Involvement of HIV-I Nucleocapsid Protein in the Recruitment of Reverse Transcriptase into Nucleoprotein Complexes Formed in Vitro*

Retroviral reverse transcription takes place within the virion core, where nucleocapsid (NC) protein (NCp) molecules cover the dimeric RNA genome. NCp thus has structural roles in the virion core but is also extensively involved in viral DNA synthesis and virion assembly. To further characterize the role of human immunodeficiency virus type 1 NCp7 during replication of the viral genome, we investigated the relationship between NCp7 and reverse transcriptase (RT) either directly or within nucleoprotein complexes in vitro . We show that NCp7 interacts directly with RT and enhances synthesis of full-length cDNA by increasing RT processivity. Using NCp7 mutants, we show that the complete amino acid sequence of NCp7 is required for functional interactions with RT. Our results suggest that NCp7 plays a role in recruitment of RT into stable and functional nucleoprotein complexes during viral DNA synthesis. Retroviral replication requires conversion of the single-stranded RNA genome into an integration-competent, double-stranded viral DNA (1). This conversion is accomplished by viral reverse transcriptase (RT), 1 which possesses RNA and DNA-dependent DNA polymerase 2 , 10 m M ZnCl 2 , and 5 m M dithiothreitol) and then diluted 2-fold with NC buffer, 1% Nonidet P-40 (Nonidet P-40) containing 4 m g of anti-NCp7 mouse monoclonal antibody HH3 (27), and incubated at 4 °C for 1 h with gentle agitation. All subsequent steps performed

Retroviral replication requires conversion of the singlestranded RNA genome into an integration-competent, doublestranded viral DNA (1). This conversion is accomplished by viral reverse transcriptase (RT), 1 which possesses RNA and DNA-dependent DNA polymerase activities, and occurs within a nucleoprotein complex derived from the virion core. The nucleoprotein complex contains the dimeric RNA genome in close association with 2000 -3000 molecules of nucleocapsid (NC) protein (2), as well as molecules of cellular tRNAs, RT, integrase, and other viral and cellular proteins (3,4). The nucleoprotein complex is a stable substructure of the viral particle (5) that protects the genomic RNA to ensure successful viral replication and dissemination (6,7). Stable nucleoprotein complexes can be generated in vitro using HIV-I NCp7, viral RNA, primer tRNA Lys,3 , and RT (8). Despite the fact that NCp7 covers the viral RNA (at a stoichiometry of about 1 NCp7 per 7 nt), offering effective protection against nucleases, reverse transcriptase is able to enter these complexes and initiate reverse transcription from tRNA Lys,3 hybridized to the primer binding site (9 -13).
NCp7 is a small basic protein that binds to both RNA and DNA. The protein has nucleic acid annealing activity and promotes rapid and extensive hybridization of complementary nucleic acid sequences. NCp7 also exhibits strand exchange activity that favors the most stable double-stranded product (14 -16). These properties are implicated in the ability of NCp7 to promote reverse transcription by stimulating both annealing of the tRNA Lys,3 to the primer binding site (9 -13) and the annealing of complementary repeat sequences of viral RNA and strong stop cDNA (17)(18)(19)(20)(21)(22) during minus strand DNA transfer. In addition, NCp7 enhances the synthesis of full-length cDNA by influencing RT processivity (8,(22)(23)(24).
The apparent functional cooperation between NCp7 and RT prompted us to closely examine possible interactions between these two viral proteins. Here we show that NCp7 interacts directly with RT, an interaction dependent upon the integrity of the nucleocapsid protein. We further show that NC/RT interactions underlie the enhancement of RT processivity within nucleoprotein complexes formed in vitro, suggesting that they are critical for DNA synthesis in vivo.
Immunoprecipitation Assays-31 pmol of NCp7 WT and 17 pmol of RT p66/p51, p66, or p51 were mixed and incubated for 30 min at room temperature in 50 l of NC buffer (20 mM Tris-HCl, pH 7, 50 mM NaCl, 0.2 mM MgCl 2 , 10 M ZnCl 2 , and 5 mM dithiothreitol) and then diluted 2-fold with NC buffer, 1% Nonidet P-40 (Nonidet P-40) containing 4 g of anti-NCp7 mouse monoclonal antibody HH3 (27), and incubated at 4°C for 1 h with gentle agitation. All subsequent steps were performed * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by grants from the French AIDS research program (ANRS) and the Mutuelle Générale de l'Education Nationale.
