©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of Human Immunodeficiency Virus Nucleocapsid Protein on Synthesis and Strand Transfer by the Reverse Transcriptase in Vitro(*)

Lorna Rodrguez-Rodrguez (1), Zenta Tsuchihashi (4), Gloria M. Fuentes (1), Robert A. Bambara (1) (3)(§), Philip J. Fay (1) (2)

From the (1)Departments of Biochemistry and (2)Medicine and the (3)Cancer Center, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 and (4)Howard Hughes Medical Institute, Stanford University, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human immunodeficiency virus (HIV) nucleocapsid protein (NC) influences HIV reverse transcriptase (RT) catalyzed strand transfer synthesis from internal regions of natural sequence RNA. In the strand transfer assay reaction in vitro, primer synthesis initiated on a donor template can transfer and be completed on an acceptor template. NC was added at concentrations up to twice that needed for 100% template coating. As the concentration of NC was increased, primer extension was stimulated until NC coated approximately 50% of the template. Stimulation was caused in part by an increase in the number of primers that sustained synthesis. Subsequent increments of NC decreased synthesis. The presence of NC also increased the efficiency of the strand transfer reaction, allowing a greater proportion of extended primers to transfer from donor to acceptor templates. Processivity of the RT on the donor template was measured using both challenged and enzyme dilution assays. NC did not alter the proportion of synthesis products that reached the end of the template, indicating little effect on processivity. This result suggests that the increase in full-length product synthesis, observed in reactions where the RT repeatedly bound the primer-template, resulted from promotion of RT reassociation by NC. Consequently, since the RT could not reassociate with the template in the processivity assay, NC could not stimulate the amount of full-length synthesis. No strand transfer was observed in dilution processivity assays, suggesting that the RT must dissociate and rebind during the transfer reaction. Stimulation of synthesis, e.g. by increased dNTP concentration, normally inhibits strand transfer. Stimulation of both synthesis and transfer by NC indicates that properties of NC that improve the transfer event prevail over the negative effects of rapid synthesis on transfer efficiency.


INTRODUCTION

The most accepted model of retroviral replication includes two distinct DNA strand transfer reactions from the end of the viral genome (Coffin et al., 1979; Gilboa et al., 1979; Temin, 1981). Strand transfer can also occur from internal regions of the genomic RNA or complementary DNA in vivo (Hu and Temin, 1990a, 1990b). This reaction very likely contributes to the high frequency of recombination observed for retroviruses (Preston et al., 1988; Roberts et al., 1988; Huber et al., 1989; Hu et al., 1990a). Internal strand transfer provides the virus with a mechanism in which synthesis can proceed even if the HIV-RT()encounters a gap on the genomic viral RNA. We have previously demonstrated that HIV-RT is capable of catalyzing internal strand transfer in vitro, from a donor template to a homologous region on an acceptor template (DeStefano et al., 1992). Internal strand transfer was found to be RNase H-dependent. It also occurred most efficiently near positions where the RT is known to pause during synthesis on the donor template. We also found that decreasing the concentration of dNTPs in the synthesis reaction, so that primer extension becomes slower, increases the efficiency of strand transfer.

HIV-NC is a 55-amino acid protein derived by proteolysis of the gag and gag-pol polyprotein precursor (Veronese et al., 1987; Tritch et al., 1991; Henderson et al., 1992). NC is a highly basic protein that contains two zinc fingers of the form Cys-X-Cys-X-His-X-Cys, flanked by regions that are rich in basic residues. It binds to the viral RNA, stimulates viral RNA dimerization (Weiss et al., 1992; De Rocquigny et al., 1992; Prats et al., 1991), and is essential for viral packaging and, therefore, infectivity (Dupraz et al., 1990; Aldovani et al., 1990). NC also enhances the annealing of the tRNA primer to the viral RNA template (Barat et al., 1989; Weiss et al., 1992; De Rocquigny et al., 1992), presumably by promoting the unwinding of the tRNA (Khan and Giedroc, 1992). This is consistent with the observation that the presence of NC can enhance reverse transcription in vitro (Weiss et al., 1992; De Rocquigny et al., 1992).

