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. 2000 Oct;74(19):8980-8.
doi: 10.1128/jvi.74.19.8980-8988.2000.

Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus- and plus-strand transfer

J Guo  1 , T Wu, J Anderson, B F Kane, D G Johnson, R J Gorelick, L E Henderson, J G Levin
Affiliations

Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus- and plus-strand transfer

J Guo et al. J Virol. 2000 Oct.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Effect of HIV-1 wild-type and SSHS NC proteins on complete removal of the tRNA primer from (−) strand DNA in reaction mixtures containing wild-type or RNase H-minus (63) RTs. (A) Nucleic acid strand transfer intermediates present in the reaction mixtures. The minus-strand donor DNA template (32 nt) is shown with a single rA attached at its 5′ end; a 17-nt RNA representing the 17 bases remaining at the 3′ end of the tRNA3Lys primer after the initial RNase H cleavage, 5′ 32P-labeled (+) SSDNA (50 nt), with the radiolabel indicated by an asterisk, and the minus-strand acceptor DNA template (48 nt) are also shown. (+) SSDNA and the minus-strand donor and acceptor DNAs are represented by filled and open rectangles, respectively; the RNA segments (rA and the 17-nt oligonucleotide) are indicated by narrow filled rectangles. This portion of the figure is taken from Fig. 7A in reference . (B and C) Primer removal in reaction mixtures containing increasing concentrations of wild-type (B) or SSHS (C) NC proteins. The amount of the plus-strand 80-nt transfer product (as determined by PhosphorImager analysis of gel data) is plotted against NC concentration. Reactions with RNase H-minus RT (RT H−) are shown in the upper panels, while those containing wild-type RT (RT H+) are shown in the lower panels. The open and solid bars represent results with RNase H-minus and wild-type RTs, respectively.
FIG. 2
FIG. 2
Effects of the zinc fingers on the kinetics of NC-facilitated annealing reactions required for HIV-1 plus- and minus-strand transfer. Annealing was performed as described in Materials and Methods. Reactions were incubated for the indicated times with wild-type or SSHS NC proteins. (A) Plus-strand transfer annealing reactions. The concentration of NC in the reaction mixtures was 0.28 μM. (A-1) The percentage of total (+) SSDNA annealed was plotted against the time of incubation. (A-2) The data in panel A-1 were replotted as semilogarithmic plots of the percentage of unannealed (+) SSDNA versus the time of incubation. (B) Minus-strand transfer annealing reactions. (B-1) The NC concentration in the reaction mixtures was 1 μM. The percentage of total (−) SSDNA annealed was plotted against the time of incubation. (B-2) The data in panel B-1 and the data from an independent experiment were replotted as semilogarithmic plots of the percentage of unannealed (−) SSDNA versus the time of incubation. Symbols: solid diamonds, solid lines, wild-type NC; open squares, dashed lines, SSHS NC. The short dashed lines in panels A-1 and B-1 represent annealing in the absence of NC and are based on data in references and , respectively.
FIG. 3
FIG. 3
Effect of HIV-1 wild-type and SSHS NC proteins on minus-strand transfer and self-priming. (A) Gel analysis. Reaction mixtures were incubated with increasing amounts of wild-type (lanes 2 to 6) or SSHS (lanes 7 to 11) NC proteins for 30 min at 37°C, according to the procedures given in Guo et al. (36) for the complete minus-strand transfer system. The DNA products were separated by gel electrophoresis in a 6% sequencing gel (36). The concentrations of NC were as follows: lane 1, no NC; lanes 2 and 7, 0.4 μM; lanes 3 and 8, 0.8 μM; lanes 4 and 9, 1.6 μM; lanes 5 and 10, 2.4 μM; and lanes 6 and 11, 3.2 μM. The positions of the primer (P), (−) SSDNA, SP products (SP), and the transfer product (T) are indicated. (B and C) PhosphorImager analysis. The gel data shown in panel A were quantified by PhosphorImager analysis, as previously described (36). The concentrations of transfer product (B) and SP products (C) are plotted against NC concentration. Symbols: solid diamonds, solid lines, wild-type NC; open squares, dashed lines, SSHS NC.
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