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. 1998 Nov;72(11):9353–9358. doi: 10.1128/jvi.72.11.9353-9358.1998

Roles of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Annealing and Initiation versus Elongation in Reverse Transcription of Viral Negative-Strand Strong-Stop DNA

Liwei Rong 1, Chen Liang 1, Mayla Hsu 1, Lawrence Kleiman 1, Patrice Petitjean 2, Hugues de Rocquigny 2, Bernard P Roques 2, Mark A Wainberg 1,*
PMCID: PMC110360  PMID: 9765488

Abstract

To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lys primer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(−)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (−)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is processed from Gag precursors and is able to bind tightly to viral genomic RNA (2, 6, 16, 17, 34, 37). Accordingly, NC can function as a portion of the Gag polyprotein precursor, essential for viral genomic packaging, and, as well, as a fully processed protein in reverse transcription steps that occur during de novo infection (4, 11, 14). At least three distinct roles are associated with NC in the context of reverse transcription: (i) it facilitates the annealing of the cognate primer of reverse transcriptase (RT), i.e., tRNA3Lys, to the primer binding site (PBS) of the viral RNA template (3, 10, 24, 35); (ii) it stimulates specific viral DNA synthesis by reducing self-primed reverse transcription (15, 25, 26) or by enhancing the processivity of RT (20, 36, 38) during the synthesis of viral negative-strand DNA; and (iii) it promotes the first template switch in RT reactions to yield a full-length negative-strand DNA product (5, 15, 30, 33, 39).

Although the ability of NC to anneal primer tRNA onto the PBS has been demonstrated mostly in in vitro systems, the biological relevance of this process is highlighted by results showing that such tRNA placement is impeded in viruses with mutated NCs (18). NC probably facilitates tRNA placement in vivo while it is part of either the Gag or Gag-Pol precursor protein.

The structures of retroviral NCs are highly conserved, and, with the exception of spumavirus NCs, they characteristically contain one or two copies of a CCHC motif that can bind Zn2+ with high affinity to form Zn fingers (7, 28). The NC of HIV-1 contains two such Zn fingers flanked by regions rich in basic residues. Mutagenesis in the Zn fingers affects both the quantity of genomic RNA that is packaged into virions and viral infectivity (1, 8, 13, 31).

The role of NC in the annealing of tRNA to the PBS is thought to involve the melting of RNA secondary structure, a concept supported by the ability of NC to unwind primer tRNA (22). However, the structure-function relationship in this process is not well understood, nor is the fact that NCs in which the Zn finger regions have been deleted retain the ability to both bind and anneal RNA in vitro (10, 24). In contrast, Zn fingers in other, nonviral nucleic acid-binding proteins can function as nucleic acid interaction domains (29).

We have studied a synthetic form of NC (i.e., NC7) (9), a naturally observed cleavage product of the Gag precursor protein, during the annealing of primer tRNA3Lys to viral genomic RNA. In doing so, we have distinguished the activities of this protein in the tRNA3Lys annealing process from that of synthesis of negative-strand strong-stop DNA [(−)ssDNA] by removing NC7 from the viral RNA template by use of proteinase K and phenol-chloroform extraction of annealed template-primer complexes; these digested complexes were then studied in reverse transcription reactions.

(This work was performed by Liwei Rong in partial fulfillment of the requirements for a Ph.D. from McGill University, Montreal, Quebec, Canada.

The manner of placement of tRNA3Lys onto the HIV-1 RNA template can affect the efficiency of elongation of reverse transcription.

To study the role of NC7 in the synthesis of (−)ssDNA, we used a cell-free reverse transcription reaction consisting of viral RNA template, human tRNA3Lys, RT (p66/51) NC7, and RT (p66/51) (36). The PBS wild-type (wt) construct was generated as previously described (2), linearized by BssHII, and used as a template in an Ambion Mega-Scripts kit (Austin, Tex.) to produce an RNA transcript. The RNA template thus generated is 251 nucleotides (nt) in length and includes HIV sequences (nt 17 to 254) as well as a short 13-nt stretch derived from the PBS wt vector. tRNA3Lys was purified in our laboratories from human placenta as previously described (21). In vitro reverse transcription initiated by tRNA3Lys from this template yielded a full-length (−)ssDNA product of 178 nt joined to the 76-nt tRNA primer. The placement of primer tRNA3Lys onto the viral RNA template was performed in a 10-μl reaction mixture containing 50 mM Tris-HCl (pH 7.2), 50 mM KCl, 5 mM MgCl2, 1 pmol of template RNA, and 1 pmol of tRNA3Lys. Various concentrations of NC7 were added into the reaction mixtures, which were then incubated at 37°C for 1 h. When placement was carried out by heat annealing, the reaction mixtures were incubated as described previously for 5 min at 85°C and then for 10 min at 55°C (27). Bovine serum albumin was used as a control protein in both heat annealing and NC placement reactions and did not have either a positive or a negative effect (data not shown).

