Identification of
 cis
 -Acting Elements That Mediate the Replication and Maintenance of Human Papillomavirus Type 16 Genomes in
 Saccharomyces cerevisiae

Kim, Kitai; Angeletti, Peter C.; Hassebroek, Elizabeth C.; Lambert, Paul F.

doi:10.1128/jvi.79.10.5933-5942.2005

Cited by 19 publications

(24 citation statements)

References 38 publications

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“…Although the molecular mechanism of how HPV16 DNA replication is limited to once per S phase in W12 cells is not known, two hypotheses can be proposed. The first posits that HPV16 DNA (17). Indirectly relevant to this point is the report that a plasmid without any known human origin of replication replicated in a once-per-S-phase mode in CHO and HeLa cells and that ORC and MCM proteins were attached to this plasmid (26).…”

Section: Discussionmentioning

confidence: 99%

Different Modes of Human Papillomavirus DNA Replication during Maintenance

Hoffmann¹,

Hirt

Bechtold³

et al. 2006

J Virol

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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.The host tissue of human papillomaviruses is the stratified epithelium. This tissue is complex in that it is composed of layered sheets of nondividing cells in various stages of terminal differentiation, with the uppermost layer being the most differentiated. Only cells of the bottom-most layer of this tissue, the basal cells, proliferate. Although the HPV life cycle begins with the infection of a basal cell, it only comes to completion when the infected cell reaches the upper layers of the epithelium. As a consequence, the HPV DNA initially finds itself in the nucleus of a proliferating cell, but later in that of a differentiating (nonproliferating) one. This sequential mixture of cell states that the virus has to contend with has undoubtedly shaped the way by which HPV replicates its DNA throughout the varying milieux during its life cycle.It is proposed that immediately after infection, the papillomavirus DNA copy number is amplified to a certain level (50 to 400) per cell (9). This first amplification replication is believed to be rapid and transient, after which the viral DNA is stably maintained at this level in subsequent divisions of the basal cell. This is thought to be achieved by maintenance replication, where the viral episomes approximately double their copy number during the S phase of the host cell cycle and segregate to the resulting two daughter cells. After differentiation of the host cell, the viral DNA undergoes another amplification step, the second amplification replication, which increases the HPV DNA copy number to several hundreds or thousands per cell. This is followed by packaging of the viral DNA into virus particles. Although this triphasic model has not been proven in its entirety, it is supported by va...

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Section: Discussionmentioning

confidence: 99%

Different Modes of Human Papillomavirus DNA Replication during Maintenance

Hoffmann¹,

Hirt

Bechtold³

et al. 2006

J Virol

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“…These transient replication assays suggest that both viral proteins E1, a DNA helicase, and E2, a transcriptional activator and auxiliary replication factor, as well as cis-acting elements, including the E1-binding site (E1BS), several E2-binding sites (E2BS), and an AT-rich region in the long control region (LCR), are all essential for efficient PV DNA replication (4,6,20,23,30,35,37,38). These studies appear to have analyzed the molecular mechanisms of viral replication in the productive stage, though they did not necessarily examine the three stages separately.In this context, it is of interest that the HPV genome lacking LCR can replicate in the absence of both E1 and E2 proteins in transient replication assays (1,16,29), and a temperaturesensitive (TS) E1 mutant of bovine papillomavirus type 1 (BPV1) can be maintained in mouse C127 cells at a nonpermissive temperature as efficiently as the wild-type BPV1 (17). These reports suggest that E1 might be dispensable for maintenance replication.…”

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confidence: 99%

“…In this context, it is of interest that the HPV genome lacking LCR can replicate in the absence of both E1 and E2 proteins in transient replication assays (1,16,29), and a temperaturesensitive (TS) E1 mutant of bovine papillomavirus type 1 (BPV1) can be maintained in mouse C127 cells at a nonpermissive temperature as efficiently as the wild-type BPV1 (17). These reports suggest that E1 might be dispensable for maintenance replication.…”

