IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors.

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

The male germline-specific protein MAPS is indispensable for pachynema progression and fertility.

Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail 1700102P08Rik, one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking Maps (Maps-/- ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal Maps-/- spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult Maps-/- spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult Maps-/- pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in Maps-/- pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.

Combined mTORC1/mTORC2 inhibition blocks growth and induces catastrophic macropinocytosis in cancer cells.

The mammalian target of rapamycin (mTOR) pathway, which plays a critical role in regulating cellular growth and metabolism, is aberrantly regulated in the pathogenesis of a variety of neoplasms. Here we demonstrate that dual mTORC1/mTORC2 inhibitors OSI-027 and PP242 cause catastrophic macropinocytosis in rhabdomyosarcoma (RMS) cells and cancers of the skin, breast, lung, and cervix, whereas the effects are much less pronounced in immortalized human keratinocytes. Using RMS as a model, we characterize in detail the mechanism of macropinocytosis induction. Macropinosomes are distinct from endocytic vesicles and autophagosomes in that they are single-membrane bound vacuoles formed by projection, ruffling, and contraction of plasma membranes. They are positive for EEA-1 and LAMP-1 and contain watery fluid but not organelles. The vacuoles then merge and rupture, killing the cells. We confirmed the inhibition of mTORC1/mTORC2 as the underpinning mechanism for macropinocytosis. Exposure to rapamycin, an mTORC1 inhibitor, or mTORC2 knockdown alone had little or reduced effect relative to the combination. We further demonstrate that macropinocytosis depends on MKK4 activated by elevated reactive oxygen species. In a murine xenograft model, OSI-027 reduced RMS tumor growth. Molecular characterization of the residual tumors was consistent with the induction of macropinocytosis. Furthermore, relative to the control xenograft tumors, the residual tumors manifested reduced expression of cell proliferation markers and proteins that drive the epithelial mesenchymal transition. These data indicate a role of mTORC2 in regulating tumor growth by macropinocytosis and suggest that dual inhibitors could help block refractory or recurrent RMS and perhaps other neoplasms and other cancer as well.

Vorinostat, a pan-HDAC inhibitor, abrogates productive HPV-18 DNA amplification.

Human papillomaviruses (HPVs) cause epithelial proliferative diseases. Persistent infection of the mucosal epithelia by the high-risk genotypes can progress to high-grade dysplasia and cancers. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and histone deacetylases (HDACs) that remodel chromatin and regulate gene expression. HDACs are also essential to remodel and repair replicating chromatin to enable the progression of replication forks. As such, Vorinostat (suberoylanilide hydroximic acid), and other pan-HDAC inhibitors, are used to treat lymphomas. Here, we investigated the effects of Vorinostat on productive infection of the high-risk HPV-18 in organotypic cultures of primary human keratinocytes. HPV DNA amplifies in the postmitotic, differentiated cells of squamous epithelia, in which the viral oncoproteins E7 and E6 establish a permissive milieu by destabilizing major tumor suppressors, the pRB family proteins and p53, respectively. We showed that Vorinostat significantly reduced these E6 and E7 activities, abrogated viral DNA amplification, and inhibited host DNA replication. The E7-induced DNA damage response, which is critical for both events, was also compromised. Consequently, Vorinostat exposure led to DNA damage and triggered apoptosis in HPV-infected, differentiated cells, whereas uninfected tissues were spared. Apoptosis was attributed to highly elevated proapoptotic Bim isoforms that are known to be repressed by EZH2 in a repressor complex containing HDACs. Two other HDAC inhibitors, Belinostat and Panobinostat, also inhibited viral DNA amplification and cause apoptosis. We suggest that HDAC inhibitors are promising therapeutic agents to treat benign HPV infections, abrogate progeny virus production, and hence interrupt transmission.

A survey of sub-inhibitory concentrations of antibiotics in the environment.

