Various native and chimeric Tat proteins and long terminal repeats were tested in human, rodent, and hybrid cell lines. Cellular factors encoded on human chromosome 12 but not present in rodent cells were shown to be necessary for HIV-1 Tat interaction with the Tat responsive RNA element and consequently for efficient trans-activation of the HIV-1 promoter. A panel of expression plasmids encoding insertion, deletion. and missense mutations in the carboxy-terminal region of the equine infectious anemia virus (EIAV) tat gene were tested for their ability to trans-activate the equine EIAV promoter or to trans-inhibit heterologous Tat proteins. A mutated basic residue resulted in a potent trans-dominant inhibitor of both EIAV and HIV-1 Tat. Other mutations in the C-terminal half of E-Tat reduced trans-activation but were not trans-dominant inhibitors. Most mutations appear to have caused conformational changes that affected the structure of the whole protein. Thus, EIAV Tat appears to contact RNA in a different way than HIV-1 Tat. The CTG initiation codon for EIAV Tat was unequivocally identified by site-directed mutagenesis of potential start codons. The translation of genes downstream of Tat in bicistronic mRNAs was shown to result from leaky ribosome scanning of the Tat CTG codon. DNase footprinting and gel mobility shift assays with nuclear extracts from monocyte cell lines were used to identify regulatory elements in the EIAV promoter that bind cellular transcription factors. In addition to an AP-1 element, the EIAV promoter is also bound by the macrophage-specific ets protein, PU.1.
