A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions

Chow, Louise T.; Duffy, Aaron A.; Wang, Hsu-Kun; Broker, Thomas R.

doi:10.4161/cc.8.9.8242

Cited by 32 publications

(28 citation statements)

References 13 publications

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“…51) and that Wee1 plays a role (52 and references therein). Our recent data showed that viral DNA amplification was initiated in cells with elevated cytoplasmic cyclin B1, the accumulation of which, however, preceded the appearance of the E1 ∧ E4 protein by some 2 or more days (50). Furthermore, the upper spinous cells with high copies of amplified viral DNA were negative for cyclin B1 but were strongly positive for the E1 ∧ E4 protein.…”

Section: Discussionmentioning

confidence: 99%

“…E1 ∧ E4 is encoded by a spliced mRNA consisting of the first 5 amino acid codons of E1 fused to the E4 open reading frame. It is an abundant cytoplasmic protein and often colocalizes with the viral major capsid protein (49,50). A number of reports concluded that in submerged cultures of cell lines, ectopically expressed E1 ∧ E4 of several HPV types can sequester cyclin B1/Cdc2 to the cytokeratin filaments, causing G 2 arrest (for a review, see Ref.…”

Section: Discussionmentioning

confidence: 99%

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Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

Banerjee

Wang

Broker

et al. 2011

Journal of Biological Chemistry

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101

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

Banerjee

Wang

Broker

et al. 2011

Journal of Biological Chemistry

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101

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“…The replication machinery induced by the ATM/ATR pathway could also be more efficient for the virus, since break-induced repair pathways do not require origin licensing proteins to reinitiate DNA replication (34). In fact, it has been shown that papillomaviruses amplify their DNA in the G 2 phase of the cell cycle in cells that have already completed S phase (6) and that efficient amplification of the viral genome requires an activated ATM response in differentiated cells (39). The DNA damage response does not seem to interfere with HPV DNA replication, since a recent study showed that transient viral replication is not inhibited by DNA damage-inducing agents, indicating that E1 and E2 can replicate the viral genome in the presence of a DNA damage response (28).…”

Section: Discussionmentioning

confidence: 99%

“…This arrest inhibits cellular DNA replication and permits only repair of DNA damage. Use of the repair machinery to replicate viral DNA would be advantageous since it would allow the virus to synthesize its DNA without competition from that of the host (6). The replication machinery induced by the ATM/ATR pathway could also be more efficient for the virus, since break-induced repair pathways do not require origin licensing proteins to reinitiate DNA replication (34).…”

Section: Discussionmentioning

confidence: 99%

The Papillomavirus E1 Helicase Activates a Cellular DNA Damage Response in Viral Replication Foci

Sakakibara

Mitra

McBride

2011

J Virol

170

234

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The papillomavirus E1 and E2 proteins are essential for viral genome replication. E1 is a helicase that unwinds the viral origin and recruits host cellular factors to replicate the viral genome. E2 is a transcriptional regulator that helps recruit the E1 helicase to the origin and also plays a role in genome partitioning. We find that when coexpressed, the E1 and E2 proteins from several papillomavirus types localize to defined nuclear foci and result in growth suppression of the host cells. Growth suppression was due primarily to E1 protein function, and nuclear expression of E1 was accompanied by activation of a DNA damage response, resulting in phosphorylation of ATM, Chk2, and H2AX. Growth suppression and ATM activation required the ATPase and origin-specific binding functions of the E1 protein and resulted in active DNA repair, as evidenced by incorporation of nucleotide analogs and detection of free DNA ends. In the presence of the E2 protein, these activities became localized to nuclear foci. We postulate that these foci represent viral replication factories and that a cellular DNA damage response is activated to facilitate replication of viral DNA.Papillomaviruses (PVs) are double-stranded DNA viruses that infect cutaneous and mucosal epithelial cells of animals. Over 200 papillomavirus types have been reported to date, and phylogenetic classification indicates that there are at least 29 genera (1). Papillomavirus genomes consist of approximately 8 kb of double-stranded DNA, which typically contains seven to eight genes. Two of these genes encode replication proteins, E1 and E2. The E1 protein is a helicase that has been shown by structural and biochemical studies to be essential for initiation of viral DNA replication. The E1 protein by itself has low affinity for the viral origin of replication, which contains specific, palindromic E1 binding sites. However, the multifunctional E2 protein binds to specific sites adjacent to the E1 binding sites and helps recruit E1 in a cooperative manner. When loaded onto the viral replication origin, the E1 and E2 proteins recruit host replication factors such as RPA, topoisomerase I, and Pol␣/primase to initiate viral DNA replication (reviewed in reference 36). The E2 protein can function both as a transcriptional transactivator and repressor of viral early genes and for some papillomavirus types has also been shown to tether the viral genome to host chromatin to maintain and partition the extrachromosomal genomes.Eukaryotic cells have many different strategies to combat viral infection. Many viruses induce a cellular DNA damage response (DDR), either indirectly by virtue of viral DNA replicative intermediates that resemble damaged DNA or directly by viral protein function (reviewed in reference 51). The host cell induces the DNA damage response in an attempt to arrest cell growth and allow repair of genomic DNA damage, thus maintaining genomic stability. Two of the major regulators of the DNA damage response are the ATM (ataxia telangiectasia mutated) and the ATR (...

