Human Papillomavirus Type 31b E1 and E2 Transcript Expression Correlates with Vegetative Viral Genome Amplification (original) (raw)
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Control of papillomavirus DNA replication and transcription
Seminars in Cancer Biology, 1996
Hallmarks of HPV infection include a restricted tropism for human epithelial cells and a viral life cycle tightly linked to the differentiation program of the host keratinocyte. This particular viral cycle has hampered the study of the HPV vegetative life cycle for decades, due to the lack of suitable in-vitro culture conditions. The tissue and differentiation dependence seems to be dictated by viral transcription rather than viral DNA replication. Indeed, viral transcription is restricted to epithelial cells of human origin, more specifically to keratinocytes. In contrast, HPV genomes can replicate in various undifferentiated cell lines regardless of their natural permissiveness to infection, as long as the viral replication proteins E1 and E2 are expressed.
Different Modes of Human Papillomavirus DNA Replication during Maintenance
Journal of Virology, 2006
Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed.
Journal of virology, 1997
The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture co...
Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes
Frontiers in Microbiology, 2021
The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely ...
Virology, 1999
The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts.
Journal of Biological Chemistry, 1994
W e have established the first homologous cell-free DNA replication system for a papillomavirus. The replication of the human papillomavirus type 11 (HPV-11) origin was achieved by using human 293 cell extracts supplemented with the HPV-11 E l and E2 proteins purified from insect cells infected with recombinant baculoviruses. Efficient replication depends on the HPV-11 origin, the HPV-11 E l and E2 proteins, as well as human DNA polymerase a, 6, replication protein A, topoisomerase I, and topoisomerase 11. High concentrations of E l protein also promoted a low level of origin-independent replication which was suppressed by the addition of the E2 protein, whereas E2 protein stimulated origin-dependent replication. W e also show that an intact E2 protein binding site was absolutely necessary for origin activity, as a strong HPV-11 origin was rendered inactive when one half-site of each of the three E2 binding sites was mutated. In contrast, there was only a relatively small reduction in this mutant origin activity when the cell extracts were supplemented with the bovine papillomavirus type 1 (BPV-1) proteins. These results suggest that the HPV-11 E2 protein plays a primary role in HPV origin recognition. Furthermore, unlike transient replication in which HPV-11 and BPV-1 viral proteins promote efficient replication of homologous and heterologous origins, efficient cell-free replication took place only with the homologous combinations. Small DNA viruses have long been used as models for higher eukaryotic DNA replication. SV40 is one of the most thoroughly studied DNA viruses, and the functions of both viral and host transacting factors and enzymes have been identified (Refs. 1 and 2; for reviews, see Refs. 3 and 4). The unchecked replication of these viruses, however, does not reflect the regulated host DNA replication that occurs once per cell cycle. Human papillomaviruses (HPVs),' which infect mucosal or cutaneous epithelium and cause hyperproliferation, have two distinct modes of DNA replication. In the basal stem cells and the parabasal transit amplifying cells of squamous epithelia or stratified epithelial raft cultures, viral DNA is maintained as low copy number extrachromosomal plasmids. Only in upper layer keratino-Grant CA36200. The costs of publication of this article were defrayed in
The role of the human papillomavirus type 18 E7 oncoprotein during the complete viral life cycle
Virology, 2005
The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the viral life cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the viral life cycle. In this study, we investigated the role of HPV18 E7 in the viral life cycle in order to gain a further understanding of this issue.
Biology of papillomavirus replication in infected epithelium
Future Virology, 2007
Human papillomaviruses complete their life cycle in differentiating epithelial cells that would not normally be competent for either cellular or viral DNA replication. To overcome this, papillomaviruses encode two groups of proteins that work together in the upper epithelial layers to amplify viral genomes. The E6 and E7 proteins play a critical role in driving differentiating epithelial cells that have left the basal layer, back into the cell cycle, in order to produce a replication-competent environment that can be used by the virus for genome amplification. Papillomavirus replication is heavily dependent on cellular replication proteins, but in addition needs the viral E1 and E2 proteins, which act to unwind viral DNA around the origin of replication, and to recruit essential cellular proteins to the replication site. Recent work using mutant viral genomes has suggested that two other viral proteins, E4 and E5, contribute to efficient replication in the upper epithelial layers, a...
Kinetics of HPV11 DNA replication after infection of keratinocytes with virions
Virus Research, 1996
Human papillomavirus type 11 (HPVI 1) normally infects keratinocytes of stratified epithelia and replicates as an episome. To untangle early events in the development of a papilloma, we have infected human keratinocytes with human papillomavirus type 11 virions and monitored replication using density labeling with bromodeoxyuridine and subsequent density centrifugation. We show that only a portion of the virus reaching the nucleus undergoes replication and that continued replication occurs without recruitment from the nonreplicating pool of virus suggesting that HPV11 replication remains confined to a small number of cells. Increasing inoculum size leads to more viral DNA reaching the nucleus but not a corresponding increase in viral replication. Subsequent papilloma development must, therefore, occur within a small subset of cells.
Journal of …, 1998
Organotypic cultures of human keratinocytes provide a useful model system to study human papillomavirus (HPV)-host cell interactions. In this study, we analyzed organotypic cultures of two HPV type 16 (HPV16) (FK16A and FK16B)-and two HPV18 (FK18A and FK18B)-transfected keratinocyte cell lines through the process of immortalization in vitro. For FK16A and FK18B cells, passages of both mortal cells in their extended life span and subsequent immortal stages were studied. Mortal cells of FK16A and FK18B showed a morphology reminiscent of mild to moderate dysplasia, whereas in their immortal descendants, severely dysplastic features were observed. Immortal FK18A cells were mildly to moderately dysplastic, while FK16B cells were severely dysplastic. The increasing degrees of dysplasia were associated with a decreasing expression of differentiation markers cytokeratin 10 and profilaggrin. All raft cultures expressed E6-E7 mRNAs in the basal layer, while the amount of viral transcripts in the suprabasal cells was in general proportional to the degree of dysplasia. In all cases, E6-E7 transcription and dysplastic features were highly correlated with cellular proliferation, as assessed by Ki-67 (MIB-1) antigen expression. Moreover, high levels of E6-E7 transcription and expression of p21cip1 protein in the basal layer seemed to be mutually exclusive. We conclude that expression of E6-E7 in the basal cells associated with increased proliferation in the absence of detectable p21cip1 protein is apparently necessary but not sufficient for immortalization, or for the loss of terminal differentiation, for which yet to be discovered additional events are required. The model system described in this study provides a valuable tool to analyze alterations in viral transcription regulation during HPV-mediated cell transformation.