Acceptor RNA Research Articles

Interaction of HIV Reverse Transcriptase with Structures Mimicking Recombination Intermediates

Interactions between human immunodeficiency virus (HIV) reverse transcriptase (RT) and structures mimicking intermediates proposed to occur during recombination (strand transfer) were investigated. One mechanism proposed for strand transfer is strand exchange in which a homologous RNA (acceptor) "invades" a donor RNA.DNA duplex (replication intermediate) on which DNA synthesis is occurring. The acceptor displaces the donor of the duplex and binds to the DNA. During exchange a transient trimeric structure forms. A model structure was designed with a replication intermediate to which an acceptor RNA was bound. The acceptor was bound to the 5'-end of the DNA over a 54-base region, whereas the donor associated with the DNA 3'-end over a 28-base region. The dimeric constituents of the trimer (acceptor RNA.DNA and donor RNA.DNA) were also constructed. The acceptor RNA.DNA formed a branched structure in this case. Results showed that RT could cleave the RNA portion of all the structures examined. Association with junction substrates was less stable as determined by off-rates. On the trimer, RT cleaved both RNAs but showed a clear preference for cleaving the donor RNA region. This preference was accentuated by HIV nucleocapsid protein (NC). Results suggest that during recombination RT generally associates with the donor-RNA portion of the trimer and the acceptor RNA is protected but not immune from cleavage. The partial protection likely allows the acceptor RNA to more easily complete strand exchange and shield this RNA to provide a means to salvage replication if the DNA were to dissociate from the cleaved donor RNA.

Nucleic Acid Conformational Changes Essential for HIV-1 Nucleocapsid Protein-mediated Inhibition of Self-priming in Minus-strand Transfer

Reverse transcription of the HIV-1 genome is a complex multi-step process. HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein that has been shown to greatly facilitate the nucleic acid rearrangements that precede the minus-strand transfer step in reverse transcription. NC destabilizes the highly structured transactivation response region (TAR) present in the R region of the RNA genome, as well as a complementary hairpin structure (“TAR DNA”) at the 3′-end of the newly synthesized minus-strand strong-stop DNA ((−) SSDNA). Melting of the latter structure inhibits a self-priming (SP) reaction that competes with the strand transfer reaction. In an in vitro minus-strand transfer system consisting of a (−) SSDNA mimic and a TAR-containing acceptor RNA molecule, we find that when both nucleic acids are present, NC facilitates formation of the transfer product and the SP reaction is greatly reduced. In contrast, in the absence of the acceptor RNA, NC has only a small inhibitory effect on the SP reaction. To further investigate NC-mediated inhibition of SP, we developed a FRET-based assay that allows us to directly monitor conformational changes in the TAR DNA structure upon NC binding. Although the majority (∼71%) of the TAR DNA molecules assume a folded hairpin conformation in the absence of NC, two minor “semi-folded” and “unfolded” populations are also observed. Upon NC binding to the TAR DNA alone, we observe a modest shift in the population towards the less-folded states. In the presence of the RNA acceptor molecule, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These measurements help to explain why acceptor RNA is required for significant inhibition of the SP reaction by NC, and support the hypothesis that NC-mediated annealing of nucleic acids is a concerted process wherein the unwinding step occurs in synchrony with hybridization.

The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates

R2 is a non-long terminal repeat (non-LTR) retrotransposon that inserts into the 28 S rRNA genes of arthropods. The element encodes two enzymatic activities: an endonuclease that specifically cleaves the 28 S gene target site, and a reverse transcriptase (RT) that can use the 3′ end of the cleaved DNA to prime reverse transcription. R2 RT only utilizes RNA templates that contain the 3′ untranslated region of the R2 element as templates in this target primed reverse transcription (TPRT) reaction. Here, detailed biochemical characterization of the R2 RT indicates that the enzyme is capable of making multiple, consecutive jumps between RNA templates. The terminal 3′ nucleotide of the “acceptor” RNA and the 5′ nucleotide of the “donor” RNA are frequently reverse transcribed in these jumps, indicating that the acceptor RNA does not anneal to the cDNA derived from the donor RNA template. These template jumps occur during TPRT as well as in non-specific extension reactions in which reverse transcription is primed by an oligonucleotide annealed to the RNA template. Analysis of these RT assays done in the absence of the target DNA also revealed that the R2 RT can initiate reverse transcription near the 3′ end of any RNA molecule using the 3′ end of a second RNA molecule as primer. Again there is no requirement for sequence complementarity between the RNA used as template and the RNA used as primer. These properties of the R2 RT differ substantially from those of retroviral RTs but have similarities to the RT of the Mauriceville retroplasmid of Neurospora crassa. We present a model which relates these unusual properties of the R2 RT to structural differences from retroviral RTs as well as correlates these properties to the likely retrotransposition mechanism of R2 and other non-LTR retrotransposons.

