My laboratory has focused on RNA splicing fidelity and regulation in oncoviral gene expression, using papillomavirus and Kaposi's sarcoma-associated herpesvirus (KSHV) RNAs as models. The pre-mRNAs from these two families of viruses are transcribed from many genes, including some viral oncogenes, that undergo RNA splicing. Because viral oncogenesis is one of the top priorities of the HIV and AIDS Malignancy Branch, the goal of my research program is to understand the molecular details regulating expression of oncogenic and/or pathogenic viral genes.Human papillomavirus type 16 (HPV16) infection, acquired via sexual transmission, is widely recognized as a leading cause of cervical cancer. High prevalence and incidence of cervical HPV infection has been observed among HIV+ women and cervical cancer has been the most common malignancy among women with AIDS in both Europe and the USA. Two viral oncoproteins, E6 and E7, of HPV16 are involved in cervical carcinogenesis and respectively, inactivate cellular tumor suppressor proteins p53 and pRb. HPV16 E6 and E7 are transcribed as a single bicistronic RNA bearing 3 exons and 2 introns, with the intron 1 in the E6 coding region. It has been believed that the E6 is expressed from the bicistronic RNA, without splicing of the intron 1. This has raised several important questions on how this intron escapes splicing, what regulates this escape, and how an RNA containing an intron is exported to cytoplasm to translate E6 protein. We have studied the mechanisms involved in this splicing regulation and found that the intron 1 splicing subjects to exon definition (size) by a cap structure on the RNA 5' end. Escaping splicing of the intron 1 results in production of unspliced E6 mRNA, whereas splicing of the intron promotes E7 expression. By limiting the RNA splicing, we identified three nuclear localization signals responsible for accumulation of HPV16 E6 in the cell nucleus, degradation of p53, and cell immortalization. Currently, we are focusing on (1) characterization of viral and cellular proteins involving the splicing regulation; (2) identification of a novel spliced isoform of the E6 and its biological function; and (3) control of HPV16 E6 and E7 expression by siRNA targeting to the bicistronic RNA. These studies will lead us to better understand oncogenesis of HPV16.KSHV infection has been associated with all forms of Kaposi's sarcoma (KS), primary effusion lymphoma PEL, and multicentric Castleman's disease and displays two viral life cycles. Among those malignancies, KS occurs frequently in patients infected with HIV. Latent KSHV infection in KS lesions and PEL-derived B cells features the highly restricted expression of only five viral genes. The lytic KSHV infection can be induced by chemicals in PEL-derived B cells with latent KSHV infection. In this lytic switch, a KSHV transactivator, ORF50, is absolutely required. ORF50 is an immediate-early gene transcribed as a polycistronic RNA with 5 exons and 4 introns. ORF50 is positioned in the virus genome with both K8 (an early gene encoding a K-bZIP protein) and K8.1 (a late gene encoding a viral envelope glycoprotein) and shares a single polyadenylation site downstream of K8.1 coding region. Accordingly, the transcripts of the three genes overlap each other and undergo extensive RNA splicing. At each gene, however, transcription is initiated from an individual promoter. Initially our focus was on removal of RNA 3' terminal introns because they are involved in the processing of all three transcripts ORF50, K8 and K8.1. We have identified and characterized the K8.1 late promoter and found its transcription start site resided in K8 exon 3 (ORF50 exon 4). We have also found that K8 exon 3 (ORF50 exon 4) contains three 5' splice sites that are alternatively selected for RNA splicing of ORF50 and K8 in KSHV+ cell lines. Selection of the nt 75838 5' splice site dictates the production of both ORF50 and K8 mRNA and stimulates removal of the intron upstream. In addition, we have demonstrated that KSHV K8 beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes the function of the K-bZIP protein encoded by K8 alpha mRNA. Recently, we have extended our research focus to KSHV ORF57, a ICP27 homolog of herpes simplex viruses that involves in regulation of RNA splicing and mediates RNA exports. We have demonstrated that the ORF57 is a phosphorylated nuclear protein bearing three nuclear localization signals and promoting viral ORF59 expression.

National Institute of Health (NIH)
Division of Clinical Sciences - NCI (NCI)
Intramural Research (Z01)
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Clinical Sciences
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Majerciak, Vladimir; Pripuzova, Natalia; McCoy, J Philip et al. (2007) Targeted disruption of Kaposi's sarcoma-associated herpesvirus ORF57 in the viral genome is detrimental for the expression of ORF59, K8alpha, and K8.1 and the production of infectious virus. J Virol 81:1062-71
Haque, Muzammel; Wang, Victoria; Davis, David A et al. (2006) Genetic organization and hypoxic activation of the Kaposi's sarcoma-associated herpesvirus ORF34-37 gene cluster. J Virol 80:7037-51
Tang, Shuang; Tao, Mingfang; McCoy Jr, J Philip et al. (2006) The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation. J Virol 80:4249-63
Majerciak, Vladimir; Yamanegi, Koji; Nie, Sarah H et al. (2006) Structural and functional analyses of Kaposi sarcoma-associated herpesvirus ORF57 nuclear localization signals in living cells. J Biol Chem 281:28365-78
Tang, S; Tao, M; McCoy Jr, J P et al. (2006) Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells. Oncogene 25:2094-104
Zheng, Zhi-Ming; Baker, Carl C (2006) Papillomavirus genome structure, expression, and post-transcriptional regulation. Front Biosci 11:2286-302
Zheng, Zhi-Ming; Tang, Shuang; Tao, Mingfang (2005) Development of resistance to RNAi in mammalian cells. Ann N Y Acad Sci 1058:105-18
Bodaghi, Sohrab; Yamanegi, Koji; Xiao, Shu-Yuan et al. (2005) Colorectal papillomavirus infection in patients with colorectal cancer. Clin Cancer Res 11:2862-7
Yamanegi, Koji; Tang, Shuang; Zheng, Zhi-Ming (2005) Kaposi's sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction. J Virol 79:14207-21
Bodaghi, Sohrab; Wood, Lauren V; Roby, Gregg et al. (2005) Could human papillomaviruses be spread through blood? J Clin Microbiol 43:5428-34

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