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Journal of Virology, October 2000, p. 8980-8988, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer

Jianhui Guo,1 Tiyun Wu,1,dagger Jada Anderson,1 Bradley F. Kane,2 Donald G. Johnson,2 Robert J. Gorelick,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 14 April 2000/Accepted 26 June 2000

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minus- and plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(+) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (-) SSDNA and 3' viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (-) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.

* Corresponding author. Mailing address: Laboratory of Molecular Genetics, NICHD, Bldg. 6B, Rm. 216, NIH, Bethesda, MD 20892-2780. Phone: (301) 496-1970. Fax: (301) 496-0243. E-mail: judith_levin{at}nih.gov.

dagger Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.

Journal of Virology, October 2000, p. 8980-8988, Vol. 74, No. 19
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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