Splicing of a Cap-proximal Human Papillomavirus 16 E6E7 Intron Promotes E7 Expression, but can be Restrained by Distance of the Intron from its RNA 5′ Cap

Zheng, Zhi-Ming; Tao, Mingfang; Yamanegi, Koji; Bodaghi, Sohrab; Xiao, Wei

doi:10.1016/j.jmb.2004.02.023

Cited by 59 publications

(88 citation statements)

References 68 publications

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“…All plasmids used in this study were previously described, as follows: pVM7 (FLAG-tagged, full-length ORF57) (35); pVM24 (FLAG-ORF57 aa 1 to 251) and its mutant derivatives pVM45 (mNLS1), pVM46 (mNLS2), pVM47 (mNLS3), pVM48 (mNLS1 ϩ 2), pVM49 (mNLS1 ϩ 3), pVM50 (mNLS2 ϩ 3), pKY15 (FLAG-ICP27), and pGS113 (myc-EBV EB2) (34); pST3 (K8␤ cDNA) and pKY3 (K8␤ cDNA with optimized intron 5Ј and 3Ј splice sites) (68); and pZMZ70 (green fluorescent protein [GFP]-human papillomavirus type 16 [HPV16] E6) and pTMF11 (GFP-human ␤-globin) (73).…”

Section: /Ebvmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Majerčiak

Yamanegi

Allemand

et al. 2008

J Virol

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122

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Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8␤ cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8␤ and production of K8␣ (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8␤ pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is a human gammaherpesvirus that is closely related to Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) (7,41,45). KSHV infection is associated with all forms of Kaposi's sarcoma, primary effusion lymphoma or body cavity-based B-cell lymphoma, and multicentric Castleman disease (20,53,55,62). Latent KSHV infection in Kaposi's sarcoma tissues and B-cell lines features the restricted expression of only five viral genes (13, 74). The lytic KSHV infection produces progeny virus from infected cells and can be induced by chemicals such as tetradecanoyl phorbol acetate (43), butyrate (39), or valproic acid (25) or by hypoxia (12) in primary effusion lymphoma-derived B cells with latent KSHV infection. Chemical induction in latently infected cells initiates the expression of a viral transactivator, ORF50, which is essential for the switch from KSHV latency to the lytic phase (32, 57).KSHV ORF57 (mRNA transcript accumulation [MTA]), which is transactivated by ORF50 (31, 63), encodes a viral early nuclear protein of 455 amino acid (aa) residues (15, 24) that is homologous to herpes simplex virus (HSV) ICP27 (IE63), EBV EB2 (SM), and HVS ORF57. KSHV ORF57 promotes the expression of ORF56 and ORF59 genes at the posttranscriptional level (24,34,35), but how ORF57 functions is poorly understood. Previous reports indicated that HSV ICP27 mediates viral intronless RNA export (...

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Section: /Ebvmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Majerčiak

Yamanegi

Allemand

et al. 2008

J Virol

Self Cite

122

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“…An 16E6E7-specific As RNA probe was synthesized by in vitro transcription and used for RPA for detection of the E6E7 RNAs, as described (Zheng et al, 2004). Antisense RNA multiprobes were prepared from a hCC-2 human cell cycle multiprobe template set (BD PharMingen, Cat.…”

Section: Rpa and Northern Blotmentioning

confidence: 99%

“…The bicistronic transcript has three exons and two introns, which undergo alternative RNA splicing to produce both the E6 and E7 proteins (Zheng et al, 2004). Several approaches have been taken to block 16E6 and 16E7 expression experimentally in cervical cancer cell lines and some promising results have been obtained.…”

Section: Introductionmentioning

confidence: 99%

“…These include viral E2-mediated suppression of the p97 promoter (Goodwin et al, 2000) and siRNA-mediated silencing of E6 or E7 expression (Jiang and Milner, 2002;Butz et al, 2003;Yoshinouchi et al, 2003). For this study, we chose an siRNA targeting a region downstream of all three alternative 3 0 splice sites in the E7 coding region (Zheng et al, 2004). Theoretically, this siRNA should be potentially more 16E6E7-suppressive.…”

Section: Introductionmentioning

confidence: 99%

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Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells

et al. 2005

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RNA interference-mediated gene silencing has the potential to block gene expression. A synthetic double-stranded small interfering RNA (siRNA) based on a sequence motif of 21 nucleotides from human papillomavirus 16 (HPV16) E6E7 bicistronic RNA was found to be a potent siRNA that suppresses expression of both the E6 and E7 oncogenes in HPV16þ CaSki and SiHa cells. When stably expressed as a short hairpin RNA in these cells, however, siRNA silencing of E6 and E7 expression was efficient only at early cell passages, but became inefficient with increased cell passages despite the continued expression of the siRNA at the same level. The loss of the siRNA function was duplicable in stable p53 siRNA cells, but not in stable lamin A/C siRNA cells, suggesting that it is gene selective. The cells resistant to siRNA function retained normal siRNA processing, duplex unwinding and degradation of the unwound sense strand and RNA-induced silencing complex formation, suggesting that loss of the siRNA function occurred at a later step. Surprisingly, the siRNA-resistant cells were found to express notably a cytoplasmic protein of B50 kDa that specifically and characteristically interacted with the unwound, antisense strand E7 siRNA. Altogether, our data indicate that a potent siRNA targeting to an essential or regulatory gene might induce a cell to develop siRNA-suppressive function.

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“…A number of elegant studies on the HPV type 16 (HPV16) transcriptome (3)(4)(5)(6)(7)(8)(9)(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.…”

mentioning

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...

show abstract

Splicing of a Cap-proximal Human Papillomavirus 16 E6E7 Intron Promotes E7 Expression, but can be Restrained by Distance of the Intron from its RNA 5′ Cap

Cited by 59 publications

References 68 publications

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Kaposi's Sarcoma-Associated Herpesvirus ORF57 Functions as a Viral Splicing Factor and Promotes Expression of Intron-Containing Viral Lytic Genes in Spliceosome-Mediated RNA Splicing

Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells

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

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