Analysis of novel human papillomavirus type 16 late mRNAs in differentiated W12 cervical epithelial cells

Milligan, Steven G.; Veerapraditsin, Thanaporn; Ahamet, Boolang; Mole, Sarah; Graham, Sheila V.

doi:10.1016/j.virol.2006.10.012

Cited by 68 publications

(106 citation statements)

References 47 publications

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“…As the only difference between these two mRNAs was that L1i mRNAs lack the 275 nt exon between SA3358 and SD3632, both L1 mRNAs should be translated efficiently into L1 protein. In addition, both L1 and L1i mRNAs have been observed in HPV-16-infected cells (Baker & Calef, 1997;Milligan et al, 2007). The role of SRp30c during HPV-16 infection may therefore be to regulate HPV-16 late mRNA splicing and to determine the ratio between L2 and L1/L1i mRNAs.…”

Section: Discussionmentioning

confidence: 99%

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

Somberg

Johansson

et al. 2011

Journal of General Virology

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Two splice sites on the human papillomavirus type 16 (HPV-16) genome are used exclusively by the late capsid protein L1 mRNAs: SD3632 and SA5639. These splice sites are suppressed in mitotic cells. This study showed that serine/arginine-rich protein 30c (SRp30c), also named SFRS9, activated both SD3632 and SA5639 and induced production of L1 mRNA. Activation of HPV-16 L1 mRNA splicing by SRp30c required an intact arginine/serine-repeat (RS) domain of SRp30c. In addition to this effect, SRp30c could enhance L1 mRNA production indirectly by inhibiting the early 39-splice site SA3358, which competed with the late 39-splice site SA5639. SRp30c bound directly to sequences downstream of SA3358, suggesting that SRp30c inhibited the enhancer at SA3358 and caused a redirection of splicing to the late 39-splice site SA5639. This inhibitory effect of SRp30c was independent of its RS domain. These results suggest that SRp30c can activate HPV-16 L1 mRNA expression via a bimodal mechanism: directly by stimulating splicing to late splice sites and indirectly by inhibiting competing early splice sites.

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Section: Discussionmentioning

confidence: 99%

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

Somberg

Johansson

et al. 2011

Journal of General Virology

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“…W12E and W12G cells were grown (2 ϫ 10 5 cells per 10-cm plate) on mitomycin C-treated J2 3T3 mouse fibroblasts (35). Differentiation was achieved as previously described (10,35). 3T3 cells were grown in DMEM and 10% donor calf serum with antibiotics as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The putative E6* proteins all share the first 44 amino acids of full-length E6 with C-terminal truncations or frame shifts into the E7 open reading frame (5). E6*I is the most abundant isoform in cervical cell lines (7)(8)(9)(10) and patient samples (11,12) and has been suggested to encode E7 (6). Although detectable in tumor samples (12), the biological function of E6*II and E6*X has not been investigated.…”

mentioning

confidence: 99%

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

McFarlane

MacDonald

Stevenson

et al. 2015

J Virol

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High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P 97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance. H uman papillomaviruses (HPV) infect mucosal and cutaneous epithelia. At least 13 so-called "high-risk" HPV (HR-HPV) infect the anogenital epithelium and can cause persistent lesions that may progress to cancer (1). For example, around 500,000 women worldwide experience anogenital HPV infection, and nearly 300,000 die per annum from cervical cancer. Increasingly, HPV infection is also being linked to oropharyngeal cancer, whereby incidence of this disease is increasing rapidly (2). HPV16 is the most prevalent HR-HPV. HPV-associated tumorigenesis is driven by increased expression of the HPV E6 and E7 oncoproteins (3). E6 promotes ubiquitin-mediated degradation of p53 to inhibit apoptosis, modulates transcription of cell cycle-related genes, induces telomerase activity, controls cell shape and polarity, and activates cap-dependent translation (4). E7 binds and degrades Rb to promote S phase entry and cell division, controls transcription of cell cycle-related genes, and acts as a mitotic mutator (4). IMPORTANCE Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that theHPV E6 and E7 oncoproteins are expressed from a polycistronic transcript that for HPV16 can potentially produce four different alternatively spliced mRNAs (E6 full length [E6fl], E6*I, E6*II, and E6*X [also called E6*III]) (5, 6). The putative E6* proteins all share the first 44 amino acids of full-length E6 with C-terminal trun...

