High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer.

The major etiological factor for cervical cancer is the high-risk human papillomavirus (HPV), which encodes E6 and E7 oncogenes. However, HPV is not sufficient and estrogen has been proposed as an etiological cofactor for the disease. Its requirement has been demonstrated in mouse models for HPV-associated cervical cancer (e.g., K14E7 transgenic mice). Although germline knockout of estrogen receptor alpha (ERα) renders mice resistant to cervical cancer, the cell type-specific requirement for ERα is not known. In this study, we demonstrate that temporal deletion of stromal ERα induced complete regression of cervical dysplasia in K14E7 mice. Our results strongly support the hypothesis that stromal ERα is necessary for HPV-induced cervical carcinogenesis and implicate paracrine mechanisms involving ERα signaling in the development of estrogen-dependent cervical cancers. This is the first evidence to support the importance of stromal ERα in estrogen-dependent neoplastic disease of the female reproductive tract.

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Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an insertion larger than 30 nt, since all sequence alterations must be embedded within the primer. Here, we describe a rapid and efficient method for creating insertions or deletions of any length at any position in a DNA molecule. This method is generally applicable, and therefore represents a significant improvement to the now widely used overlap extension PCR method.

Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5 end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3 end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the ␤5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the ␤5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the ␤5 region into its own position in a mutant in which the sequences including the ␤5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the ␤5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.Hepadnaviruses infect the liver tissue of their mammalian and avian hosts, resulting in acute and chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (5). Prototypic members of the family include human hepatitis B virus (HBV), woodchuck hepatitis virus, and duck hepatitis B virus (DHBV). Hepadnaviruses have a DNA genome which replicates through an RNA intermediate via reverse transcription (5, 20). Genome replication of hepadnaviruses, catalyzed by a viral reverse transcriptase, involves the conversion of the single-stranded RNA genome into double-stranded DNA through a complex series of reactions. The strategy of hepadnaviral reverse transcription parallels that of retroviruses in many respects (30). For instance, both of its reverse transcription reactions require multiple template switching events for the completion of viral genome replication. These template switching events are mediated primarily through complementarity between donor sites and acceptor sites present in the terminal redundancy region of the RNA genomes (2,19,29).Despite its fundamental similarity to retroviral reverse transcription, many features of hepadnaviral reverse transcription are distinct. Hepadnaviral reverse transcription occurs inside nucleocapsids after encapsidation of the pregenomic RNA (pgRNA) (5, 20). A stem-loop structure (i.e., ε) near the 5Ј end of the pgRNA serves not only as an encapsidation signal (6, 10, 11, 21) but also as the initiation site for minus-strand DNA synthesis (19,23,29,32). Consequently, unlike the case for retroviruses, the initiation of minus-strand DNA synthesis is mechanistically coupled to encapsidation of the pgRNA. Fu...