Optimization of a Weak 3' Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

doi:10.1128/JVI.74.13.5902-5910.2000

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Journal of Virology, July 2000, p. 5902-5910, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Optimization of a Weak 3' Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

Zhi-Ming Zheng,^* Jesse Quintero, Eric S. Reid, Christian Gocke, and Carl C. Baker

Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received 28 January 2000/Accepted 7 April 2000

Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirustype 1 (BPV-1), a switch in 3' splice site utilization from anearly 3' splice site at nucleotide (nt) 3225 to a late-specific3' splice site at nt 3605 is essential for expression of the majorcapsid (L1) mRNA. Three viral splicing elements have recentlybeen identified between the two alternative 3' splice sites andhave been shown to play an important role in this regulation.A bipartite element lies approximately 30 nt downstream of thent 3225 3' splice site and consists of an exonic splicing enhancer(ESE), SE1, followed immediately by a pyrimidine-rich exonic splicingsuppressor (ESS). A second ESE (SE2) is located approximately125 nt downstream of the ESS. We have previously demonstratedthat the ESS inhibits use of the suboptimal nt 3225 3' splicesite in vitro through binding of cellular splicing factors. However,these in vitro studies did not address the role of the ESS inthe regulation of alternative splicing. In the present study,we have analyzed the role of the ESS in the alternative splicingof a BPV-1 late pre-mRNA in vivo. Mutation or deletion of justthe ESS did not significantly change the normal splicing patternwhere the nt 3225 3' splice site is already used predominantly.However, a pre-mRNA containing mutations in SE2 is spliced predominantlyusing the nt 3605 3' splice site. In this context, mutation ofthe ESS restored preferential use of the nt 3225 3' splice site,indicating that the ESS also functions as a splicing suppressorin vivo. Moreover, optimization of the suboptimal nt 3225 3' splicesite counteracted the in vivo function of the ESS and led to preferentialselection of the nt 3225 3' splice site even in pre-mRNAs withSE2 mutations. In vitro splicing assays also showed that the ESSis unable to suppress splicing of a pre-mRNA with an optimizednt 3225 3' splice site. These data confirm that the function ofthe ESS requires a suboptimal upstream 3' splice site. A surprisingfinding of our study is the observation that SE1 can stimulateboth the first and the second steps ofsplicing.

^* Corresponding author. Present address: Bldg. 10/Rm. 13N240, National Cancer Institute, National Institutes of Health, HIVand AIDS Malignancy Branch, 9000 Rockville Pike-Bethesda, MD 20892.Phone: (301) 594-1382. Fax: (301) 480-8250. E-mail: zhengt{at}dce41.nci.nih.gov.

Journal of Virology, July 2000, p. 5902-5910, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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