In Vitro Strand Transfer from Broken RNAs Results in Mismatch but Not Frameshift Mutations

An in vitro system to compare the fidelity of strand transfers from truncated vs full-length RNAs was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. All RNAs were derived from the N-terminal portion of the α-lac gene. On full-length donors, transfers occurred when DNAs migrated to the acceptor prior to being completed on the donor. On donors that were truncated, most transfers occurred after DNAs reached the end of the donor. Transfer products were amplified by PCR and used to replace the corresponding region in a vector containing the α-lac gene. Transformed Escherichia coli were screened for α-complementation by blue–white phenotype analysis, with white colonies scored as those with errors in α-lac. These errors were derived from RT synthesis and strand transfer. The mutant colony frequency approximately doubled for transfer products derived from truncated donors (0.026 ± 0.005 vs 0.053 ± 0.011 (three experiments ± SD), for full-length vs truncated, respectively). The increases resulted from additional non-template-directed bases (mostly thymidines) added to the DNAs before transfer. Sequence analysis of DNAs synthesized on truncated donors showed that about 60% had additions (20/34); however, those without additions transferred at a much higher rate than those with. Transfer of the DNAs with additions always resulted in substitutions; no frameshifts were observed. Results are consistent with RT adding nontemplated nucleotides at template termini. Transfer and subsequent extension of these products is severely inhibited relative to products without additions. The potential relevance of these findings to retrovirus replication is discussed.

Human immunodeficiency virus reverse transcriptase base misincorporations can promote strand transfer.

A system to determine if HIV-reverse transcriptase (RT) base misincorporations can promote strand transfer was constructed. A donor RNA, on which RT-directed DNA synthesis was initiated, shared homology over a 119 base internal region with an acceptor RNA, to which DNAs initiated on the donor could transfer. Products completed on the donor in the presence or absence of acceptor were isolated and PCR was used to amplify these DNAs. PCR products were ligated into a vector which had this same region (near the N-terminus of the alpha-lac gene) removed. Transformed E. coli were screened in an alpha-complementation assay by blue-white phenotype analysis with white colonies scored as those with errors in plasmid-derived alpha-lac. The frequence of white colonies +/- standard deviations was 0.031 +/- 0.006 and 0.0037 +/- 0.009, for plasmids with inserts derived from donor-directed products synthesized with 100 microM dNTPs in the presence and absence of acceptor template, respectively. Statistical analysis indicated a lower white colony frequency in the presence of acceptor (p = 0.0025). The lower frequency with acceptor implies that a portion of the errors made on the donor are transferred to the acceptor suggesting that base misincorporations can induce strand transfer.

Copy-choice recombination by reverse transcriptases: reshuffling of genetic markers mediated by RNA chaperones.

Copy-choice recombination efficiently reshuffles genetic markers in retroviruses. In vivo, the folding of the genomic RNA is controlled by the nucleocapsid protein (NC). We show that binding of NC onto the acceptor RNA molecule is sufficient to enhance recombination, providing evidence for a mechanism where the structure of the acceptor template determines the template switch. NC as well as another RNA chaperone (StpA) converts recombination into a widespread process no longer restricted to rare hot spots, an effect maximized when both the NC and the reverse transcriptase come from HIV-1. These data suggest that RNA chaperones confer a higher genetic flexibility to retroviruses.

The effect of mutations in the HIV-1 nucleocapsid protein on strand transfer in cell-free reverse transcription reactions.

