Termination and reinitiation signals of bacteriophage phi X174 rolling circle DNA replication

The nucleotide sequence requirements for termination and reinitiation of rolling circle DNA replication within the 30-bp phi X174 origin region were studied. Plasmids were constructed which contained a complete and a partial phi X174 origin region in the same orientation. The partial origin consisted of the first 16, 24, 25, 26, 27, or 28 bp of the origin region. Plasmids harboring a complete origin region are subject to rolling circle DNA replication and packaging of single-stranded plasmid DNA into phage coats in phi X174 or G4 phage infected cells. The plasmids with a complete and partial origin region were tested in these in vivo transduction systems. The results lead to the following conclusions: The phi X174 and G4 in vivo transduction systems are useful in studying termination and reinitiation of rolling circle DNA replication. The first 24 bp of the origin region are sufficient for termination of a round of rolling circle DNA replication coupled to DNA packaging. The first 16 bp, however, are not recognized as a termination signal. Reinitiation of rolling circle DNA replication coupled to DNA packaging on a partial origin region occurs with low frequency.     

Human papillomavirus-16 and -18 replication in esophagus squamous cancer cell lines does not require sheterologous E1 and E2 proteins

Human papillomaviruses (HPVs) are doublestranded DNA viruses that replicate in the nuclei of squamous epithelial cells. HPVs can be classified into high-risk (e.g., types 16, 18, 31, and 33) or low-risk (e.g., types 6, 11, and 30), depending on their association with benign or malignant tumors. We recently described the association of HPV-16 and -18 with esophagus squamous cell cancer. HPV replication was studied in representative cell lines derived from esophagus cancers. HPV-16 and -18 genomes were independently transiently transfected into HCE-4 and HCE-7 cell lines with and without E1 and E2 genes under heterologous promoters. Southern blot analysis demonstrated that these cell lines support viral replication. However, heterologous E1 and E2 are not required for HPV replication. These findings suggest that specific host nuclear factors in esophageal squamous epithelial cells may support HPV replication. © 1995 Wiley-Liss, Inc.     

Multiple human papillomavirus genes affect the adeno-associated virus life cycle

The risk of cervical cancer, one of the most prevalent cancers in the world, is determined by two viruses. Human papillomavirus (HPV) is the main risk factor for developing cervical cancer. However, although little known, it is well substantiated that the human Parvovirus adeno-associated virus type 2 (AAV), and its encoded Rep78 protein, interacts with HPV and lowers the risk of cervical cancer. HPV also contributes to AAV inhibition by serving as a helper virus for AAV and stimulating higher AAV replication levels. Here we surveyed four HPV-16 early genes, E1, E2, E6 and E7, for their ability to increase/decrease the basal level of AAV replication in stratifying squamous epithelium (the epithelial raft culture system). It was found that the HPV-16 E1, E2 and E6 genes were able to help/enhance AAV-2 replication in epithelial raft cultures. Under these conditions, with all the HPV genes being expressed from the AAV p5 promoter, E1 appeared to have the strongest enhancing effect on AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of E1 mutants showed that the carboxy-half of E1, the putative helicase/ATPase domain, was the main contributor of helper activity. These data are important for understanding the HPV-AAV interaction and its effect on modifying cervical cancer risk. These data also suggest the possibility that the identified HPV helper genes may be useful in the generation of recombinant (r)AAV virions for gene therapy, as rAAV is increasing in popularity for such purposes.     

The full-length E1E4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1E4 protein. To determine the role of E1E4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1E4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1E4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1E4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1E4 expression, and to HPV16 where only C-terminal truncations in E1E4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1E4 proteins.     

Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication

Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.     

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.     

Human papillomavirus DNA is present in a subset of unselected breast cancers

OBJECTIVE: The major molecular events in the genesis of most breast cancers are unknown. However, human papillomaviruses (HPV) have been reported to be found in a significant portion of breast cancers of women with concomitant cervical intraepithelial neoplasia III. To investigate a potential HPV-breast cancer link, we carried out a small survey to identify HPV in unselected, general breast cancer tissues. STUDY DESIGN/METHODS: Deoxyribonucleic acid (DNA) was isolated from 17 breast cancer tissues (and one cervical swab) taken from our local, randomly selected patient population. Two different previously characterized broad-spectrum primer sets (targeting the E6/E7 or L1 regions) were used to amplify HPV DNA, and another primer set was used to amplify the ColE1/pBR322 origin of replication by polymerase chain reaction amplification. The polymerase chain reaction product DNA was analyzed by dot blot hybridization with HPV-16, -18, -31, or pRB322 DNA probes. Total cellular DNA was also analyzed by one- and two-dimensional Southern blot analysis. Finally, the E6/E7 polymerase chain reaction products were cloned, sequenced, and compared to previously cloned HPV types. RESULTS: Polymerase chain reaction/dot blot analysis by both the HPV E6-E7 and L1 primer sets identified the same 6 out of 17 (35%) breast cancers as being HPV positive. ColE1/pBR322 origin targeted polymerase chain reaction/dot blot analysis failed to identify plasmid contamination. One- and two-dimensional Southern blot analysis showed that the breast cancers specimens contained significant levels of HPV DNA and that the viral DNA was largely episomal. The sequences of the HPV clones demonstrated that HPV-16, -18, and possibly type 11 were present within the breast cancer specimens. Furthermore, the HPV sequences cloned from the cervical swab and breast cancer of the same patient were found to be identical. CONCLUSIONS: These data suggest that HPV may be associated with a significant subset of breast cancers, and further suggest that additional studies are warranted.     

Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.     

The history of the HSV amplicon: from naturally occurring defective genomes to engineered amplicon vectors

We have derived the HSV amplicon vector in 1981/1982 after elaborate experience with "defective viruses", arising spontaneously in viral stocks propagated at high multiplicities of infection (m.o.i.). The defective viruses were found to contain large concatemeric genomes with repeat units of limited complexity. We employed cloned defective genome repeats to generate the "amplicon" vectors, which in the presence of helper virus replicate to produce packaged large concatemeric genomes, transmissible to uninfected cells. The cloned amplicons were then employed to fine map and analyze the signals essential for amplicon propagation: (i) A DNA replication origin, producing concatemeric genomes by rolling circle replication. Three DNA replication origins were identified in the HSV genome. (ii) Signals termed pac-1 and pac-2, directing a measuring function for coordinate cleavage of the concatemeric genomes and their packaging as full-size (150 kb) genomes. Using amplicons, foreign genes of large sizes could be linked to less than 1 kb of the cis-acting HSV DNA sequences and become amplified in packaged defective genomes, transmissible to new cells. The transgenes are expressed efficiently, due to sequence reiterations. Large quantities of vectors can be produced in vitro. The amplicons are attractive vectors for use as non-integrating gene delivery vectors. The packaging signals pac-1 and pac-2 are well conserved in different herpesviruses and amplicons with a DNA replication origin and cleavage and packaging signals have been produced in additional herpesviruses. Depending on amplicon-host cell combination, the vectors can be employed with and without mutated helper virus(es) to obtain high gene expression, and desired effect on the target cell. In the absence of helper virus, the defective virus produced is limited for spread in the targeted cells. We expect that new vectors employing state of the art transgenes, will be developed to generate amplicon based concatemeric defective viruses capable of efficient expression of these genes.     

Identification of domains of the HPV11 E1 protein required for DNA replication in vitro

The HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase alpha primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol alpha primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol alpha primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol alpha primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol alpha primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353-649) that binds to pol alpha primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol alpha primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol alpha primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol alpha primase.     

Papillomavirus E1 Proteins: Form,Function, and Features

The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.