Purification of RBF-2, a transcription factor with specificity for the most conserved cis-element of naturally occurring HIV-1 LTRs

The combination of high turnover and error-prone reverse transcription resu lts in naturally occurring human immunodeficiency virus-1 long terminal rep eats that differ considerably from the prototype sequence. Although no tran scription-factor-binding site esca pes mutation, the only mutated site that appears to be invariably compensated by co-occurrence of its duplication i s the RBE III site, a target for the transcription factor RBF-2. In this wo rk, we characterize RBF-2 further by biochemical purification. RBF-2 was pu rified by chromatography on heparin agarose and Mono-O ion exchange chromat ography, followed by affinity chromatography on mutant and wild-type RBE II I oligonucleotide columns. The purified RBF-2 preparation contained 4 major and 1 minor polypeptides of 50, 100, 110, 120 and 125 kD, as detected by s ilver staining in SDS-PAGE gels. UV cross-linking revealed a specific 100-k D species, indicating that this protein likely represents the DNA-binding c omponent of a complex. A second factor with DNA-binding specificity similar to that of RBF-2, called RBF-B, was also identified by heparin-agarose fra ctionation, which suggests that effects of the RBE III cis-element may be m ediated by a combination of factors in vivo.