The human papillomavirus E7 protein is phosphorylated at the two serines in positions 31/32, which are part of a consensus sequence for casein kinase II (CKII). In this study, we have investigated the effect of CKII phosphorylation site mutations, all of which lead to unphosphorylated E7 proteins. The replacement of the two serines by uncharged alanine residues drastically reduced the ability of E7 to cotransform primary cells with ras, whereas negatively charged aspartic acid at the same positions produced only a slight effect. This difference was not reflected in the plO5Rb binding or the E2 promoter transactivation capability of these two mutants.Mutations that changed the CKII consensus without altering the serine residues also resulted in a loss of phosphorylation and transformation. This indicated that negative charge at positions 31/32 provided either by phosphorylation or by a negatively charged amino acid is necessary for efficient transformation without significantly affecting plO5Rb binding or transactivation.Human papillomavirus type 16 (HPV16) is believed to be involved in the etiology of human cervical cancer (1). In tumors, the viral DNA is frequently integrated (2) and only the early reading frames E6 and E7 are expressed (3). Recent studies have demonstrated that the expression of E6 together with E7 is necessary for the transformation of primary human cells (4-6). In addition, it was shown that the expression of the E7 open reading frame (ORF) in the absence of E6 is capable of transforming rodent fibroblast cell lines (7-10).The E7 protein shares several functional properties with the ElA protein of adenovirus type 5 in that E7 is able to cooperate with an activated ras gene product in the transformation of primary rodent cells (11-13) and it can transactivate the adenovirus E2 promoter (12). In addition, it was recently shown that the E7 protein, like ElA, participates in a complex with the retinoblastoma (Rb) gene product plO5Rb (Rb;.The E7 ORF of HPV16 encodes a nuclear, zinc-binding phosphoprotein with a calculated molecular mass of 11 kDa (17)(18)(19). The E7 protein is phosphorylated in vivo at only one site, 20). In this region, which lies just C terminal to the Rb binding region (residues 17-26), a consensus sequence for phosphorylation by casein kinase II (CKII; Asp-Ser-Ser-Glu-Glu-Glu-Asp-Glu; residues 30-37) can be found. In this study, we have explored the effects of sitespecific mutations within the CKII phosphorylation region on cotransformation, transactivation, and in vivo interaction with Rb.

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Human papillomaviruses (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b LI ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

In mouse Ltk-cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.The expression of mouse mammary tumor virus (MMTV) in mice as well as in tissue culture cells is regulated by steroid hormones (18,23,25,28,38). Studies utilizing homologous and heterologous cell lines revealed that the strong stimulation of viral RNA synthesis could be attributed to the action of glucocorticoids alone. The regulation is mediated by a hormone-receptor complex; it is very rapid and independent of simultaneous protein synthesis (30, 34). The hormone-receptor complex binds specifically to sequences in the long terminal repeat (LTR) of MMTV DNA (9,11,26,27,32). In transfection experiments it was shown that the same DNA region is involved in glucocorticoidregulated transcription (7,13,16,17,19). Upon deletion of some of these sequences, the hormonal effect on MMTV transcription is lost (2,7,21). It was demonstrated that the sequences required for the hormone response reside between -100 and -200 nucleotides upstream from the initiation site for viral RNA synthesis (2, 21). Larger DNA fragments containing this sequence but lacking the MMTV promoter were shown to confer hormone inducibility to other, normally non-hormone-responsive promoters (3, 21). As a result of hormonal treatment, the rate of viral RNA synthesis increases, as measured by pulse labeling (12,29,36,37,40), and higher levels of stable mRNA accumulate. From these studies it was deduced that the rate of initiation of viral RNA synthesis is increased, but they could not exclude the possibility of rapid metabolic effects of glucocorticoids, which may alter for example the stability of the MMTV mRNA. To further study the molecular events which are caused by hormone-receptor binding to DNA, it was therefore necessary to apply an approach which looks more directly at initiation of transcription. In the experiments presented here we measured the number of RNA polymerase II molecules transcribing MMTV DNA with and without hormone treatment. With the sa...