The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas. In these cancers, the viral DNAs are often integrated into the host genome so that expression of the El and the E2 genes is lost, suggesting that disruption of these regulatory genes plays an important role in carcinogenic pgression. Previous studies defiming the viral genes affecting PV-16 transformation functions have used the "prototype" viral genome, which was cloned from a human cervical carcinoma and later discovered to harbor a mutation in the El gene. In this study, we have corrected this mutation and have evaluated the effect of mutations of either the El or the E2 gene on the efficiency of HPV-16 immortalization of human keratinocytes. Mutation of either the El gene or the E2 gene in the background of a "wild-type" HPV-16 genome markedly increased immortalization capacity. Mutations were also generated in the E2-binding sites located upstream of the P,7 promoter, which directs synthesis of the viral E6 and E7 transforming genes. E2 negatively regulates the Pg. promoter through bindin at adjacent sites. Surprly, the mutation of these sites only partially relieved the negative effect of E2 on viral immortalization, implicating additional mechaniss in theE2 repression of viral immortalization functions. Our results provide genetic evidence that the El and E2 gene products each can repress HPV-16 immortalization and support the hypothesis that a selective growth advantage is provided by integration of the viral-genome in a manner that causes the loss of expression of either El or E2.A subgroup of human papillomaviruses (HPVs) is associated with genital intraepithelial neoplasias and cervical carcinomas. The two viruses found in most of these lesions are HPV types 16 (HPV-16) and 18 (HPV-18). HPV-16 DNA and HPV-18 DNA can immortalize primary human keratinocytes in culture (1-3) and can induce histological abnormalities in organotypic cultures similar to those seen in clinical lesions in vivo (4, 5). Previous analyses have shown that the expression of both the E6 and E7 genes of the "high-risk" HPVs is necessary for the efficient immortalization of primary human keratinocytes (5-7). Furthermore, the E6 and E7 genes are consistently expressed in the HPV-positive cervical cancers and in cell lines derived from such cancers (8-13), supporting the hypothesis that expression of E6 and E7 plays a role in cervical carcinogenesis. Analyses of the state of the viral DNA in HPV-positive lesions have generally correlated the presence of extrachromosomal viral genomes in benign preneoplastic lesions and of integrated viral DNA in cancers (8,(13)(14)(15)(16) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P9 and HPV-18 Plos promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P10s promoter in infected human genital epithelial cells.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, El and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the El open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding El and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral El and/or E2 genes required for DNA replication.

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Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious for gene transfer in vitro, improvement in the efficiency of vector delivery in vivo may aid several gene therapy applications. One major obstacle is the lack of high-affinity viral receptors on the surface of certain cells that are targets for gene therapy. In principle, incorporation of avid, cell-specific ligands into the virion could markedly improve vector entry into the desired tissues. We have developed a strategy for addressing this issue in the lung by biopanning differentiated, ciliated airway epithelial cells against a phage display library. The peptide with the most effective binding was coupled to the surface of an adenovirus using bifunctional polyethylene glycol (PEG) molecules. The chemically modified adenoviral vector was able to effect gene transfer to well-differentiated human airway epithelial cells by an alternative pathway dependent on the incorporated peptide. Coupling of PEG to the surface of the virus also served to partially protect the virus from neutralizing antibodies in vitro. These experiments will aid in the design of improved adenoviral vectors with the capacity for more specific and efficient delivery of therapeutic genes to desired target tissues. We have used a novel method for enhancing gene delivery to target cells by coupling a biologically selected peptide to the surface of an adenovirus with bifunctional PEG molecules. Modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Incorporation of the CFTR gene in a similarly modified vector resulted in correction of defective Cl- transport in well-differentiated epithelial cultures established from human cystic fibrosis (CF) donors. The presence of PEG molecules on the surface of the virus served, in addition, to reduce antibody neutralization. Modification of adenoviruses with PEG/peptide complexes can serve to partially overcome the barrier of inefficient gene transfer in some cell types and some of the adverse immunological responses associated with gene delivery by these vectors.