After the placement of tRNA3Lys onto the viral RNA template by either NC7 or heat annealing, single-nucleotide extension by incorporation of [α-32P]dCTP, mediated by 50 ng of RT, was performed in a total volume of 20 μl. One microliter of a 2 mM mixture of all four deoxynucleoside triphosphates was added to achieve further extension to generate full-length (−)ssDNA. Reactions were terminated at 16 min by adding EDTA (pH 8.0) at a final concentration of 50 mM. The amounts of both full-length (−)ssDNA and total cDNA products were quantified by molecular imaging using a program provided by the manufacturer (Bio-Rad, Mississauga, Ontario, Canada).

In order to understand the role of NC7 during the synthesis of (−)ssDNA, we studied the effects of varying concentrations of this protein (i.e., 5, 15, 30, and 45 pmol, corresponding to 1 molecule of NC7 per 50, 17, 8, and 6 nt residues, respectively) in comparison to reactions in which tRNA3Lys had been placed onto the RNA template by heat annealing. The proportion of full-length (−)ssDNA product relative to total cDNA generated was calculated as a measure of the efficiency of elongation of reverse transcription. The results in Fig. 1A (lanes 1 to 5) show that the use of increasing concentrations of NC7 in the placement of tRNA3Lys onto viral RNA resulted in an increased proportion of full-length (−)ssDNA in comparison to total cDNA product. The data show an increase in the amount of total cDNA product up to an NC7 concentration of 15 pmol (Fig. 1A, lanes 1 to 3), followed by a decline when higher NC7 concentrations were used (Fig. 1A, lanes 4 and 5). However, at these higher NC7 concentrations, the production of nonspecific DNA products was also decreased, while the proportion of full-length (−)ssDNA that was made relative to total cDNA product continued to increase. At an NC7 concentration of 45 pmol, more than two-thirds of the total cDNA product was present as full-length (−)ssDNA. Therefore, the efficiency of elongation was generally proportional to the amount of NC7 used to form the tRNA3Lys-RNA template complex (see Fig. 1A, graph).

FIG. 1.

FIG. 1

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Dose-response curve of (−)ssDNA synthesis. (A) Complexes of tRNA and viral RNA template were formed by either wt NC7 (lanes 1 to 5), mutated H23C NC (lanes 6 to 10), or mutated ddNC (lanes 11 to 15), and reactions were carried out for 16 min as described in Materials and Methods. The concentrations of wt or mutated NCs used in these reactions were 0 (lanes 1, 6, and 11), 5 (lanes 2, 7, and 12), 15 (lanes 3, 8, and 13), 30 (lanes 4, 9, and 14), and 45 (lanes 5, 10, and 15) pmol per reaction. The proportions of full length (−)ssDNA compared with total cDNA product in these reactions are shown in the graph. (B) Primer-template complexes were generated by heat annealing. Varying amounts of wt NC7 (lanes 2 to 5), mutated H23C NC (lanes 6 to 9), or mutated ddNC (lanes 10 to 13) were added during the primer elongation phase of these reactions. Lane 1, control reaction performed without NC. Lanes 2, 6, and 10, lanes 3, 7, and 11, lanes 4, 8, and 12, and lanes 5, 9, and 13, reactions performed with 5, 15, 30, and 45 pmol of NC per reaction, representing occlusion by one NC molecule of 50, 17, 8, and 6 nt, respectively. Band positions represent the first nucleotide added at the 3′ end of tRNA3Lys, and relative quantification of amounts of product was carried out by molecular imaging, by using a program that provides counts per minute equivalents, as suggested by the manufacturer (Bio-Rad Instruments).

Because NC7 is thought to exert its effect on the efficiency of viral cDNA synthesis through disruption of the secondary structure of the RNA template (12), we also studied tRNA3Lys-RNA template complexes that had been generated by heat annealing. In this circumstance, varying concentrations of NC7 were added only later, during the primer elongation stage of these reactions. The results in Fig. 1B show that the presence of NC7 in these reactions, even at very high concentrations, resulted in only a modest increase in the yield of full-length (−)ssDNA. Furthermore, the addition of NC7, at times after the heat annealing of tRNA3Lys to viral RNA, never yielded full-length (−)ssDNA products that represented more than 25% of total cDNA synthesis. Therefore, the increased efficiency of elongation in the synthesis of (−)ssDNA, in reactions in which NC7 was used to promote the formation of the primer-template complex, cannot be attributed solely to the role of NC7 during primer elongation but must also involve aspects of NC7 function during the placement of tRNA3Lys onto the viral RNA template.