‡ Supported by a grant from the Direction Régionale de l'Industrie et de la Recherche en Rhône-Alpes and ANRS.
For dot blot analysis, 62 pmol of NCp7 WT and NCp7 mutants were blotted onto nitrocellulose. The membrane was dried, and proteins were cross-linked to it by UV radiation (0.1 J/cm 2 ). The membrane was blocked in HBB, 5% milk powder at room temperature for 4 h, washed with HBB, probed with 0.62 pmol/ml RT in HBB at 4°C overnight, and washed three times with phosphate-buffered saline. Bound proteins were detected by incubation with anti-RT polyclonal antibodies and ECL.
Reverse Transcription Assay-Reactions were performed basically as described previously (9,17,19). Primer tRNA Lys,3 was heat-annealed to the primer binding site of 5Ј-RNA to avoid annealing differences due to the use of NCp7 WT or NCp7 mutants (1 pmol of tRNA and 1 pmol of 5Ј-RNA, 2 min at 90°C, followed by a 20-min incubation at 70°C). Template-primer tRNA Lys,3 complexes were added to 29.6 pmol, 59.2 pmol, or 118.4 pmol of NCp7 WT or NCp7 mutants (see figure legends for NCp7 to nucleotide ratios) and incubated for 5 min at 37°C in a 15-l final volume. Reverse transcription was started by adding RT p66/p51 (1 pmol; the RT:nucleic acid ratio thus being 1:1) and dNTPs (at 0.25 mM each) in 30 mM Tris-Cl, pH 8.2, 60 mM NaCl, 3 mM MgCl 2 , 5 mM dithiothreitol in a 25-l final volume and incubated at 37°C for 30 min. Reactions were stopped with 10 mM EDTA, 1% SDS and heated for 3 min at 65°C, and proteins were extracted by phenol and phenol/chloroform. Nucleic acid was precipitated with ethanol, recovered by centrifugation, dissolved in 95% formamide, heat-denatured for 2 min at 95°C, and resolved by 5% PAGE, 7 M urea in 40 mM Tris-borate, pH 8.3. Alternatively, primer-5Ј-RNA complexes were formed by incubating 1 pmol of 5Ј-RNA and 1 pmol of tRNA Lys,3 in the presence of 29.6, 59.2, or 118.4 pmol of NCp7 WT or NCp7 mutants (see figure legends for NCp7:nucleotide ratios) for 15 min at 37°C in a 15-l final volume. Reactions were started by the addition of RT and dNTPs as above.
A radiolabeled primer DNA complementary to TAR (1 pmol) was annealed to the TAR region of a 3Ј-RNA (1 pmol) by heat (2 min at 95°C and then 30 min at 65°C). The primer-template complex was added to 46, 92, or 184 pmol of NCp7 WT or NCp7 mutants (see figure legends for NCp7:nucleotide ratios) and incubated for 5 min at 37°C in a 15-l final volume. Reverse transcription was as above, and reaction products were resolved by 6% PAGE, 7 M urea. Alternatively, primer-template complexes were formed by incubating 1 pmol of 3Ј-RNA and 1 pmol of TAR oligo in the presence of 46, 92, or 184 pmol of NCp7 WT or NCp7 mutants (see figure legends for NCp7:nucleotide ratios) for 15 min at 37°C in a 15-l final volume. Reactions were initiated upon the addition of RT and dNTPs as above. Quantification was by phosphor-imaging densitometry.
Formation of HIV-I Nucleoprotein Complexes-Primer-templates were annealed as described above and incubated in the presence or absence of NCp7 WT or NCp7 mutants (92 pmol for TAR primer-3Ј-RNA and 59.2 pmol for tRNA Lys,3 -5Ј-RNA, respectively; see figure legends for NCp7:nucleotide ratios) at 37°C for 5 min, to form nucleoprotein complexes. 0.5 pmol of RT was then added either before the centrifugation step (4°C for 5 min, 14,000 ϫ g; Fig. 5A, method 1) or after centrifugation directly to the pellet (Fig. 5A, method 2) and incubated at 37°C for 5 min. After incubation, complexes were recentrifuged, and the supernatant was separated from the pellet. Reverse transcription as-says were then performed on supernatant (S) and pellet (P) fractions (S, P, S1, S2, and P2; see Fig. 5A, methods 1 and 2), as above. Quantification was by phosphor-imaging densitometry.