NC also binds to other nucleic acids, although less avidly. The order of binding affinities is single-stranded RNA > single-stranded DNA > double-stranded DNA (Surovoy et al., 1993; You and McHenry, 1993). The nucleic acid binding site size of mature NC is 7 nucleotides (You and McHenry, 1993). The role of the zinc fingers in nucleic acid binding has been studied. Mutation experiments show that the basic amino acids around the zinc fingers, but not the zinc fingers themselves, are essential to carry on the nucleotide binding by the protein (Lapadat-Tapolsky et. al., 1993; De Rocquigny et al., 1992). On the other hand, at least one zinc finger is needed for RNA packaging (South et al., 1991; Gorelick et al., 1993; De Rocquigny et al., 1992). The role of the zinc ion in the reactions involving NC is not yet well understood (Karpel et al., 1987; Delahunty et al., 1992; Rice et al., 1993).

At high concentrations, NC can form large co-aggregates consisting of NC and nucleic acids (Lapadat-Tapolsky et al., 1993; Tsuchihashi and Brown, 1994). It is believed that NC coats the viral RNA template, possibly to protect it from cellular RNases or to stabilize unwound strands in a similar fashion as single-stranded DNA binding protein does in Escherichia coli (Kornberg and Baker, 1991). NC promotes strand exchange from a double-stranded DNA to a single-stranded DNA favoring the most stable duplex (Tsuchihashi and Brown, 1994). NC has a complex effect on properties of nucleic acids that is concentration dependent. At low concentrations, NC promotes annealing of complementary DNA strands, while at higher concentrations it promotes melting of double strands (Lapadat-Tapolsky et al., 1993; Tsuchihashi and Brown, 1994). NC was shown to stimulate the annealing of the HIV R region sequences 3000-fold (You and McHenry, 1994). These findings argue for a possible role of NC in strand transfer events, which necessarily involve both of these processes.

In this report we demonstrate that NC slightly enhances the efficiency of internal strand transfer reactions by HIV-RT. At low NC concentrations synthesis is stimulated. NC-promoted melting of secondary structures is likely to contribute to this stimulation. At higher concentrations, NC inhibits synthesis. We also demonstrate that NC does not significantly alter the processivity of primer elongation by the RT.


EXPERIMENTAL PROCEDURES

Materials

Recombinant HIV-RT with native primary structure was provided by the Genetics Institute (Cambridge, MA). The enzyme has a specific activity of 40,000 units/mg. One unit is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37 °C using poly(rA)-oligo(dT) as template primer. HIV-NC was chemically synthesized by the Louisiana State University Medical Center Core Laboratories. The sequence of the mature NC used for synthesis was that of the first 55 amino acids of the NC precursor protein described by Khan and Giedroc(1992). The peptide was kept under reducing conditions, and aliquots were stored in 10% 2-mercaptoethanol. The peptide concentration was determined by quantitative amino acid analysis, performed by the Cornell University Peptide Facility. Identity of the peptide was confirmed by amino acid composition analysis. A molecular weight of 6444 was determined by electrospray mass spectrometry.

Aliquots of both HIV-RT and NC were stored at -70 °C, and a fresh aliquot was used for each experiment. T4 polynucleotide kinase, T7 RNA polymerase, placental RNase inhibitor, RNase-free DNase I, rNTPs, restriction enzymes, and G25 and G50 Sephadex (RNA) columns were obtained from Boehringer Mannheim; dNTPs were from Pharmacia Biotech. Inc. Radiolabeled compounds and Nensorb 20 cartridges were from DuPont NEN. The 20-nucleotide-long DNA primer and the 68-nucleotide-long DNA acceptor template were synthesized by Genosys, Inc. (Houston, TX). All other chemicals were from Sigma.

Methods

Strand Transfer Reactions

The amount of HIV-NC necessary to coat 100% of the templates used was calculated based on one molecule of NC binding to every seven nucleotides (You and McHenry, 1993). NC was preincubated for 5 min with 0.5 nM primer-template and 50 nM acceptor template (Fig. 1) in 50 mM Tris-HCl (pH8), 80 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA (buffer A), 2% 2-mercaptoethanol, and 1 mM rNTPs, all given as final reaction concentrations. Then HIV-RT (0.4 units, unless otherwise specified) was added and then incubated for 5 additional min. The reaction was started by adding MgCl and dNTPs, in buffer A, to a final concentration of 6 mM and 100 µM, respectively (except where indicated). In some reactions, heparin trap was also added at this point, to a final concentration of 0.4 mg/ml. The NC concentrations ranged from 0 to twice the concentration calculated to obtain a 100% template-coating level, or from 0 to 990 nM, respectively. The reactions were performed in a final volume of 10 µl, at 37 °C for 30 min, unless otherwise specified. The reactions were terminated by addition of an equal volume of 2 gel loading buffer (90% formamide, 10 mM EDTA (pH 8), 0.1% xylene cyanol, 0.1% bromphenol blue).


