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Journal of Virology, August 1999, p. 6307-6318, Vol. 73, No. 8
Department of Biochemistry, Case Western
Reserve University School of Medicine, Cleveland, Ohio 44106-4935
Received 16 December 1998/Accepted 15 April 1999
Predicted secondary-structure elements encompassing the primer
binding site in the 5' untranslated region of Rous sarcoma virus (RSV)
RNA play an integral role in multiple viral replications steps
including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol.
66:2464-2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648-7656, 1997). These elements include the U5-Leader stem, U5-IR
stem-loop, and U5-T
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Changes in Rous Sarcoma Virus RNA Secondary
Structure near the Primer Binding Site upon tRNATrp
Primer Annealing
C interaction region. Limited digestion of the 5'
untranslated region of wild-type and mutant RSV RNAs with structure-
and/or sequence-specific RNases detects the presence of the U5-Leader
stem and the U5-IR stem-loop. When a tRNATrp primer is
annealed to wild-type RNAs in vitro, limited nuclease mapping indicates
that the U5-IR stem becomes partially unwound. This is not observed
when mutant RNAs with altered U5-IR stem-loop structures are
substituted for wild-type RNAs. The U5-Leader stem also becomes
destabilized when the tRNA primer is annealed to either wild-type or
mutant RNA fragments. Nuclease mapping studies of tRNATrp,
as well as the viral RNA, indicate that the U5-T
C helix does form in
vitro upon primer annealing. Collectively, these data suggest that the
various structural elements near the RSV primer binding site undergo
significant changes during the process of primer annealing.
*
Corresponding author. Present address: School of
Medicine, Northwestern University, 303 East Chicago Ave., Chicago, IL
60611. Phone: (312) 503-0336. Fax: (312) 503-7654. E-mail:
j_leis{at}nwu.edu.
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