Nucleoprotein complexes, obtained by adding RT before centrifugation ( Fig. 5A, method 1), were also analyzed by Western blotting. Briefly, supernatant and pellet fractions, obtained as described above, were diluted to 20 l with sample buffer, and proteins were resolved by 10 -15% step gradient SDS-PAGE. After electroblotting, proteins were analyzed by immunoblotting with anti-NCp7 and anti-RT polyclonal antibodies using the ECL system. Quantification was by laser-scanning densitometry.

Interactions between HIV-I RT and
NCp7-Interactions between NCp7 and RT were examined using three separate approaches. In Fig. 1 (lane 1), using mouse mAb against NCp7, we show efficient co-immunoprecipitation of the RT p66/p51 heterodimer. As shown in Fig. 1, lanes 2 and 3, NCp7 WT was able to interact with both p66 and p51 subunits independently. Since both RT and NCp7 have strong affinities for nucleic acid, the same experiment was repeated in the presence of nucleases as a control. The interaction between NCp7 WT and RT was not affected by incubation with RNase A or RQ1 DNase, ruling out any implication of contaminating nucleic acids in this interaction (data not shown).
Second, interactions between RT and NCp7 WT or NCp7 mutants (see Table I) were tested by far Western blotting. Fig.  2A shows a positive signal for NCp7 WT, NCp7-(13-64) and NCp7-(1-55) incubated with RT (lanes 1-3). In contrast, even after lengthy exposure, no signal was detected for the NCp7 1-72 Zn Ϫ mutant (lane 4). No direct cross-reaction between anti-RT and NC proteins was observed (data not shown).
Finally, since denaturing conditions might alter the binding properties of NCp7 during the far Western analysis, RT/NCp7 interactions were also tested under native conditions using a dot blot method (Fig. 2B). NCp7-(1-55) gave a signal equivalent to that of NCp7 WT (lane 3), suggesting that the C-terminal 17 nucleotides of NCp7 are not involved in the interaction with In conclusion, the combined data of Figs. 1 and 2 provide evidence that NCp7 interacts directly with both subunits of RT, an interaction implicating the zinc fingers and the N-terminal domain of the former.
RT/NC Interactions Influence RT Recruitment into Nucleoprotein Complexes-Reverse transcription-competent nucleoprotein complexes mimicking HIV-I nucleocapsid structures can be assembled in vitro (8). We analyzed the ability of the NCp7 mutants to form high molecular mass nucleoprotein complexes containing radiolabeled 32 P-5Ј-viral RNA, focusing our attention on those mutants that were found to interact with RT (Fig. 2, A and B). After incubation with the radiolabeled 5Ј-RNA, reaction mixtures were centrifuged to separate high molecular weight complexes (pellet fractions) from uncomplexed material (supernatant) (Fig. 3, Method 1), and the amount of labeled RNA in each fraction was determined. As summarized in Table II, NCp7 WT and NCp7-(1-55) exhibited similar ability to form high molecular mass complexes at physiological stoichiometry (protein:nt ratio of 1:7), while at lower concentrations (protein:nt ratios of 1:28 and 1:21) WT NC protein remained the most effective. The NCp7-(13-64) mutant was less efficient than the WT at forming nucleoprotein complexes even at high protein:nt ratios. These results may largely be explained by a lower affinity of the NCp7 mutants for RNA (data not shown), although NC/NC interactions, crucial for nucleoprotein complex formation (8), may also be implicated.
To investigate RT recruitment into nucleoprotein complexes, we analyzed by Western blotting the protein composition of supernatant and pellet fractions obtained from complexes formed in the presence of limiting amounts of RT (RT:primer ratio of 1:2) using the 3Ј-RNA and the TAR (Ϫ) primer (Fig. 3,  Method 1). As summarized in Table III, when complexes were formed with NCp7 WT and NCp7-(1-55) using an occluded site size of 7 nt for NC, 80 -90% of NC was found in the sedimented fraction, while this value was only 47% for the NCp7-(13-64) mutant. RT was predominantly found in the pellet fraction in the case of NCp7 WT (over 60%), while for NCp7-(1-55) most was present in the supernatant, with only 40% remaining in pellet fraction. When NCp7-(13-64) was used, about 47% of NC protein was present in the pellet fraction, while for RT this value was about 30%.
