Human Papillomavirus Transcription

Chowand, Louise T.; Broker, Thomas R.

doi:10.1007/978-0-387-36523-7_7

Cited by 9 publications

(11 citation statements)

References 192 publications

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“…The relatively low abundance of this RNA is in accordance with L2 being a minor capsid protein. In this regard, we hypothesize that the more abundant singly spliced transcript (nucleotides 929^3434), which spans E1^E4, E5, and the entire L region (29,30), is not a message for L2, because L2 would be the third ORF, an unfavorable position for protein translation. We suggest that this long transcript is the splicing intermediate for the twice-spliced (nucleotides 929^3434 to 3696^5613) mature bicistronic mRNA encoding E1E4 and L1 (Table 1, species g).…”

Section: Discussionmentioning

confidence: 99%

“…Using E1F1/E4R or E1F1/ E5R primer set, a major band and a minor band (Fig. 5 B and C, bands 1 and 2) were detected; they correspond to the E1^E4, E5 transcript with a splice at nucleotides 929^3434 (Table 1, species a) and the E5 transcript with the splice at nucleotides 929^3465 (Table 1, species b), respectively (29,30). A product with the splice nucleotides 929^3506 was not observed.…”

Section: Phenotypes Of the E6 Null Mutant And Its Complementation In mentioning

confidence: 94%

“…HPV mRNA splicing patterns are conserved among different HPVs (review in ref. 29). To investigate the reasons for the scarce late protein expression by E6 mutants, we compared viral RNA levels by using semi quantitative RT-PCR followed by agarose gel electrophoresis (Fig.…”

Section: Phenotypes Of the E6 Null Mutant And Its Complementation In mentioning

confidence: 99%

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

Kho

Wang

Banerjee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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.human papillomavirus DNA amplification | trans complementation | HPV transcripts P apillomaviruses are small DNA viruses with a protein capsid harboring a double-stranded circular genome of ∼7,900 bp. Dozens of human papillomavirus (HPV) types are tropic for the anogenital tract (1). HPV-16 and -18 and closely related genotypes are high-risk (HR) types and at a low frequency, can cause cancers, in which the viral E6 and E7 oncogenes are invariably overexpressed. HPV-6 and -11 are low-risk (LR) types and induce benign anogenital warts and laryngeal papillomas (review in ref. 2). Typically, viral DNA is maintained as low copy nuclear plasmids in basal and parabasal keratinocytes, and vegetative amplification depends on squamous differentiation (review in ref.3). Because viral DNA replication requires the host DNA replication machinery, the role of the HPV E7 protein is to promote S-phase reentry in differentiated cells that have withdrawn from the cell cycle. It does so by destabilizing the p130 protein (4, 5), a pocket protein related to the retinoblastoma susceptibility protein, a major tumor suppressor. The HR but not the LR HPV E7 protein also destabilizes retinoblastoma susceptibility protein (6). Both HR and LR HPV E6 proteins inactivate the transcription regulatory activities of another major tumor suppressor, p53 (7-10), abrogating its control over cell cycle ar...

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Section: Discussionmentioning

confidence: 99%

Section: Phenotypes Of the E6 Null Mutant And Its Complementation In mentioning

confidence: 94%

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

Kho

Wang

Banerjee

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Secondly, none of the HPV genotypes can be propagated in conventional cell cultures because their RNA transcription and DNA amplification take place only in differentiated strata of squamous epithelia (reviewed in ref. 3). Rather, viral genomes are recovered from patient specimens as cloned recombinant DNAs.…”

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confidence: 99%

“…Indeed, a dedicated mRNA encodes E1^E4 at early times while a bicistronic mRNA encodes both E1^E4 and L1 at late times. 3 The delayed expression of L1 relative to E1^E4 would suggest that high E1^E4 protein could not have caused cell death. In agreement, we did not detect apoptotic cells based on tissue histology, nor did we detect cleaved caspase 3 in these raft cultures.…”

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confidence: 99%

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

Chow¹,

Duffy²,

Wang³

et al. 2009

Cell Cycle

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

Wang¹,

Duffy²,

Broker³

et al. 2009

Genes Dev.

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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.[Keywords: Human papillomavirus infection program; organotypic cultures of primary human keratinocytes; Cre-loxP-mediated recombination in vivo; cell cycle regulation; immortalization-and replication-defective E6 mutant; p53 protein stabilization] Supplemental material is available at http://www.genesdev.org.

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Human Papillomavirus Transcription

Cited by 9 publications

References 192 publications

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

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

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

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

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