A CELLULAR 65-KDA PROTEIN RECOGNIZES THE NEGATIVE REGULATORY ELEMENT OF HUMAN PAPILLOMAVIRUS LATE MESSENGER-RNA

Papillomavirus late gene expression is tightly linked to the different iation state of the host cell. Levels of late mRNAs are only in part c ontrolled by regulation of the late promoter, other posttranscriptiona l mechanisms exist that reduce the amount of late mRNA in undifferenti ated cells, Previously we described a negative regulatory element (NRE ) located upstream of the human papillomavirus type 16 late poly(A) si te, We have delineated the NRE to a 79-nt region in which a G+U-rich r egion was the major determinant of NRE activity. UV-crosslinking assay s identified a prominent nuclear protein of 65 kDa as the only factor in close contact with the NRE, and a complex of at least five proteins , including the 65-kDa protein, was enriched on NRE-RNA. Binding of th e 65-kDa protein was depleted by preincubation with poly(U) Sepharose in high salt, a property characteristic of the U2 small nuclear ribonu cleoprotein auxiliary factor U2AF(65) and bacterially expressed U2AF(6 5) exhibited NRE binding. The 65-kDa protein bound to the G+U-rich NRE 3' half which shows homology to the B2P2 sequence a known U2AF(65) bi nding site in the alpha-tropomyosin gene, and the G+U-rich element can be replaced by B2P2 in the binding assay, Treatment of cells with pho rbol 12-myristate 13-acetate reduced binding of the 65-kDa protein, in duced NRE binding of a cytoplasmic protein, and relieved the NRE block on reporter gene expression.