Reverse Transcription within Nucleoprotein Complexes Formed in Vitro-Reverse transcription was analyzed within nucleoprotein complexes using an occluded site size of 7 nt for NC. Primer-template complexes were formed by heat annealing and then added to NC proteins. Nucleoprotein complexes were formed in the presence of a limiting amount of RT and sedimented, and supernatant and pellet fractions were tested for RT activity by adding dNTPs and Mg 2ϩ (Fig. 3, Method 1). When NCp7 WT and NCp7-(1-55) were used to form the complexes, reverse transcription took place only in the pellet frac-  . 186 pmol of NCp7 WT or NCp7 mutants was subjected to 12-20% gradient SDS-PAGE and then blotted onto nitrocellulose (A), or 62 pmol was directly blotted onto nitrocellulose (B). Both membranes were incubated with 3.1 pmol of RT prior to revelation of NCp7⅐RT complexes using an anti-RT polyclonal antiserum.

FIG. 3. Flow chart of nucleoprotein complex formation.
Nucleoprotein complex formation was as described under "Experimental Procedures." Method 1 describes the formation of complexes resulting from direct incubation of RNA-primer with NC protein and RT, followed by centrifugation. Method 2 describes the formation of complexes where RNA-primer was incubated with NC protein and centrifuged, and RT was added to the pellet. Afterwards, incubation complexes were recentrifuged, and supernatant and pellet fractions were used in reverse transcription assays as described under "Experimental Procedures." NCp7/RT Interactions in Viral DNA Synthesis tion (Fig. 4, lanes 2 and 3 and lanes 18 and 19), indicating that sedimented complexes were fully active. In contrast, when NCp7-(13-64) was used, reverse transcription took place in both fractions (Fig. 4, lanes 11 and 12), reflecting less extensive complex formation. Using a similar system, we tested whether complexes formed in the absence of RT were accessible to RT added after sedimentation (Fig. 3, Method 2). Nucleoprotein complexes formed in the absence of RT were sedimented, incubated with RT, and resedimented. The pellet obtained (P2) and supernatant of both centrifugation steps (S1 and S2) were tested for RT activity by adding dNTPs and Mg 2ϩ as above. In all cases we observed reverse transcription in P2, indicating that nucleoprotein complexes were indeed accessible to RT (Fig. 4, lanes 4 -7, 14 -16, and 20 -22). With NCp7 WT and NCp7-(1-55), there was no reverse transcription activity in S2, whereas for NCp7-(13-64) a low level of reverse transcription was observed in the S2 fraction, probably highlighting the instability of nucleoprotein complexes formed by this NC mutant.
Influence of NCp7 Mutants on RT Processivity in Vitro-The effects of NCp7/RT interactions on RT activity were further analyzed in a reverse transcription assay using a 415-nt RNA corresponding to the 5Ј-end of the HIV-I genome as template and a synthetic radiolabeled 32 P-tRNA Lys,3 as primer. This system mimics the synthesis of strong stop cDNA (Ϫ), the first step of viral DNA synthesis. Fig. 5A shows almost no elongated products in the absence of NCp7 and a striking enhancement of RT polymerization in the presence of NCp7 WT (lanes 2-4). RT processivity, expressed as the relative percentage of full-length single-stranded cDNA divided by the sum of intermediate products and full-length single-stranded cDNA (raw data from phosphor-imaging densitometry), was optimal at a protein-:nucleotide ratio of 1:14 (approximately 12-fold stimulation; compare lanes 1 and 4) although visibly enhanced even at 1:28. Above a ratio of 1:14, we found strong inhibition of cDNA synthesis (lane 5 and data not shown). Similar results were observed with NCp7-(1-55) (lanes 10 -13). In contrast, NCp7-(13-64), while stimulating RT activity with an accumulation of the full-length product at NCp:nt ratios up to 1:7 did not dramatically improve RT processivity (lanes 6 -9) at the same protein concentration as NCp7 WT.
The effect of NCp7 WT and mutants on reverse transcription was also tested using a 646-nt RNA, corresponding to the 3Ј-end of the HIV-I genome and a 56-nt primer, corresponding to TAR (Ϫ) mimicking elongation after minus strand DNA transfer (Fig. 5B). In this system, we did not observe marked inhibition of cDNA synthesis, even at high NCp7 concentration. FIG. 4. Minus strand DNA synthesis in nucleoprotein complexes. 3Ј-HIV-I RNA and radiolabeled TAR (Ϫ) primer were annealed by heat and used to form high molecular mass complexes in the presence or absence of NCp7 WT or NCp7 mutants. Incubation conditions, purification of the DNA products, and analysis by urea/PAGE were as described under "Experimental Procedures." The gray arrowhead indicates products representing primer-template complexes resistant to denaturation. DNA markers are indicated on the left in nt; full-length product (FL-cDNA) and the 32 P-TAR (Ϫ) primer are shown on the right. The protein:nucleotide ratio was 1:7. Separation of pellet and supernatant fractions was as in Fig.  3. T, total reaction without a centrifugation step. P and S are the pellet and supernatant fractions, respectively (method 1 in Fig. 3). P2, S1, and S2 are the pellet and supernatant fractions obtained as described in method 2 of Fig. 3. The data are representative of three independent experiments.

