The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit.

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

Human papillomavirus detection by hybrid capture and its possible clinical use.

AIMS: To determine the sensitivity of the hybrid capture method for human papillomavirus (HPV) detection and potential clinical uses as a screening method for the identification of cervical intraepithelial neoplasia. METHODS: The presence of oncogenic types of HPV was tested for in samples taken from the cervix at colposcopy, and compared with detection by polymerase chain reaction (PCR) in 60 patients. Both sets of results were corrected with the pathology determined by biopsy and smear cytology. RESULTS: Hybrid capture detection showed 86% agreement with PCR. Eighty three percent of CIN 3 lesions, 62% of CIN 2, 59% of CIN 1 and 21% of normal controls were positive for oncogenic HPV types. CONCLUSION: The hybrid capture detection method is reliable, sensitive, and easy to use. The addition of HPV testing to cytological screening would detect a greater proportion of cervical dysplasia with a higher false positive rate.

Modulation of transcriptional regulatory properties of p53 by HPV E6.

The E6 protein encoded by human papillomavirus type 16 (HPV16), a genital virus with oncogenic potential, can target cellular p53 for rapid degradation following the formation of a complex including the two proteins. Some studies suggest that the E6 proteins encoded by HPV6 and 11, viral types which are normally limited to benign lesions, may also interact with p53, although the association is weaker than that seen with HPV16 E6. The present study demonstrates that E6 proteins from HPV16 and HPV6 can modulate the transcriptional regulatory functions of p53 in several cell types. A series of E6 mutants was used to show that association between E6 and p53 is necessary for this activity and that E6 proteins which retain the ability to associate with p53 but show no detectable degradation activity in vitro can, to some extent, abrogate p53 mediated transcriptional trans-regulation. This activity is augmented, however, by the ability of the E6 protein to target bound p53 for rapid degradation. These results suggest that some degree of modulation of p53 function is necessary in the normal viral life cycle but also demonstrate a correlation between the efficiency of this activity and oncogenic potential of the virus.