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“…The major promoters in HPV16 are the early promoter (p97), located in the locus control region (LCR), and the differentiation-inducible promoter (p670), with additional promoters in the LCR and within E4 and E5, potentially generating late transcripts. HPV transcription is polycistronic, undergoing differential splicing to produce numerous viral mRNAs (at least 13 transcripts from 8 HPV16 genes in W12E cells [11]). Previous analyses considered only coding RNA, and it is possible that new transcription start sites (TSSs) are obscured by prominent promoters.…”

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confidence: 99%

Characterization of Novel Transcripts of Human Papillomavirus Type 16 Using Cap Analysis Gene Expression Technology

Taguchi¹,

Nagasaka²,

Kawana³

et al. 2015

J Virol

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We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression. Cervical cancer is the third most common female cancer worldwide, with 530,000 new cases and 275,000 deaths annually (1), and infection by one of a subset of high-risk human papillomaviruses (HPVs) is a prerequisite for its development (2).A number of elegant studies on the HPV type 16 (HPV16) transcriptome (3-10) have shown that transcription in all HPVs occurs unidirectionally from at least two promoters and involves multiple splice sites. The major promoters in HPV16 are the early promoter (p97), located in the locus control region (LCR), and the differentiation-inducible promoter (p670), with additional promoters in the LCR and within E4 and E5, potentially generating late transcripts. HPV transcription is polycistronic, undergoing differential splicing to produce numerous viral mRNAs (at least 13 transcripts from 8 HPV16 genes in W12E cells [11]). Previous analyses considered only coding RNA, and it is possible that new transcription start sites (TSSs) are obscured by prominent promoters. Therefore, we have used cap analysis gene expression (CAGE) for high-throughput transcriptome analysis (12-16) CAGE specifically determines transcriptional start sites (TSS) genome wide, by capping 5= ends of RNA transcripts, reverse transcribing them, and mapping cDNAs to the reference genome to identify .To assess all potential TSSs in HPV16-infected cell lines, we performed CAGE analysis using cervical carcinoma-derived CaSki and SiHa cells (21,22), cervical intraepithelial neoplasia grade 1 (CIN1)-derived W12E (20863) cells, containing episomal copies of HPV16 (3, 23) (3T3 feeder cells removed by trypsinization), and a biopsy specimen from a CIN1 cervical lesion (approved by the Institutional Review Board, University of Tokyo Hospital). From each sample, 5 g RNA was extracted using a miRNeasy kit (Qiagen). RNA quality was assessed by the use of a Bioanalyzer (Agilent) and standardized at an RNA integrity number (RIN) of Ͼ7.0. Quantitation by NanoDrop analysis confirmed that the A 260 /A 290 and A 260 /A 230 ratios were Ͼ1.7.First-strand cDNAs were transcribed to the 5= end of capped RNAs and attached to CAGE "bar code" tags (Table 1), and the sequenced CAGE tags were mapped to the HPV16 genome and the human hg19 genomes using BWA software (v0.5.9), discarding ribosomal or non-A/C/G/T base-containing RNAs (24). CAGE tags were normalized by the total number of tags per sample mapped to the human genome and are indicated as tags per m...

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Analysis of novel human papillomavirus type 16 late mRNAs in differentiated W12 cervical epithelial cells

Cited by 68 publications

References 47 publications

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

Serine/arginine-rich protein 30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

Characterization of Novel Transcripts of Human Papillomavirus Type 16 Using Cap Analysis Gene Expression Technology

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