Antibiotics are poorly metabolized, and can enter the environment via human waste streams, agricultural run-off and pharmaceutical effluent. We consequently expect to see a concentration gradient of antibiotic compounds radiating from areas of human population. Such antibiotics should be thought of as pollutants, as they can accumulate, and have biological effects. These antibiotic pollutants can increase rates of mutation and lateral transfer events, and continue to exert selection pressure even at sub-inhibitory concentrations. Here, we conducted a literature survey on environmental concentrations of antibiotics. We collated 887 data points from 40 peer-reviewed papers. We then determined whether these concentrations were biologically relevant by comparing them to their minimum selective concentrations, usually defined as between 1/4 and 1/230 of the minimum inhibitory concentration. Environmental concentrations of antibiotics surveyed often fall into this range. In general, the antibiotic concentrations recorded in aquatic and sediment samples were similar. These findings indicate that environmental concentrations of antibiotics are likely to be influencing microbial ecology, and to be driving the selection of antibiotic resistant bacteria.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins.

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10-14-10-17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

Targeting DNA Damage Response as a Strategy to Treat HPV Infections.

Mucosotropic human papillomaviruses (HPVs) cause prevalent anogenital infections, some of which can progress to cancers. It is imperative to identify efficacious drug candidates, as there are few therapeutic options. We have recapitulated a robust productive program of HPV-18 in organotypic raft cultures of primary human keratinocytes. The HPV E7 protein induces S phase reentry, along with DNA damage response (DDR) in differentiated cells to support viral DNA amplification. A number of small molecule inhibitors of DDR regulators are in clinical use or clinical trials to treat cancers. Here, we used our raft culture system to examine effects of inhibitors of ATR/Chk1 and ATM/Chk2 on HPV infection. The inhibitors impaired S-phase reentry and progression as well as HPV DNA amplification. The Chk1 inhibitor MK-8776 was most effective, reducing viral DNA amplification by 90-99% and caused DNA damage and apoptosis, preferentially in HPV infected cells. We found that this sensitivity was imparted by the E7 protein and report that MK-8776 also caused extensive cell death of cervical cancer cell lines. Furthermore, it sensitized the cells to cisplatin, commonly used to treat advanced cervical cancer. Based on these observations, the Chk1 inhibitors could be potential effective agents to be re-purposed to treat the spectrum of HPV infections in single or combination therapy.

Destabilization of TIP60 by human papillomavirus E6 results in attenuation of TIP60-dependent transcriptional regulation and apoptotic pathway.

The TIP60 tumor suppressor is a histone acetyltransferase involved in transcriptional regulation, checkpoint activation, and p53-directed proapoptotic pathways. We report that human papillomavirus (HPV) E6 destabilizes TIP60 both in vivo and in vitro. TIP60 binds to the HPV major early promoter and acetylates histone H4 to recruit Brd4, a cellular repressor of HPV E6 expression. Both low- and high-risk HPV E6 destabilize TIP60, thereby derepressing their own promoter. Destabilization of TIP60 by HPV E6 also relieves cellular promoters from TIP60-initiated repression and abrogates p53-dependent activation of apoptotic pathway. Degradation of TIP60, therefore, allows low- and high-risk HPV to promote cell proliferation and cell survival.

microRNAs are biomarkers of oncogenic human papillomavirus infections.

Cellular and viral microRNAs (miRNAs) are the transcriptional products of RNA polymerase II and are regulated by transcriptional factors for their differential expression. The altered expression of miRNAs in many cancer types has been explored as a marker for possible diagnosis and therapy. We report in this study that oncogenic human papillomaviruses (HPVs) induce aberrant expression of many cellular miRNAs and that HPV18 infection produces no detectable viral miRNA. Thirteen abundant host miRNAs were specifically regulated by HPV16 and HPV18 in organotypic raft cultures of foreskin and vaginal keratinocytes as determined by miRNA array in combination with small RNA sequencing. The increase of miR-16, miR-25, miR-92a, and miR-378 and the decrease of miR-22, miR-27a, miR-29a, and miR-100 could be attributed to viral oncoprotein E6, E7, or both, all of which are known to target many cellular transcription factors. The examination of 158 cervical specimens, including 38 normal, 52 cervical intraepithelial neoplasia (CIN), and 68 cervical cancer (CC) tissues, for the expression of these eight miRNAs showed a remarkable increase of miR-25, miR-92a, and miR-378 with lesion progression but no obvious change of miR-22, miR-29a, and miR-100 among the HPV-infected tissues. Further analyses indicate that an expression ratio ≥1.5 of miR-25/92a group over miR-22/29a group could serve as a cutoff value to distinguish normal cervix from CIN and from CIN to CC.