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“…Modifications to the organotypic culture conditions to induce differentiation provided the opportunity to propagate wild-type and mutant HPV genomes; thus, the ability to study papillomavirus genetics was achieved, at least for HR-HPVs (12,15,16,19,36,37), although the yield of virions from stably transfected cells has been low. Introducing adenovirus recombinants carrying the HPV genome flanked by loxP sites and Cre recombinase into PHKs grown in raft cultures also allowed the production of infectious HPV virions (31), and an improved method for efficient production and passaging of HPV-18 has been described (7,49). Also, cell lines have been established from mild dysplasia that harbored episomal HPV genomes, and these lines allow viral DNA amplification and packaging when grown in organotypic cultures (45,46).…”

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confidence: 99%

Development of a Cellular Assay System To Study the Genome Replication of High- and Low-Risk Mucosal and Cutaneous Human Papillomaviruses

Geimanen

Isok-Paas

Pipitch

et al. 2011

J Virol

113

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We found that recircularized high-risk (type 16 and 18) and low-risk mucosal (type 6b and 11) and cutaneous (type 5 and 8) human papillomavirus (HPV) genomes replicate readily when delivered into U2OS cells by electroporation. The replication efficiency is dependent on the amount of input HPV DNA and can be followed for more than 3 weeks in proliferating cell culture without selection. Cotransfection of recircularized HPV genomes with a linear G418 resistance marker plasmid has allowed subcloning of cell lines, which, in a majority of cases, carry multicopy episomal HPV DNA. Analysis of the HPV DNA status in these established cell lines showed that HPV genomes exist in these cells as stable extrachromosomal oligomers. When the cell lines were cultivated as confluent cultures, a 3-to 10-fold amplification of the HPV genomes per cell was induced. Two-dimensional (2D) agarose gel electrophoresis confirmed amplification of mono-and oligomeric HPV genomes in these confluent cell cultures. Amplification occurred as a result of the initiation of semiconservative two-dimensional replication from one active origin in the HPV oligomer. Our data suggest that the system described here might be a valuable, cost-effective, and efficient tool for use in HPV DNA replication studies, as well as for the design of cell-based assays to identify potential inhibitors of all stages of HPV genome replication.Due to their association with distinctive human cancers, human papillomaviruses (HPVs) are widely studied. Papillomaviruses have been phylogenetically grouped into genera, species, types, subtypes, and variants (11), and more than 100 HPV types have been identified thus far. The best-characterized ␣-genus HPVs are associated with infections of the mucosal epithelia that lead to the induction of benign tumors. These viruses are divided into high-risk types (e.g., high-risk HPV type 16 [HR-HPV-16] and -18), which have the capability of inducing anogenital malignancies, and low-risk types (e.g., LR-HPV-6 and -11), which induce hyperproliferative mucosal lesions and are rarely associated with malignancy. Mucosal HPV infections tend to clear on their own, but in some cases, latent infection could be established and may persist for years. Cutaneous ␤-genus HPV infections are highly prevalent in the general population and tend to persist (14). Vaccines based on virus-like particles made up of the capsid protein L1 have been developed against and Gardasil/Silgard [Merck Research Laboratories]). Because these virus types are responsible for only a portion of all HPV-induced malignant and benign tumors, a clear need exists for vaccines or antivirals against a broader spectrum of pathogenic HPV types.Despite the differences in viral pathogenesis, progeny virion production invariably depends on cell differentiation and occurs only in terminally differentiated keratinocytes. HPVs require the host's replication machinery to reproduce their genomes, and these viruses have developed a unique replication strategy that is adapted to keratinocyte di...

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A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions

Cited by 32 publications

References 13 publications

Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes

The Papillomavirus E1 Helicase Activates a Cellular DNA Damage Response in Viral Replication Foci

Development of a Cellular Assay System To Study the Genome Replication of High- and Low-Risk Mucosal and Cutaneous Human Papillomaviruses

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