Figure 1: Schematic representation of substrates used in the strand transfer reactions. The donor template is 142 nucleotides long. It was primed with a 5`-P-labeled DNA oligonucleotide 20 nucleotides long. The products of full-length synthesis over the donor template are 108 nucleotides in length. The acceptor template has an area of homology to the donor template of 27 nucleotides in the region indicated. The products of full-length synthesis after transfer to the acceptor template are 118 nucleotides long.



RNA/DNA Hybridization

A specific 20-nucleotide-long DNA primer was labeled with P at the 5`-end, using T4 polynucleotide kinase. The hybrids were prepared by mixing the RNA donor transcript with the 5`-labeled DNA primer at an approximately 1:10 ratio of 3`-termini in 50 mM Tris-HCl, 0.1 mM EDTA, 80 mM KCl buffer. This mixture was heated at 65 °C for 15 min and then cooled slowly to room temperature. The primer-template complex was then passed through a G50 Sephadex (RNA) column to remove unbound primers. The concentration of primer-template was determined by measuring the P specific activity present in the labeled primer before and after passing the templates through the column, assuming 100% hybridization. The purified primer-templates were shown by native gel electrophoresis to contain less than 1% unannealed primers.

Quantitation of Nucleic Acids

The concentration of unlabeled RNA transcript was estimated by titration with a known amount of 5`-P-labeled DNA primer (0.025 pmol), using the conditions of the strand transfer reactions. The primer-templates were either extended with murine leukemia virus RT or just separated on a native gel. The concentration of RNA was estimated from the level of RNA required to extend 50% of a known concentration of primers assuming 100% hybridization. The RNA concentration was also determined by estimating the amount of RNA required to hybridize 50% of a known concentration of radiolabeled primer. The concentrations of the DNA primers and of the acceptor templates were determined by spectrophotometry.

Quantitation of Primer Extension Products

All quantitation was obtained with a PhosphorImager (Molecular Dynamics).

Runoff Transcript

T7 RNA polymerase-catalyzed runoff transcription was performed as specified by the Boehringer Mannheim manufacturer's protocol. Plasmid pBSM13() (DeStefano et al., 1992) was linearized using BstN1 and then used for the runoff transcriptions. The transcript was treated with RNase-free DNase I and was then gel purified by electroelution.

Gel Electrophoresis

Denaturing 8% sequencing gels (19:1 acrylamide/bisacrylamide) containing 7 M urea were prepared and subjected to electrophoresis as described (Sambrook et al., 1989)


RESULTS

Effect of NC on Strand Transfer Reactions

NC has been shown to promote annealing and melting of nucleic acids. These are important steps in the strand transfer reaction. Initial experiments addressed whether NC had an effect on internal strand transfer reactions. We measured strand transfer using a donor substrate consisting of an RNA segment to which a DNA oligonucleotide was annealed (Fig. 1). Full-length extension of the primer on this template resulted in a product 108 nucleotides in length. Also present was a DNA acceptor template 68 nucleotides long, with a 27-nucleotide region that was exactly homologous to the donor template beginning 31 nucleotides beyond the primer. Strand transfer and completion of synthesis on the acceptor template resulted in a product 118 nucleotides long.

Increasing concentrations of NC were added to the strand transfer assay reaction described under ``Experimental Procedures.'' Fig. 2shows that as the concentration of NC was increased, the number of primers that started synthesis increased by 11-79% as determined by PhosphorImager analysis of the total amount of radioactivity in elongated primers. The stimulation of primer initiation reached a plateau at NC concentrations that would coat more than 50% of the template. With subsequent increments of NC addition, the number of primers that underwent synthesis declined.


Figure 2: Effect of NC on the level of strand transfer. Reverse transcription was performed for 30 min as described under ``Experimental Procedures.'' NC concentration was titrated from that needed to coat 200% of the templates (lane1), 100% (lane2), 50% (lane3), 25% (lane4), 12.5% (lane5), 6.2% (lane6), 3.1% (lane7), and 1.5% (lane8), to no NC (lane9). The length in nucleotides of full-length donor template-directed DNA synthesis products (F) and products produced by strand transfer events (T) are indicated. The region where the donor and acceptor templates were homologous is indicated.



