1. Papillomavirus infection and viral gene expression. Human papillomavirus type 16 (HPV16) or 18 (HPV18) infection, acquired primarily via sexual transmission, is widely recognized as a leading cause of cervical and anal cancer. Infection with oncogenic HPV in other tissues could also lead to development of cancer. For example, we recently demonstrated that colorectal oncogenic HPV infection is common in patients (51%) with colorectal cancer. High prevalence and incidence of cervical HPV infection has been observed among HIV-positive and immunodeficient women. Cervical cancer has been the most common malignancy among women with AIDS in both Europe and the United States. Two viral oncoproteins, E6 and E7, of HPV16 and HPV18 are involved in cervical carcinogenesis and are known to destabilize cellular tumor suppressor proteins p53 and pRb, respectively. In HPV16 and HPV18, 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. Splicing of the intron 1 in the E6E7 bicistronic pre-mRNAs is highly efficient and the majority of the spliced transcripts in cancer tissues and cervical cancer cell lines are E6*I, a spliced product without intron 1. We have demonstrated that the E6*1 is the authentic mRNA for E7 translation, while a small portion of the bicistronic RNAs with retention of the intron 1 encode E6 oncoprotein. This conclusion was based on our specific siRNA approaches that selectively silence the expression of each viral oncogene. During virus life cycle which is tightly regulated by keratinocyte differentiation, we found that a cellular RNA splicing factor, SRp20, which is also differentiately expressed, controls virus early-to-late switch. Overexpression SRp20 in cervical cancer promotes viral E6 and E7 expression, whereas a decreased SRp20 level promotes viral L1 expression and a productive HPV infection. Surprisingly, we characterized SRp20 as an oncoprotein which is required for cancer cell growth and tumor induction. Recently, we showed that high-risk HPV infection also deregulates the expression of tumor-suppressive miR-34a and p18Ink4c through viral oncoprotein E6. Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth and viral oncoproteins are partially responsible for this deregulation. 2. KSHV Gene expression and post-transcriptional regulation. KSHV is a lymphotropic DNA tumor virus that induces Kaposi sarcoma (KS), primary effusion lymphoma (PEL) or body cavity-based B-cell lymphoma, and multicentric Castleman disease (MCD). Among those malignancies, KS occurs frequently in patients infected with HIV. PEL and MCD feature an increased levels of cytokines (IL6 and IL10). Latent KSHV infection in KS lesions and PEL-derived B cells can be reactivated as lytic KSHV infection by chemicals or hypoxia. In this lytic switch, a KSHV transactivator, ORF50, is absolutely required. Our initial study demonstrated that ORF50 is positioned in the same locus of the virus genome along 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. There are two major isoforms of spliced RNA products, α(exclusion of K8 intron 2 or ORF50 intron 3) and β(inclusion of K8 intron 2 or ORF50 intron 3) in KSHV lytic infection. We demonstrated that KSHV K8βis derived from a splicing intermediate and antagonizes K8α-mediated induction of p21 and p53 and blocks K8α-CDK2 interaction. We further showed that KSHV ORF57 promotes RNA splicing of K8βto produce K8α. KSHV ORF57 is a ICP27 homolog of herpes simplex viruses and is essential for KSHV replication and virus production. Demonstration of that ORF57, a mRNA transcript accumulator (MTA), is a viral splicing factor was a surprise since its homologs in other herpesvirus are all splicing suppressive in reported studies. In the analysis of the gene structure and expression of KSHV ORF56 (viral primase), ORF57, ORF58 (EB virus BMRF2 homology) and ORF59 (viral DNA polymerase processing factor), we demonstrated that both ORF56 and ORF59 are expressed as bicistronic RNAs that subject to ORF57 up-regulation. In our genome-wide search for ORF57 RNA target, we identified that ORF57 regulates the expression of more than 16 KSHV genes at the posttranscriptional level. Strikingly, we found that, through interacting with targeted RNA and microRNA pathway, ORF57 promotes IL6 expression by interrupting microRNA-mediated translational repression of IL6, which is necessary for cancer cell growth.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIASC010357-09
Application #
7969832
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2009
Total Cost
$1,163,601
Indirect Cost
Name
National Cancer Institute Division of Clinical Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Massimelli, Maria J; Majerciak, Vladimir; Kruhlak, Michael et al. (2013) Interplay between polyadenylate-binding protein 1 and Kaposi's sarcoma-associated herpesvirus ORF57 in accumulation of polyadenylated nuclear RNA, a viral long noncoding RNA. J Virol 87:243-56
Bodaghi, Sohrab; Jia, Rong; Zheng, Zhi-Ming (2009) Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites. Virology 386:32-43
Wang, Xiaohong; Wang, Hsu-Kun; McCoy, J Philip et al. (2009) Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6. RNA 15:637-47
Jia, Rong; Liu, Xuefeng; Tao, Mingfang et al. (2009) Control of the papillomavirus early-to-late switch by differentially expressed SRp20. J Virol 83:167-80
Majerciak, Vladimir; Zheng, Zhi-Ming (2009) Kaposi's sarcoma-associated herpesvirus ORF57 in viral RNA processing. Front Biosci 14:1516-28
Jia, Rong; Zheng, Zhi-Ming (2009) Regulation of bovine papillomavirus type 1 gene expression by RNA processing. Front Biosci 14:1270-82
Wang, Xiaohong; Tang, Shuang; Le, Shu-Yun et al. (2008) Aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth. PLoS One 3:e2557
Majerciak, Vladimir; Yamanegi, Koji; Allemand, Eric et al. (2008) 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. J Virol 82:2792-801