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Journal of Virology, February 2001, p. 1864-1869, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.1864-1869.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Kaposi's Sarcoma-Associated Herpesvirus vCyclin Open
Reading Frame Contains an Internal Ribosome Entry Site
Lara
Bieleski and
Simon J.
Talbot*
Laboratory for Clinical and Molecular
Virology, University of Edinburgh, Edinburgh EH9 1QH, United
Kingdom
Received 15 September 2000/Accepted 29 November 2000
We have previously examined the transcription and splicing of open
reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma
cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and
latency-associated nuclear antigen [LANA]) are transcribed from a
common transcription start site in BCP-1 cells during both latency and
the lytic cycles. The resulting transcript is spliced to yield a
5.32-kb message encoding LANA, vCyclin, and vFLIP and a
1.7-kb bicistronic message encoding vCyclin and vFLIP. To
investigate whether the vFLIP protein could be expressed from this
vCyclin/vFLIP message, we utilized a bicistronic luciferase
reporter system. The genes for Renilla and firefly
luciferases (which utilize different substrates) were cloned in tandem
downstream from a T7 RNA polymerase promoter. Fragments of DNA
immediately upstream from the initiating codon of vFLIP were cloned
between the two luciferase genes. The relative expression of the two
luciferases, one directed by the putative internal ribosome entry site
(IRES) sequences and the other by cap-dependent ribosome scanning, was
used to compare the activities of the different DNA fragments. A
minimum fragment of 233 bp within the coding region of
vCyclin was found to direct efficient expression of the
downstream cistron (firefly luciferase). The activity of this IRES was
orientation dependent and unaffected by methods used to inhibit
cap-dependent translation. This is the first demonstration of an IRES
element encoded by a DNA virus and may represent a novel mechanism
through which KSHV controls protein expression.
*
Corresponding author. Mailing address: Laboratory for
Clinical and Molecular Virology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom. Phone: 44 131 6507938. Fax: 44 131 6506511. E-mail: s.talbot{at}ed.ac.uk.
Journal of Virology, February 2001, p. 1864-1869, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.1864-1869.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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