Of the primers that initiated synthesis, the proportion that reached the end of either the donor template or acceptor template was also increased as the NC concentration was augmented (). However, the proportion of initiated primers extended to pause sites in the region of homology between the donor and acceptor templates was the same or less in the presence of NC. This is consistent with the reported ability of NC to melt secondary structures (Khan and Giedroc, 1992; Tsuchihashi and Brown, 1994; You and McHenry, 1994).

We define transfer efficiency as the quantity of DNA primers extended to the end of the acceptor template (T), divided by the sum of the quantity extended to the end of the donor template (F) plus those extended to the end of the acceptor (T). The quantities of these synthesis products on the gel were determined with the PhosphorImager. shows the transfer efficiencies calculated for each reaction shown in Fig. 2. The efficiency of transfer was slightly increased in the presence of NC. This increase was concomitant with changes in band intensity in the region of homology between donor and acceptor. A slight increase in transfer efficiency occurred even at concentrations of NC that inhibit primer initiation. At high concentrations of NC, the absolute number of primers initiated was reduced. However, the proportion of initiated primers that underwent full-length synthesis over both donor and acceptor templates was higher than when NC was not present. Therefore, in addition to altering the number of primers that initiate, NC influences transfer efficiency.

Effect of Znon NC Simulation of Strand Transfer

NC has two Zn finger domains. Some NC functions are not Zn dependent, such as increasing tRNA unwinding and stimulation of initiation of synthesis (Khan and Giedroc, 1992; De Rocquigny et. al., 1992). The role of the Zn fingers or of Zn ions with respect to internal strand transfer reactions is not known. We tested whether the presence of Zn has an effect on the stimulation of strand transfer by NC. We compared the effect of ZnCl on strand transfer reactions at different concentrations of NC (Fig. 3). Note that the synthetic NC used here is not initially chelated with Zn. We observed that NC continues to stimulate strand transfer in the presence of ZnCl. Furthermore, the concentrations of NC needed to achieve maximum stimulation of strand transfer are slightly lower in the presence of ZnCl. A likely possibility is that the presence of Zn protects the NC from inactivation as a result of denaturation. The effect would be to increase the concentration of active protein. This interpretation is consistent with the NMR observations, which show that NC is more stable in the presence of ZnCl (Morellet et al., 1992; Surovoy et al., 1993). The remaining experiments discussed in this paper have been performed both in the presence and absence of ZnCl. For brevity we are showing only those performed in its presence. In a series of five independent experiments in the presence of ZnCl strand transfer efficiency increased from 28 to 33% in reactions with NC at 10% of template-coating level compared with reactions with no NC. Application of a matched pairs t test indicated a significance of this difference with p = 0.03.


Figure 3: Influence of ZnCl on NC activity. Reverse transcription was performed with decreasing concentrations of NC in the presence or absence of ZnCl (50 µM) as indicated. The length in nucleotides of full-length donor template-directed DNA synthesis products (F) and products produced by strand transfer events (T) are indicated. Amounts of NC used were 200% template-coating level (lanes1 and 7), 100% (lanes2 and 8), 50% (lanes3 and 9), 10% (lanes4 and 10), 5% (lanes5 and 11), and none (lanes6 and 12).



Fig. 4presents a time course experiment of the transfer reaction in the presence and absence of NC. Previous work has shown that the standard transfer reaction is slower than synthesis over the donor template, resulting in an increase in transfer efficiency with time (DeStefano, 1994). Here transfer efficiency reached a maximum by 30 min, in the presence or absence of NC.


Figure 4: Time course of strand transfer reaction in the presence and absence of NC. Reverse transcription was performed in the presence of 50 nM NC and in the absence of NC as indicated in the figure. The reaction was stopped at different points in time as indicated. The positions of full-length donor directed DNA synthesis products (F) and transfer products (T) are indicated.



Effect of NC on HIV-RT Processivity

Processivity is the length of primer extension that occurs each time the RT initiates synthesis and before it dissociates. Here primer extension is not a simple distribution but a series of products extended to pause sites and over the full length of the template. Consequently processivity cannot readily be quantitated numerically. Instead we can compare the distribution of products made by a single round of processive synthesis. Fig. 2shows that NC at low concentrations has a stimulatory effect on overall end product synthesis. This suggests that NC at particular concentrations may increase the ability of HIV-RT to remain associated with the template until full-length synthesis is accomplished, increasing processivity.