TABLE III Relative amount of proteins in nucleoprotein complexes
Nucleoprotein complexes were formed in the presence of NCp7 WT or NCp7 mutants and RT (Fig. 3, Method 1), centrifuged, and analyzed by Western blotting as described under "Experimental Procedures." S.D. was calculated from three independently quantified experiments. RT

NCp7/RT Interactions in Viral DNA Synthesis
However, as with the 5Ј-RNA-tRNA Lys,3 system (Fig. 5A), NCp7 WT and NCp7-(1-55) significantly increased RT processivity (Fig. 5B, lanes 2-4 and 8 -10, respectively), while NCp7-(13-64) was noticeably less effective in this regard (lanes 5-7). Similar experiments were carried out using NCp7 WT in the presence of heparin in order to sequester dissociated RT, thus ensuring a single RT binding event. The results obtained (data not shown) were very much consistent with those shown in Figs. 5, A and B.

DISCUSSION
Current data suggest that NCp7 is an essential co-factor of reverse transcriptase in vitro (22)(23)(24). Its RNA binding and annealing activities (14 -16) are necessary for primer tRNA Lys,3 hybridization to the primer binding site as well as minus strand DNA transfer (18,19,21,22). Recent work in our laboratory also supports an important role for NCp7 during the reverse transcription step of viral replication in vivo (28 -30).
In vitro reconstitution of functional nucleoprotein complexes showed that their stability is achieved through the binding of NCp7 to the RNA template and NCp7/NCp7 interactions (8). The coating of RNA by NC and protein/protein interactions within the resulting complex most likely account for the protection of viral RNA from RNase degradation (8). While NC/NC interactions may contribute to nucleoprotein complex stability and protection of the RNA template, it remains difficult to reconcile these properties with the widely reported ability of NC to enhance RT activity (22)(23)(24), which requires access to the primer and template during replication. Possible interactions between NCp7 and RT have been inferred in the past (18,21,31), but the nature of this interaction and its implication in the co-operation of RT and NCp7 during reverse transcription are poorly understood. For these reasons, the focus of the present work was on putative NCp7/RT interactions within nucleoprotein complexes.
Using three independent methods to probe protein/protein interactions in vitro, we show that NCp7 and RT are capable of direct interaction (Figs. 1 and 2). This interaction is independent of nucleic acid integrity, since it remains unaffected by FIG. 5. Effect of NCp7 mutants on RT processivity with 5-HIV-I RNA and 3-RNA template. RNA templates and primers were annealed by heat and used in reverse transcription assays in the presence or absence of NCp7 WT or mutants. After incubation, the purification of DNA products and analysis by urea/PAGE were as described under "Experimental Procedures." Numbers on the right correspond to DNA size markers in nt, and arrows on the left indicate intermediate products. A, reverse transcription of 5Ј-HIV RNA using synthetic 32 P-tRNA Lys,3 as primer. Strong stop cDNA (ss-cDNA) and primer tRNA Lys,3 are indicated on the right. Protein:nt ratios used were 1:28, 1:21, 1:14, and 1:7 for each NCp7 mutant. B, reverse transcription of 3Ј-HIV RNA using TAR (Ϫ) as primer. Full-length product (FL-cDNA) and the 32 P-TAR (Ϫ) primer are indicated on the right, and the gray arrowhead indicates primer-template complexes resistant to denaturation. Protein:nucleotide ratios used were 1:14, 1:7, and 1:3.5 for each NCp7 mutant. RT processivity, analyzed in the presence of NCp7 WT or mutants, was expressed as relative percentage of full-length product (ss-cDNA and FL-cDNA, respectively, for A and B) divided by the total amount of elongated primer (full-length primer plus intermediate products). Quantification was by phosphor-imaging densitometry. To facilitate comparison, the greatest stimulation due to NCp7 WT was normalized to 100% (ratios 1:14 and 1:7, respectively, for A and B). The data are representative of four independent experiments. treatment with either RNase or DNase (data not shown). NCp7 interacts with both RT subunits independently (Fig. 1) and requires the presence of both its zinc fingers (Table I, Fig. 2, A  and B). The N-terminal domain of NCp7 also seems to be implicated, since the mutant NCp7-(13-64) interacts less efficiently with RT (Fig. 2B).
