 to p robe a HeLa cDNA library was used. We identified a cDNA, designated TA R DNA-binding protein (TDP-43), which encodes a cellular factor of 43 kDa that binds specifically to pyrimidine-rich motifs in TAR. Antibody to TDP-43 was used in gel retardation assays to demonstrate that endo genous TDP-43, present in HeLa nuclear extract, also bound to TAR DNA. Although TDP-43 bound strongly to double-stranded TAR DNA via its rib onucleoprotein protein-binding motifs, it did not bind to TAR RNA exte nding from +1 to +80. To determine the function of TDP-43 in regulatin g HTV-1 gene expression, in vitro transcription analysis was performed . TDP-43 repressed in vitro transcription from the HIV-1 long terminal repeat in both the presence and absence of Tar, but it did not repres s transcription from other promoters such as the adenovirus major late promoter. In addition, transfection of a vector which expressed TDP-4 3 resulted in the repression of gene expression from an HIV-1 provirus . These results indicate that TDP-43 is capable of modulating both in vitro and in vivo HIV-1 gene expression by either altering or blocking the assembly of transcription complexes that are capable of respondin g to Tat.