The role of NC7 in formation of a tRNA3Lys-RNA binary complex with potential to yield high levels of full-length (−)ssDNA product.

To further verify a role for NC7 in primer placement and the synthesis of (−)ssDNA, tRNA3Lys was placed onto the viral RNA template by use of varying concentrations of NC7 or by heat annealing. Next, the proteins in these reactions were eliminated by the addition of 1 μl of proteinase K (5 mg/ml) at 37°C for 15 min, following which both the proteinase K and undigested residual NC were extracted with phenol-chloroform. Then the binary tRNA-RNA template complexes in the liquid phase were precipitated at −20°C for 6 h by using an equivalent volume of isopropanol. After precipitation, the tRNA3Lys-RNA template complex was redissolved in the initial reaction buffer, and reactions continued in the presence of RT and [α-32P]dCTP.

The results in Fig. 2 show that NC7 had again acted in a concentration-dependent manner during tRNA3Lys placement to promote primer elongation, compared with results obtained when primer-template complexes had been generated by heat annealing. Concentrations of NC7 of >30 pmol, i.e., saturating levels, were especially active in this regard. Figure 2, lanes 3 and 4, shows that overall levels of total cDNA product dropped significantly when a concentration of 5 or 15 pmol of NC7 was used and that no full-length (−)ssDNA products were generated under these conditions. In this experiment, NC7 had been eliminated from the template-primer complex by exposure to proteinase K and chloroform-phenol; primer elongation occurred in the presence of the annealed tRNA3Lys-viral RNA complex, RT, and deoxynucleoside triphosphates only. Therefore, differences among reactions with regard to the generation of full-length (−)ssDNA can be attributed only to qualitative differences between the types of template-primer complex formed in the presence of varying concentrations of NC7, with the highest efficiency occurring when NC7 was used at saturating conditions.

FIG. 2.

FIG. 2

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Efficiency of elongation of tRNA-template complexes formed with varying concentrations of NCs. tRNA3Lys was placed onto the viral RNA template in the presence of varying concentrations of NC7 to achieve the NC7/nucleotide ratios described in the legend to Fig. 1. The primer-template complexes were then treated with proteinase K and phenol-chloroform as described in Materials and Methods, after which the reactions were reconstituted with recovered primer-template complex. Lane 1, control reaction representing the elongation efficiency of the primer-template complex formed by heat annealing. Lanes 2 to 6, elongation efficiencies of complexes formed by the addition of 0, 5, 15, 30, and 45 pmol of NC7 per reaction, respectively. Band positions represent the first nucleotide added to the 3′ end of tRNA3Lys; relative quantification was carried out by molecular imaging. The proportion of (−)ssDNA relative to the total amount of cDNA produced in each reaction is presented in the graph.

The NC7 Zn fingers are important in the formation of primer-template complexes with the potential to participate in highly efficient elongation.

We next examined whether structural elements within NC7 might be involved in formation of the tRNA3Lys primer-RNA template complex, with potential to participate in efficient elongation reactions. Toward this end, we studied the effects of two NCs with mutations in the Zn finger motifs, including a variant containing a His-to-Cys modification in the first Zn finger (i.e., H23C NC) and a variant termed ddNC, in which both of the Zn fingers are deleted (9). Previous studies showed that a truncated form of NC, containing amino acids 13 to 64 and including both the Zn finger motifs as well as the H23C substitution, could no longer participate in annealing (8, 32). In contrast, ddNC that was devoid of both Zn fingers did retain both nucleic acid binding and annealing activities (10, 24).

Figure 1B shows results obtained when either wt or mutated NC was added to RT reactions in which primer-template complexes had been formed by heat annealing. The data show that the addition of either wt or mutated NC during elongation only slightly increased the proportion of full-length (−)ssDNA relative to total cDNA product and that H23C NC was able to promote the synthesis of (−)ssDNA at low concentrations (5 pmol). In contrast, when the annealing of primer to template was performed in the presence of NCs, far less full-length (−)ssDNA was observed in reactions in which the mutated forms of NC were used (Fig. 1A, lanes 6 through 15).

We next performed single-base-extension experiments to determine whether the inefficient generation of full-length (−)ssDNA was due to decreased levels of tRNA3Lys primer-RNA template complexes that had been formed by the mutated NCs. The results in Fig. 3 show that reactions that used wt NC7 (lanes 6 to 10) or either of the two types of mutated NC (lanes 11 to 15 and 16 to 20) yielded similar levels of single-base-extended product, although, in each case, less product was generated than that seen when heat annealing was used for purposes of primer-template formation (Fig. 3, lanes 1 to 5). These findings are consistent with earlier reports that NCs with Zn finger deletions retain annealing activity (10, 24).