In order to examine the effect of NC on RT processivity we performed strand transfer reactions in the presence of a compound that traps and inactivates RT that is not bound to polynucleotide substrate. A suitable trapping compound is heparin (Peliska and Benkovic, 1992). The appropriate concentration of heparin trap was chosen so that if the heparin is mixed with the primer-template and then the RT is added, no subsequent synthesis is observed. We used the minimal concentration of heparin that would serve as a complete inhibitor of unbound RT. During the course of the experiment, the primer-template was incubated with the RT first, and then the reaction was started by adding Mg, dNTPs, and the heparin trap. In this fashion we were able to determine the length of products synthesized by the RT during a single binding event. In the presence of ZnCl, NC stimulated synthesis at a 10% template coating level and was slightly inhibitory at a 50% coating level (Fig. 3). The reactions were performed at these two levels of NC. The products of synthesis were resolved by gel electrophoresis (Fig. 5A). At the stimulatory level of NC the total number of primers initiated was only slightly higher than in the absence of NC. At the inhibitory level of NC the total number of primers initiated was lower than in the absence of NC. The time course progression of primer elongation was slightly greater at the stimulatory level of NC. Interestingly, the positions of pausing are distributed differently with the addition of NC. At the highest NC level there was greater pausing over the first half of the template and proportionally less in the homologous region than in the absence of NC. However, the proportion of extended primers that underwent full-length synthesis was the same with either concentration of NC as well as without NC.


Figure 5: A, time course of primer extension in a challenged processivity assay. Internal strand transfer reactions were performed as described under ``Experimental Procedures'' in the presence of a heparin trap. Inhibitory (250 nM) and stimulatory (50 nM) concentrations of NC were tested and compared with reactions in the absence of NC (0 nM). The reactions were stopped at the indicated intervals. LaneC represents a control reaction in which the heparin trap was added to the reaction prior to the RT. No synthesis occurred, indicating that the trap was effectively inhibiting free RT from binding to the template. The same control reaction shows no synthesis in the presence of NC (data not shown). The positions of full-length donor-directed DNA synthesis products (F) are indicated. Products produced by strand transfer events (T) are not detectable. B, NC titration in the presence and absence of a heparin trap. Strand transfer reactions were performed in the presence () and absence () of heparin trap. The radioactivity in full-length products of synthesis was determined by PhosphorImager analysis. The amount of full-length product synthesized in the absence of NC was used as a control. The amounts of radioactivity in full-length products of synthesis using different concentrations of NC are represented as percent of control (y axis). The different concentrations of NC utilized are indicated as a percentage of template coating levels (x axis).



An effect of NC on processivity could be masked if the heparin trap were competing away the NC from the templates. We ruled out this possibility by performing strand transfer reactions over a range of NC concentrations in the presence or absence of heparin. At the concentration of heparin used, the stimulatory effect of NC starts appearing at the same NC concentrations in the presence and absence of heparin (Fig. 5B). This demonstrated that the heparin was not altering the amount of NC bound to the template. We note that NC concentration for maximum stimulation varies somewhat between experiments (Tsuchihashi and Brown, 1994), possibly because of differences in oxidation of NC aliquots.

We also studied the effect of NC on RT processivity in the absence of a trap. This was accomplished by performing RT dilution experiments (Fig. 6). We diluted the RT to a level of 1 RT/20 templates. At this level of RT, 90% of the primers do not sustain any synthesis during the course of the reaction (Fig. 6, lanes6, 12, and 18). Consequently, it was unlikely that after completion of one round of synthesis on a primer-template, an RT would have returned to the same primer-template during the time of the reaction. This is because unreacted primer-templates are in large excess over RTs. Therefore, the lengths of extension of primers that have undergone synthesis represent elongation from a single RT binding event. At this RT dilution, the distribution of the products of synthesis was the same in the presence and absence of NC. PhosphorImager analysis of all the extended primers shows that the same proportion of primers reached the end of the template whether NC was present or not. Evidently, NC has no detectable effect on processivity.


Figure 6: Dilution processivity assay. Amounts of RT used were 2 units (lanes1, 7, and 13), 1 unit (lanes2, 8, and 14), 0.2 units (lanes3, 9, and 15), 0.1 units (lanes4, 10, and 16), 0.024 units (lanes5, 11, and 17) and 0.012 units (lanes6, 12, and 18).



