The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

Toots, Märt; Männik, Andres; Kivi, Gaily; Ustav, Mart; Ustav, Ene

doi:10.1371/journal.pone.0116151

Cited by 28 publications

(70 citation statements)

References 57 publications

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“…This observation suggests the presence of a possible splicing enhancer that facilitates 929^3434 splicing. On the other hand, we failed to detect any splicing activity from pre-mRNAs 3 and 4, and 7 and 8, Other, minor promoters, P 498/520/586 and P 1193/1202/1207 , identified both in productive HPV18 infection (24) and in U2OS cells transfected with an HPV18 plasmid (25), are also shown. The promoter P 3036/3385 , identified by in vitro transcription and chloramphenicol acetyltransferase (CAT) assays in HeLa cells (87) and in U2OS cells transfected with an HPV18 plasmid (25) but not confirmed in natural HPV18 infection, is represented by a dashed arrow.…”

Section: Resultsmentioning

confidence: 68%

“…The individual ORFs are shown above the linearized HPV18 genome, along with various RNA splicing isoforms below. The transcription map originated from the Zheng laboratory (24) and has been updated to include less abundant RNA isoforms from two recent publications (25,26), with additional support from the current study. Exons (thick lines) and introns (thin lines) are illustrated for each RNA species derived from alternative promoter usage and alternative RNA splicing, with splice site positions numbered by nucleotide positions in the virus genome.…”

Section: Resultsmentioning

confidence: 99%

“…1) (24,26,27). With other, weak splice sites recently identified in HPV18-transfected U2OS cells (25), the updated HPV18 transcription map ( Fig. 1) is one of the most complex transcription maps in human papillomaviruses.…”

mentioning

confidence: 84%

“…Although the molecular mechanisms that regulate alternative RNA splicing of bovine papillomavirus type 1 (BPV-1) (4-11) and HPV16 pre-mRNA transcripts (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) have been extensively studied in the past, a full transcription map of HPV18 in productive infection, the second most prevalent high-risk HPV genotype in association with cervical cancer (23), was constructed only recently (24), and the mechanistic regulation of HPV18 RNA splicing remains poorly investigated. HPV18 pre-mRNAs are transcribed mainly from a major early promoter, P 55/102 , or a major late promoter, P 811 , although a few other, weak promoters exist in the virus genome (24,25). The HPV18 genome encodes at least five 5= splice sites and eight 3= splice sites.…”

mentioning

confidence: 99%

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Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Ajiro

Tang

Doorbar

et al. 2016

J Virol

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Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic premRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. IMPORTANCEExpression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.H igh-risk human papillomavirus (HPV) is associated with more than 95% of cervical cancer, 40 to 90% of anogenital cancer, and 10 to 60% of oropharyngeal cancer with geographic variation (1). Two major polycistronic pre-mRNAs, early and late pre-mRNAs, are transcribed from the high-risk HPV genome. Alternative RNA splicing of the HPV polycistronic pre-mRNAs plays a crucial role in regu...

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Section: Resultsmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 84%

mentioning

confidence: 99%

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Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Ajiro

Tang

Doorbar

et al. 2016

J Virol

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“…Genetic analyses have revealed that E8^E2C inhibits genome replication of HR-HPV16, -18, and -31 in undifferentiated cells, and there is also evidence that E8^E2C limits productive replication of HPV16 in differentiated cells (8)(9)(10)(11). The HPV16, -18, and -31 E8^E2C proteins bind to the viral origin of replication and are potent repressors of transcription and the E1/ E2-dependent replication (7,9,(11)(12)(13)(14). Both activities require the conserved E8 part, which recruits cellular corepressor molecules (14)(15)(16).…”

Section: Hpv16 Replicates In Differentiating Epithelia and Can Cause mentioning

confidence: 99%

Characterization of the Human Papillomavirus 16 E8 Promoter

Straub¹,

Fertey²,

Dreer³

et al. 2015

J Virol

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Persistent infections with certain human papillomaviruses (HPV) such as HPV16 are a necessary risk factor for the development of anogenital and oropharyngeal cancers. HPV16 genomes replicate as low-copy-number plasmids in the nucleus of undifferentiated keratinocytes, which requires the viral E1 and E2 replication proteins. The HPV16 E8^E2C (or E8^E2) protein limits genome replication by repressing both viral transcription and the E1/E2-dependent DNA replication. How E8^E2C expression is regulated is not understood. Previous transcript analyses indicated that the spliced E8^E2C RNA is initiated at a promoter located in the E1 region upstream of the E8 gene. Deletion and mutational analyses of the E8 promoter region identify two conserved elements that are required for basal promoter activity in HPV-negative keratinocytes. In contrast, the transcriptional enhancer in the upstream regulatory region of HPV16 does not modulate basal E8 promoter activity. Cotransfection studies indicate that E8^E2C inhibits, whereas E2 weakly activates, the E8 promoter. Interestingly, the cotransfection of E1 and E2 induces the E8 promoter much more strongly than the major early promoter, and this is partially dependent upon binding of E2 to Brd4. Mutation of E8 promoter elements in the context of HPV16 genomes results in an increased genome copy number and elevated levels of viral early and late transcripts. In summary, the promoter responsible for the expression of E8^E2C is both positively and negatively regulated by viral and cellular factors, and this regulatory circuit may be crucial to maintain a low but constant copy number of HPV16 genomes in undifferentiated cells. IMPORTANCEHPV16 replicates in differentiating epithelia and can cause cancer. How HPV16 maintains its genome in undifferentiated cells at a low but constant level is not well understood but may be relevant for the immunological escape of HPV16 in the basal layers of the infected epithelium. This study demonstrates that the expression of the viral E8^E2C protein, which is a potent inhibitor of viral replication in undifferentiated cells, is driven by a separate promoter. The E8 promoter is both positively and negatively regulated by viral proteins and thus most likely acts as a sensor and modulator of viral copy number.

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Analysis of Human Papillomavirus Genome Replication Using Two‐ and Three‐Dimensional Agarose Gel Electrophoresis

et al. 2017

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This unit includes the necessary information to conduct neutral/neutral and neutral/alkaline two-dimensional and neutral/neutral/alkaline three-dimensional agarose gel electrophoresis. The methodology has been optimized over the years to gain a better outcome from the hard-to-interpret signals of human papilloma virus replication intermediates obtained from two- and three-dimensional agarose gels. Examples of typical results and interpretation of replication intermediate patterns are included, and the outcomes of multiple-dimension assays are assessed using previously published experimental data. © 2017 by John Wiley & Sons, Inc.

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The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

Cited by 28 publications

References 57 publications

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements

Characterization of the Human Papillomavirus 16 E8 Promoter

Analysis of Human Papillomavirus Genome Replication Using Two‐ and Three‐Dimensional Agarose Gel Electrophoresis

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