Protein/protein and protein/nucleic acid interactions in active nucleoprotein complexes were analyzed in vitro using a centrifugation method to separate material not participating in high molecular mass complexes. NC mutants described herein show markedly different abilities to bind RNA and participate in high molecular mass complexes. NCp7 mutants NCp7-(13-64) and NCp7-(1-55) show 1.5-2-fold decreased recruitment of RNA into complexes relative to WT NCp7 (Table II), although for NCp7-(1-55) the difference is most evident at low NC concentrations. The reduced ability of these NCp7 mutants to promote high molecular mass complexes containing RNA may be partly explained by RNA affinity differences. A filter-binding assay, measuring RNA affinity directly, was in good agreement with the order and magnitude of differences between the NCp7 mutants (data not shown). The variation in RNA affinity between NCp7 mutants NCp7-(1-55) and NCp7-(13-64) is in agreement with previous data (32). Moreover, differences observed in nucleoprotein complex formation may also be due to defective NCp7/NCp7 interactions. We previously showed that the N-terminal domain of NCp7 is important for cDNA synthesis and nucleocapsid morphogenesis in vivo (28), in agreement with evidence demonstrating N-terminal domain involvement in NC/NC interactions (8). Thus, at least for NCp7-(13-64), which lacks this domain, anomalous NC/NC interactions may contribute to its attenuated capacity to form complexes. Work is in progress to better define the regions in nucleocapsid protein involved in NC/NC and NC/RNA interactions.
When we analyzed nucleoprotein complexes in the presence of limiting amounts of RT, we found that the relative quantities of RT co-sedimented with the complexes were lower for NCp7-(1-55) and NCp7-(13-64) relative to NCp7 WT (Table III). This variation may be accounted for by nucleic acid affinity differences outlined above and/or the relative capacities of these NC mutants to recruit RT. For NCp7-(1-55), interaction with RNA is more labile than WT, possibly explaining why RT is retained in complexes to a lesser extent. For NCp7 13-64, both NC/RNA and NC/RT interactions are less efficient, resulting in weak retention of RT in high molecular weight complexes (pellet fraction, Table III). In summary, RT recruitment into nucleoprotein complexes is influenced by the stability of NC/RT interactions as well as the sum of a complex interplay implicating NC/nucleic acid, NC/NC, and RT/nucleic acid interactions.
Reverse transcription within nucleoprotein complexes was possible when a limiting amount of RT was added after sedimentation (Figs. 3 and 4), indicating that unlike nucleases, RT can access the primer-template complex even when coated by NC protein (8). However, only NCp7 WT allowed full recovery of reverse transcription activity in the pellet fraction under these conditions (Fig. 4). Furthermore, NCp7 enhances RT processivity, in agreement with recent reports (22)(23)(24). In our hands, NCp7 WT and NCp7-(1-55) enhance RT processivity, while NCp7-(13-64) is generally much less efficient in systems based on either 5Ј-or 3Ј-HIV-I RNA (Fig. 5, A and B).
Taken together, results presented herein allow us to propose an extended model for the enhancement of RT processivity within nucleoprotein complexes. Coupled to the well documented unwinding activity of NC protein (evidenced by its inhibition of self-priming; see Refs. 12, 20, and 24) NCp7/RNA interactions as well as NCp7/NCp7 and NCp7/RT interactions act synergistically to form functional nucleoprotein complexes, possibly giving rise to local increases in RT concentration. We speculate that NCp7 coating the RNA template acts as a bridge for additional RT/RNA interactions. These may have a stabilizing effect on RT, making it more likely to remain bound to the template and facilitating renewed RT recruitment if/when elongation is prematurely terminated.
The distinct properties observed in this work between NCp7 WT (NCp7-(1-72)) and NCp7-(1-55) are of interest, since both forms are reportedly observed in mature viruses (2). The coexistence of both forms may be of importance, their properties permitting a form of regulation in which distinct activities are utilized at different stages of viral DNA synthesis.