Requirement for RT Dissociation for Strand Transfer Reactions

When reactions were performed in the presence of a trap (Fig. 5) or with very diluted RT (Fig. 6) strand transfer was very inefficient. The transfer product is slightly longer than the full-length product on the donor template. Therefore, it is possible but unlikely that the RT cannot synthesize that distance in one round of processive synthesis. Instead, it is probable that dissociation of the RT invariably accompanies the transfer process. Reassociation of the RT would then be necessary for completion of synthesis of the transfer product on the acceptor template. Since reassociation is prevented under the conditions for measuring processivity, essentially no full-length transfer product is observed.

Effect of dNTP Concentration on Internal Strand Transfer in the Presence of NC

Internal strand transfer efficiency was increased when concentrations of dNTPs were lowered from 50 to 1 µM (DeStefano et al., 1992). This concentration of dNTPs slows synthesis and would be expected to have a similar effect as pausing in the transfer zone. This suggests that pausing promotes transfer simply by increasing the time that the RT and primer terminus are in the homologous region. We investigated whether lowering the dNTP concentration in the reaction would influence the effect of NC on the strand transfer event. Once again we observed that at lower concentrations of dNTPs strand transfer efficiency was improved (Fig. 7). Furthermore, stimulation of the transfer reaction continues to be observed at lower dNTP concentrations. The stimulatory action of NC should allow the RT to move faster through the region of acceptor homology. The low concentration of dNTPs should slow movement of the RT over the whole template including the homologous region. It appears that the stimulation of movement of the RT over the transfer zone by NC was counterbalanced by the low dNTP concentration. When the RT movement was slowed by low dNTPs the increase in transfer efficiency produced by NC was even greater than at standard dNTP concentration.


Figure 7: Effect of dNTP concentration on strand transfer reactions. The concentration of dNTPs was titrated from 150 µM to 0.2 µM. Strand transfer efficiency was assessed by quantitating the products of synthesis over the donor and acceptor templates using a PhosphorImager. Strand transfer efficiency was calculated as described under ``Results.'' Strand transfer efficiency was plotted against dNTP concentration. Strand transfer in the presence () and absence () of NC was compared.




DISCUSSION

We have investigated the effect of HIV-NC on the HIV-RT-catalyzed internal strand transfer reactions from a heteropolymeric RNA donor template to a complementary acceptor template. NC slightly enhanced the internal strand transfer reactions from the template used. Previous work has correlated enhanced strand transfer with reaction conditions that slow the progression of synthesis through the region of homology between the two templates. However, properties of NC suggest that it should melt secondary structure in the template. NC is known to promote both melting and reannealing of double-stranded DNA. These characteristics of the NC are consistent with an increased rate of synthesis that occurs as the concentration of NC is raised. If primers were elongated more rapidly over the region of homology, the efficiency of transfer should have been reduced. The observation of increased transfer indicates that other features of the NC-coated template improve transfer efficiency. This improvement is sufficiently large to overcome the effects of faster primer elongation in the homologous region and results in a net increase in transfer efficiency.

NC has a complex effect on properties of nucleic acids, which is concentration-dependent. Tsuchihashi et al. (1993) and Herschlag et al.(1994) reported that at high concentrations, NC inhibited multiple turnover and single turnover of a hammerhead ribozyme by shutting down the cleavage step. At lower concentrations NC enhanced the multiple turnover of the ribozyme by increasing the annealing of the substrate RNA and by catalyzing the dissociation of the product after cleavage by the ribozyme. Similarly, Tsuchihashi and Brown(1994) showed complete melting of short double-stranded DNA at high NC concentrations, while at lower concentrations, NC stimulated renaturation. You and McHenry(1994) reported a very large stimulation of annealing of complementary RNA and DNA segments of the HIV R region, which seems to be caused by melting of secondary structure in these segments. Dib-Hajj et al. (1993) have evidence that two types of saturated NC-polynucleotide complexes can be formed that differ in apparent site size: n = 8 versusn = 14. The smaller site size binding mode was also observed by You and McHenry(1993). The formation of the smaller and larger site size complexes occurs at higher and lower concentrations of NC, respectively. The functional effects of NC that we have observed are explainable by a progressive weakening of the stability of double helices with increasing concentration of NC. However, a relationship between site size and NC effects cannot be ruled out.

