1. The full transcription map of cottontail rabbit papillomavirus in tumor tissues.
- Author
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Jiang, Pengfei, Majerciak, Vladimir, Hu, Jiafen, Balogh, Karla, Meyer, Thomas J., Cam, Maggie, Shearer, Debra, Lanza, Matthew, Christensen, Neil, and Zheng, Zhi-Ming
- Subjects
MOLECULAR biology ,NUCLEIC acid hybridization ,HUMAN papillomavirus ,PAPILLOMAVIRUS diseases ,ALTERNATIVE RNA splicing - Abstract
Cottontail rabbit papillomavirus (CRPV), the first papillomavirus associated with tumor development, has been used as a powerful model to study papillomavirus pathogenesis for more than 90 years. However, lack of a comprehensive analysis of the CRPV transcriptome has impeded the understanding of CRPV biology and molecular pathogenesis. Here, we report the construction of a complete CRPV transcription map from Hershey CRPV-induced skin tumor tissues. By using RNA-seq in combination with long-reads PacBio Iso-seq, 5′ and 3′ RACE, primer-walking RT-PCR, Northern blot, and RNA in situ hybridization, we demonstrated that the CRPV genome transcribes its early and late RNA transcripts unidirectionally from at least five distinct major promoters (P) and polyadenylates its transcripts at two major polyadenylation (pA) sites. The viral early transcripts are primarily transcribed from three "early" promoters, P
90 , P156 , and P907 and polyadenylated at nt 4368 by using an early polyadenylation signal (PAS) at nt 4351. Like other low-risk human papillomaviruses and animal papillomaviruses, CRPV E6 and E7 transcripts are transcribed from three separate early promoters. Transcripts from two "late" promoters, P7525 , and P1225 , utilize either an early PAS for E1^E4 or a late PAS at 7399 for L2 and L1 RNA polyadenylation at nt 7415 to express capsid L2 and L1 proteins respectively. By using the mapped four 5′ splice sites and three 3′ splice sites, CRPV RNA transcripts undergo extensive alternative splicing to produce more than 33 viral RNA isoforms for production of at least 12 viral proteins, some of which without codon optimization are expressible in rabbit RK13 and human HEK293T cells. The constructed full CRPV transcription map in this study for the first time will enhance our understanding of the structures and expressions of CRPV genes and their contribution to molecular pathogenesis and tumorigenesis. Author summary: Papillomavirus infections induce benign warts and cancers. Naturally occurring infections in animal models have played a pivotal role in studying human papillomavirus pathogenesis. The cottontail rabbit papillomavirus (CRPV), the first reported papillomavirus, has been used widely for understanding the pathogenesis of papillomavirus-associated diseases and cancer. The current study uses the state-of-the-art RNA-seq and PacBio Iso-seq in combination with 5′ and 3′ RACE, primer-walking RT-PCR, Northern blot, and RNA in situ hybridization to determine genome-wide transcription and RNA structures of individual CRPV transcripts in Hershey CRPV-induced rabbit skin wart tissues. As a result, we have constructed the full transcription map from CRPV-induced rabbit skin warts and further determined the relative abundance and spatial localization of identified viral transcripts in these warts. The CRPV genome uses three early and two late viral promoters, four splice donor and three splice acceptor sites, and two viral PAS to express at least 33 RNA isoforms and twelve viral proteins. Most of their open reading frames were cloned for ectopic expression in this study. The constructed CRPV transcript map in this report will provide an important resource to advance our understanding of papillomavirus biology and pathogenesis. [ABSTRACT FROM AUTHOR]- Published
- 2024
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