Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid-and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.[Keywords: Human papillomavirus infection program; organotypic cultures of primary human keratinocytes; Cre-loxP-mediated recombination in vivo; cell cycle regulation; immortalization-and replication-defective E6 mutant; p53 protein stabilization] Supplemental material is available at http://www.genesdev.org.

Human papillomaviruses (HPV)2 are small double-stranded DNA tumor viruses of considerable medical importance. They infect epithelial tissues, causing benign hyperproliferative lesions. More than 120 HPV genotypes have been cloned from patient specimens. The mucosotropic HPVs are broadly grouped into high-risk (HR) or low-risk (LR) types (1). Persistent infections of the cervix, penis, anus, and the oropharyngeal epithelium by HR HPV-16, HPV-18, and related types can at a low frequency progress into high grade dysplasias and cancers. Infections by LR HPV types 6 and 11 cause 90% of benign genital warts and all the laryngeal papillomas, and these infections rarely progress to cancers. For both HR and LR HPVs, infection initiates in basal epithelial keratinocytes through wounding and is established during healing, whereas the viral productive program is tightly linked to squamous differentiation (for a review, see Ref.2). Because the differentiated spinous cells would normally have withdrawn from the cell cycle and viral DNA replication is dependent on the host DNA replication machinery, the roles of the viral E6 and E7 oncoproteins are to reestablish a milieu in the differentiated keratinocytes supportive for viral DNA amplification. Briefly, E7 proteins of HR and LR HPV types promote S phase reentry in the differentiated strata (3, 4). They do so by destabilizing p130, a pRB related pocket protein, which prevents S phase reentry by the differentiated cells (5, 6). The mechanisms by which E6 enables efficient viral DNA amplification are not yet understood.The viral life cycle has recently been recapitulated with high efficiency in organotypic (raft) cultures of primary human keratinocytes (PHKs) grown at the liquid medium-air interface. In these PHKs, HPV-18 genomic plasmids were efficiently generated in vivo by Cre-loxP-mediated excision recombination from a transfer vector (7). As in patient specimens, the doublestranded HPV genomic plasmid is maintained at low copy numbers in the basal keratinocytes, and extensive amplification occurs in the mid and upper spinous strata. Moreover, viral DNA initiates amplification in G 2 -arrested cells, following host DNA replication, as revealed by the accumulation of highly elevated cytoplasmic cyclin B1 protein (7). As the viral DNA amplifies, the E7 activity progressively diminishes and eventually ceases in the upper strata; keratinocytes then exit the cell cycle, as evidenced by the gradual reappearance of p130 and disappearance of the proliferating cell nuclear antigen, a protein induced by E7. Being an E2F-responsive gene, cyclin B1 disappears when E7 activity ceases. These cells then transit to the late phase of infection and express the capsid proteins for progeny virion morphogenesis. Here, we show that E7 alone is necessary and sufficient to induce prolonged G 2 following S phase reentry in differentiated keratinocytes of raft cultures and have investigated the mechanisms that mediate this arrest.

The gene status of ALDH2*2/*2 alone can tremendously but not completely (as thought previously) protect against development of alcohol dependence. Individuals carrying the combinatorial genotype of ADH2*2/*2-ALDH2*2/*2 are at the least risk for the disease in East Asians. Physiological tolerance or innate insensitivity to the accumulation of blood acetaldehyde following alcohol ingestion may be crucial for the development of alcoholism in individuals homozygous for ALDH2*2.

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1.human papillomavirus DNA amplification | trans complementation | HPV transcripts P apillomaviruses are small DNA viruses with a protein capsid harboring a double-stranded circular genome of ∼7,900 bp. Dozens of human papillomavirus (HPV) types are tropic for the anogenital tract (1). HPV-16 and -18 and closely related genotypes are high-risk (HR) types and at a low frequency, can cause cancers, in which the viral E6 and E7 oncogenes are invariably overexpressed. HPV-6 and -11 are low-risk (LR) types and induce benign anogenital warts and laryngeal papillomas (review in ref. 2). Typically, viral DNA is maintained as low copy nuclear plasmids in basal and parabasal keratinocytes, and vegetative amplification depends on squamous differentiation (review in ref.3). Because viral DNA replication requires the host DNA replication machinery, the role of the HPV E7 protein is to promote S-phase reentry in differentiated cells that have withdrawn from the cell cycle. It does so by destabilizing the p130 protein (4, 5), a pocket protein related to the retinoblastoma susceptibility protein, a major tumor suppressor. The HR but not the LR HPV E7 protein also destabilizes retinoblastoma susceptibility protein (6). Both HR and LR HPV E6 proteins inactivate the transcription regulatory activities of another major tumor suppressor, p53 (7-10), abrogating its control over cell cycle ar...

Site-directed and random coupling of antibodies (Abs) was performed using aminated silica surfaces as substrates. The site-directed coupling linked the Ab, a monoclonal anti-fluorescein IgG1 (9-40), to the surface via 3400 Dalton (Da) poly(ethylene oxide) (PEO) spacers. The hydrazide end groups of these spacers were attached to aldehyde groups in the hinge region of the oxidized Ab to yield surfaces with an Ab concentration of 2.1 pmol/cm 2 . The random coupling, in turn, linked the Ab via its available primary amines directly to the glutaraldehyde-activated surface with a yield of 3.8 pmol/cm 2 . Despite a nearly twofold difference in Ab concentration, the two surfaces bound similar amounts of the FL-BSA antigen (0.56 vs 0.55 pmol/cm 2 ), while their nonspecific uptake of protein was 0.11 vs. 0.21 pmol/cm 2 , respectively, reflecting the protein repellent quality of PEO-coated surfaces. Fab′ fragments of the 9-40 Ab were also linked to the same tethers. Here, the succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) difunctional coupling reagent was attached to the PEO-hydrazide via its succinimide end and to the carboxy-terminal thiol of the Fab′ via its maleimide end. The concentration of reactive groups was varied by mixing difunctionalized PEO (PEO-(HZ) 2) with monofunctionalized polymer (CH3O-PEO-HZ) prior to surface attachment. At 100% PEO-(HZ)2 the FL-BSA (Ag) binding was 0.77 pmol/cm 2 while the nonspecific binding was 0.058 pmol/cm 2 . Progressive dilution of the reactive PEO chains to 25% led to the remarkable binding of 0.87 pmol/cm 2 of Ag with 0.037 pmol/cm 2 of nonspecific binding. Competitive release from these various surfaces showed more favorable kinetics for the Fab′ surface with 25% active tethers.