We have shown that the effect of NC on synthesis stimulation is also concentration-dependent. At low concentrations, NC increases RT synthesis, while at high concentrations it inhibits synthesis. Part of the stimulation is likely to result from the ability of NC to melt secondary structures (Lapadat-Tapolsky et al., 1993; Tsuchihashi and Brown, 1994; You and McHenry, 1994). We have observed nonuniform changes in the pattern of paused intermediates in the presence of NC. It is believed that the binding of NC to nucleic acids is not specific with respect to sequence. The only evidence so far of preferential NC binding to a specific sequence has been the interaction with the putative HIV packaging (PSI) domain (Dannull et al., 1994). This suggests that NC could also interact with other specific viral sequences that have not yet been identified. Some sequence specificity of binding could explain the nonuniform effect of NC on RT pausing over different regions of the template.

Primer extension is inhibited at high levels of NC. One possibility is that as the concentration of NC approaches a value that can coat the majority of the single-stranded polymer, large amounts of free NC become available to displace the primer from the template.

Another explanation for the inhibitory effect of NC is that it may promote the dissociation of RT prior to initiation of synthesis from the primer template. NC forms coaggregates, large complexes involving interaction with itself and nucleic acids (Tsuchihashi and Brown, 1994; Lapadat-Tapolsky et al., 1993). At high NC concentrations, excess protein could aggregate at the recessed 3`-end and thereby hinder the binding of the RT. Recent evidence argues in favor of this possibility. If the primer-template-RT-dNTP complex is formed before NC is added to the reaction, synthesis occurs and the NC stimulates melting of secondary structures.()When NC is added before the complex is formed, it inhibits initiation of primer elongation. It is possible that the RT encounters difficulty in moving through the first few nucleotides if NC is aggregated over the primer region. We are presently studying this possibility.

We determined that neither the stimulatory nor the inhibitory effects of NC on RT synthesis were due to an effect on RT processivity. At both tested concentrations of NC, the proportion of primers that were extended to the end of the template was the same as in the absence of NC. We obtained similar results whether we used a heparin trap or diluted the RT enough to observe the results of a single RT binding event. It appears from NC-induced changes in product distribution under conditions where it stimulates synthesis that NC melts some of the template secondary structures and not others. Indeed, the large stimulation of annealing of R region sequences suggests that NC is particularly effective at altering secondary structure of this sequence (You and McHenry, 1994). On such a template, processive synthesis may increase in response to NC. This would be expected since the likelihood of the RT to dissociate from the template is high at pause sites (Klarmann et al., 1993). The results of our processivity assays in the presence of inhibitory levels of NC suggest that NC does not inhibit synthesis by increasing the dissociation of the RT from the template.

Examination of the time course of primer extension in processivity assays shows little effect of NC on the rate of movement of the RT over the template. However, there is a large stimulation of synthesis of full-length products observed in the standard assays in which the RT is allowed to carry out multiple cycles of synthesis, dissociation, rebinding, and further synthesis. This suggests that a step in the reaction that occurs in the standard assay but not the processivity assay is being stimulated by NC. A likely possibility is the rebinding of the primer-template. The presence of the protein may create the appropriate environment to facilitate nearby RTs to rebind the primer-template faster. Another possibility is that nonspecific binding of the RT to portions of the template away from the primer terminus is discouraged, allowing more RT to be available for productive binding.

We propose that the stimulation of strand transfer by NC is the net result of opposing activities of this protein. On one hand, observed stimulation of synthesis by NC should inhibit strand transfer. On the other hand, strand exchange capacities of the NC should stimulate transfer. Furthermore, we have presented evidence that NC improves the rebinding of the RT to the template after dissociation, a process that should also promote strand transfer.

  
Table: Quantitation of products of synthesis

The concentration of NC necessary to coat 100% of all templates in the reaction was calculated as 495 nM. The counts of all primers elongated per reaction were determined by PhosphorImager and are shown in the second row. The relative amounts of products that underwent full-length synthesis over either the acceptor or donor templates are shown, labeled T and F respectively. Transfer efficiency is shown in the last row (for details see ``Results'').



FOOTNOTES

*
This research was supported by National Institutes of Health Grants GM 49573 and AI 01146, Cancer Center Core Grant CA11198, and the James P. Wilmot Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-275-3269; Fax: 716-271-2683.

The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; NC, nucleocapsid protein.

X. Ji and B. D. Preston, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Jasbir Seehra and John McCoy, representing Genetics Institute, for the generous gift of HIV-RT.


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