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confidence: 99%

The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

Egawa¹,

Nakahara²,

Ohno³

et al. 2012

J Virol

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dPapillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16. P apillomaviruses (PVs) are small, double-stranded DNA viruses that infect stratified squamous epithelium. Human PVs (HPVs) are very important causative agents for various lesions, ranging from verrucas to cancer. Among them, a subset of HPVs, the so-called high-risk types such as type 16 and 18, are associated with more than 90% of all cervical carcinomas as primary etiological factors (45). PVs establish long-term persistent infections in squamous epithelium, and the viral life cycle is tightly linked with the differentiation state of the host keratinocytes (7).PV genome is replicated and amplified in three different stages: establishment, maintenance, and productive stages of the life cycle. In the establishment stage, soon after infection of the basal layer keratinocytes, a single or a few initial copies of the viral genome amplify and establish residence as multicopy circular extrachromosomal elements (episomes) in the nucleus. In the maintenance stage, the viral genomes in each affected cell replicate approximately once in a cell cycle in proliferating basal layer keratinocytes. Then, in the productive stage, they are exponentially amplified in terminally differentiating keratinocytes and packaged into progeny virions. It is important to understand the molecular mechanisms underlying this triphasic model for development of new therapies against HPV-infected lesions, such as cervical intraepithelial neoplasias, which can progress to cervical cancer.The regulation of viral DNA replication is thought to differ in these three distinct stages of the viral life cycle. Studies of lesions experimentally induced by rabbit oral papillomavirus (ROPV) infection showed that the genome copy number of ROPV is low in the basal layer and increases up to four orders of magnitude during the terminal differentiation of host keratinocytes (21). This corresponds to more than 13 rounds of continuous replication of the viral ...

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“…We also lack efficient technologies for encapsidating mutant genomes for use in infection studies, something that can be achieved with a number of other viral systems. Previous studies have established that HPVs can replicate in yeast (4)(5)(6). Further developed methodologies are described here, which allow not only replication, but also the amplification and encapsidation of target HPV genomes in yeast.…”

Section: Introductionmentioning

confidence: 99%

“…Details regarding standard yeast mating techniques can be found in the Current Protocols in Molecular Biology manual (14). 5 An alternate technique for transformation of plasmids into yeast is by means of the standard LiAc and PEG 8000 method, as described by Schiestl et al (17). An important tip here is to use yeast that are actively dividing in mid-log phase for optimal transformation efficiency.…”

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confidence: 99%

Replication and Encapsidation of Papillomaviruses in Saccharomyces cerevisiae

Angeletti

Human Papillomaviruses

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SummaryImprovements in methodologies to recapitulate and study particular biological functions of the papillomavirus life cycle have led to great advances in our knowledge of these viruses. Described in this chapter are techniques that allow low-copy and high-copy replication of full-length human papillomavirus (HPV) genomes, as well as assembly of virus-like particles, in Saccharomyces cerevisiae (yeast). This system has several distinct advantages that make it an attractive complement to the well-established raft-culturing system. First, yeast are inexpensive, rapid, and simple to culture in the lab. Second, they provide an ever-widening array of genetic tools to analyze HPV functions -most recently notable, the yeast ORF-deletion library. Third, yeast provide a potentially highefficiency means to produce large quantities of infectious virus in a short time frame. Fourth, assembly of HPV virus in yeast allows encapsidation of mutant genomes, since previous studies have shown that no viral ORF is required for replication of full-length HPV in yeast.

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Identification of cis -Acting Elements That Mediate the Replication and Maintenance of Human Papillomavirus Type 16 Genomes in Saccharomyces cerevisiae

Cited by 19 publications

References 38 publications

Different Modes of Human Papillomavirus DNA Replication during Maintenance

Different Modes of Human Papillomavirus DNA Replication during Maintenance

The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

Replication and Encapsidation of Papillomaviruses in <I>Saccharomyces cerevisiae</I>

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