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Journal of Virology, June 1999, p. 4794-4805, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Molecular Requirements for Human Immunodeficiency
Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and
Endogenous Reverse Transcription Systems
Tiyun
Wu,1
Jianhui
Guo,1
Julian
Bess,2
Louis E.
Henderson,2 and
Judith
G.
Levin1,*
Laboratory of Molecular Genetics, National
Institute of Child Health and Human Development, Bethesda, Maryland
20892,1 and AIDS Vaccine Program,
SAIC-Frederick, NCI-Frederick Cancer Research and Development Center,
Frederick, Maryland 217022
Received 29 December 1998/Accepted 9 March 1999
We have developed a reconstituted system which models the events
associated with human immunodeficiency virus type 1 (HIV-1) plus-strand
transfer. These events include synthesis of plus-strand strong-stop DNA
[(+) SSDNA] from a minus-strand DNA donor template covalently
attached to human tRNA3Lys, tRNA primer removal, and
annealing of (+) SSDNA to the minus-strand DNA acceptor template.
Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near
the 3' end of tRNA3Lys reconstitutes the 18-nucleotide
primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and
in HIV-1 endogenous reactions indicated another major termination site:
the pseudouridine at nucleotide 55. In certain HIV-1 strains,
complementarity between nucleotides 56 to 58 and the first three bases
downstream of the PBS could allow all of the (+) SSDNA products to be
productively transferred. Undermodification of the tRNA may be
responsible for termination beyond the methyl A. In studies of tRNA
removal, we find that initial cleavage of the 3' rA by RNase H is not
sufficient to achieve successful strand transfer. The RNA-DNA hybrid
formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or
trans). Alternatively, the nucleic acid chaperone activity
of nucleocapsid protein (NC) can catalyze this destabilization. NC
stimulates annealing of the complementary PBS sequences in (+) SSDNA
and the acceptor DNA template. Reverse transcriptase also promotes
annealing but to a lesser extent than NC.
*
Corresponding author. Mailing address: Laboratory of
Molecular Genetics, NICHD, Building 6B, Room 216, NIH, Bethesda,
MD 20892. Phone: (301) 496-1970. Fax: (301) 496-0243. E-mail:
judith_levin{at}nih.gov.
Journal of Virology, June 1999, p. 4794-4805, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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