FIG. 3.

FIG. 3

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Single-base extension of tRNA-template complexes. By monitoring the incorporation of [α-32P]dCTP, reactions were observed for varying times, i.e., 0.5 min (lanes 1, 6, 11, and 16), 1 min (lanes 2, 7, 12, and 17), 4 min (lanes 3, 8, 13, and 18), 16 min (lanes 4, 9, 14, and 19), and 32 min (lanes 5, 10, 15, and 20), and we were able to distinguish the rates at which reactions had been initiated from different tRNA-template complexes. Complexes had been formed either by heat annealing or in the presence of wt NC, H23C mutated NC, or mutated ddNC, at a concentration of 30 pmol per reaction. The densities of single-base-extension products were determined on the basis of molecular imaging.

To test the hypothesis that the NCs containing mutations in the Zn fingers might be less able than wt NC to promote subsequent extension, reaction mixtures containing primer-template and NC were subjected to proteinase K digestion and phenol-chloroform extraction as described above, in protocols in which both wt and mutated NC peptides were used at saturating conditions, i.e., 30 pmol per reaction. The results in Fig. 4 show that the NC that contained a mutation in the first Zn finger, i.e., H23C NC, was about 2.5 times less efficient than wt NC7 with regard to the generation of a template-primer complex able to participate in the highly efficient synthesis of (−)ssDNA. In contrast, hardly any full-length (−)ssDNA product was formed in reactions performed with the NC peptide in which both Zn fingers were deleted (Fig. 4, lane 3), and RT reactions were blocked after the formation of short cDNA products. Significant arrest was noted in these reactions at nucleotide positions +1 to +5.

FIG. 4.

FIG. 4

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Effects of mutations within the Zn finger domains of NC on reverse transcription elongation. Either wt or mutated NC was used to generate primer-template complexes; these NCs were then eliminated from the complexes by digestion with proteinase K and extraction with phenol-chloroform as described in Materials and Methods. The primer elongation step was performed in the absence of NC. Quantification of products was carried out as described in the legend to Fig. 1.

Conclusions.

The role of NC7 in reverse transcription is thought to result from the denaturation of RNA template secondary structure during the elongation phase of the reaction (12). However, we have shown that the use of NC7 to promote complex formation between tRNA3Lys and the RNA template led to a higher proportion of full-length (−)ssDNA in the total cDNA product than that obtained when a heat-annealing procedure was used. In contrast, the increased elongation efficiency of RT reactions in the presence of NC7 is not due solely to the activity of NC7 during primer extension but is also related to the manner of placement of the tRNA3Lys primer onto the HIV RNA template. The addition of different amounts of NC7 at times after the heat-annealed formation of template-primer complex had little or no effect on the subsequent efficiency of elongation.

The fact that elongation efficiency was lower in reactions in which NC7 was removed provides direct evidence that NC is involved in the formation of a tRNA3Lys-RNA binary complex with potential to yield high levels of full-length (−)ssDNA. Thus, our data complement the notion that NC plays a role in the disruption of the spatial structure of the RNA template during both tRNA placement and elongation; however, an additional role is also evident with regard to promotion of efficient elongation.

Our results show that the Zn finger motifs are especially important for the formation of tRNA3Lys-RNA template complexes that are necessary for fully efficient reverse transcription reactions during the elongation of (−)ssDNA synthesis. These observations are consistent with a recent report that mutations within the Cys-His motif are relatively unimportant in the genomic placement of tRNA3Lys in vivo but are important for extension from the tRNA primer (18).

In order to attain full-length synthesis of HIV-1 (−)ssDNA from tRNA3Lys annealed to the viral RNA template, at least two distinct reaction phases are necessary, i.e., initiation, to generate products extended by 3 to 5 nt, and elongation, to yield longer cDNA products (19, 23). Our study shows that NC7 also promotes the transition from initiation to elongation (Fig. 2). NC devoid of both Zn fingers resulted in a primer-template complex that could yield only initiation products, i.e., products extended by 3 to 5 nt, and other short DNA products, but not fully synthesized (−)ssDNA (Fig. 4, lane 3). This helps to explain why the tRNA-template complex formed by NC7 yielded a higher proportion of full-length (−)ssDNA products than complexes formed by heat annealing (Fig. 2, lane 1).

Acknowledgments

This research was supported by grants to M.A.W. from the Medical Research Council of Canada.

We thank Matthias Götte, Xuguang Li, and Yudong Quan for helpful suggestions for the performance of this work.

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