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.

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.

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures.

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.

Conditioned Media From Adipose-Derived Stromal Cells Accelerates Healing in 3-Dimensional Skin Cultures.

Wound healing involves a number of factors that results in the production of a "closed" wound. Studies have shown, in animal models, acceleration of wound healing with the addition of adipose-derived stromal cells (ADSC). The cause for the positive effect which these cells have on wound healing has not been elucidated. We have previously shown that addition of ADSC to the dermal equivalent in 3-dimensional skin cultures accelerates reepithelialization. We now demonstrate that conditioned media (CM) from cultured ADSC produced a similar rate of healing. This result suggests that a feedback from the 3-dimensional epithelial cultures to ADSC was not necessary to effect the accelerated reepithelialization. Mass spectrometry of CM from ADSC and primary human fibroblasts revealed differences in secretomes, some of which might have roles in the accelerating wound healing. Thus, the use of CM has provided some preliminary information on a possible mode of action.

Phylogenetic considerations in designing a broadly protective multimeric L2 vaccine.

While the oncogenic human papillomavirus (HPV) types with the greatest medical impact are clustered within the α9 and α7 species, a significant fraction of cervical cancers are caused by α5, α6, and α11 viruses. Benign genital warts are caused principally by the α10 viruses HPV6 and HPV11. In an effort to achieve broad protection against both cervical cancer- and genital wart-associated types, we produced at high levels in bacteria a multimeric protein (α11-88x8) fusing eight polypeptides corresponding to a protective domain comprising L2 residues ∼11 to 88 derived from HPV6 (α10), HPV16 (α9), HPV18 (α7), HPV31 (α9), HPV39 (α7), HPV51 (α5), HPV56 (α6), and HPV73 (α11) and a truncated derivative with the last three units deleted (α11-88x5). Mice were immunized three times with α11-88x8 or α11-88x5 adjuvanted with alum or the licensed HPV vaccines and challenged intravaginally with HPV6, HPV16, HPV26, HPV31, HPV33, HPV35, HPV45, HPV51, HPV56, HPV58, or HPV59 pseudovirions. The α11-88x5 and α11-88x8 vaccines induced similarly robust protection against each HPV type tested and indistinguishable HPV16-neutralizing antibody titers. Passive transfer of α11-88x8 antisera was protective. Further, rabbit antisera to α11-88x8 and α11-88x5 similarly neutralized native HPV18 virions. These findings suggest that immunologic competition between units is not a significant issue and that it is not necessary to include a unit of L2 derived from each species to achieve broader protection against diverse medically significant HPV types than is achieved with the licensed HPV vaccines.

The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo.

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

Human papillomavirus (HPV) E7 induces prolonged G2 following S phase reentry in differentiated human keratinocytes.

The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G(2) phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181-194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862-4873), also elicited extensive G(2) responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G(2)/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr(14) or Tyr(15), and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser(216), unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser(1981)) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G(2) phase in the differentiated cells following S phase induction.

Nonconserved lysine residues attenuate the biological function of the low-risk human papillomavirus E7 protein.

The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures.

Construction of a full transcription map of human papillomavirus type 18 during productive viral infection.

Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ∼15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5' rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3' RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3' ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5' RACE and primer walking techniques. Five 5' splice sites (donor sites) and six 3' splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.