Interactions between the nucleocapsid protein (NC) and reverse transcriptase of HIV-1 have been shown to promote the initiation of reverse transcription. We assayed the effect of NC on later events, using a strand transfer system with donor and acceptor HIV RNA templates and found that the presence of NC resulted in increased synthesis of full-length strand-transferred (FLST) DNA. This effect also occurred with mutated forms of NC that lacked both zinc fingers, or that contained a point mutation (histidine-->cysteine) at amino acid 23. In contrast, NC-derived proteins containing only the proximal or distal zinc fingers, or lacking the N- and C-termini, were all unable to catalyze the synthesis of FLST DNA. Band-shift assays using both the mutated and wild-type forms of these proteins revealed that all the NC proteins promoted strand association between (-) strong-stop DNA [(-)ssDNA] and acceptor RNA. The zinc finger motifs were dispensable for full-length processive reverse transcription, and the N- and C-termini were required; however, all NC domains were dispensable for association of (-)ssDNA and acceptor RNA. This suggests that annealing is a less stringent reaction than DNA polymerization.

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription.

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys)primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys)was either substituted (S) or deleted (Delta). We used either natural tRNA(3)(Lys)or an 18 nt DNA as primer and measured the efficiency of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA(3)(Lys)increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA. Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.

Correlation of deformability at a tRNA recognition site and aminoacylation specificity.

The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.

Nonhomologous RNA recombination in bovine viral diarrhea virus: molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease.

Four bovine viral diarrhea virus type 2 (BVDV-2) pairs consisting of cytopathogenic (cp) and noncp BVDV-2 were isolated during an outbreak of mucosal disease. Comparative sequence analysis showed that the four noncp BVDV-2 isolates were almost identical. For the cp BVDV-2 isolates, viral subgenomic RNAs were shown by Northern blot to have a length of about 8 kb, which is about 4.3 kb shorter than the genome of noncp BVDV. Cytopathogenicity and the expression of NS3 were both strictly correlated to the presence of viral subgenomic RNAs. By reverse transcription-PCR, Southern blot analysis, and nucleotide sequencing, a set of 11 unique subgenomes was identified with up to 5 different subgenomes isolated from one animal. To our knowledge, this is the first report on isolation of a set of pestiviral subgenomes from individual animals. Common features of the BVDV-2 subgenomic RNAs include (i) deletion of most of the genomic region encoding the structural proteins, as well as the nonstructural proteins p7 and NS2, and (ii) insertion of cellular (poly)ubiquitin coding sequences. Three subgenomes also comprised 15 to 75 nucleotides derived from the 5' part of the NS2 gene. Comparisons of the obtained nucleotide sequences revealed that the different BVDV-2 subgenomes evolved from the respective noncp BVDV-2 by RNA recombination. The presence of short regions of sequence similarity at several crossing-over sites suggests that base pairing between the nascent RNA strand and the acceptor RNA template facilitates template switching of the BVDV RNA-dependent RNA polymerase.

Kinetic Analysis of the Effect of HIV Nucleocapsid Protein (NCp) on Internal Strand Transfer Reactions

The mechanism of HIV reverse transcriptase (RT) catalyzed strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions on RNA templates in the presence and absence of HIV nucleocapsid protein (NCp) was investigated. Two different systems each consisting of DNA-primed RNA donor (on which primer extension initiated) and acceptor (to which DNAs initiated on the donor could transfer) templates were used to determine kinetic parameters of strand transfer. The donor and acceptor shared an internal region of homology where homologous strand transfer could occur. The rate of strand transfer at various acceptor concentrations was determined by monitoring the production of transfer products over time. These rates were used to construct Lineweaver-Burk plots. In each system, NCp increased the Vmax about 3-fold while the Km for acceptor template was decreased severalfold. NCp's effects on RT extension ranged from no effect to inhibition depending on the primer-template used. The lowered Km shows that NCp increases the affinity of the acceptor template for the transferring DNA. Vmax increases despite the inhibition of RT extension. The increased Vmax implies a stimulatory mechanism that cannot be mimicked by high acceptor concentrations. Therefore, NCp does not act by merely increasing the effective concentration of nucleic acids.

The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription.

The mechanism of reverse transcription was analyzed in vitro with RNA templates and the reverse transcriptase (RT) enzyme of human immunodeficiency virus type 1 (HIV-1). In particular, we analyzed the mechanism of actinomycin D (ActD) mediated inhibition of the strand transfer step, in which the newly synthesized cDNA, termed the (-) strand strong stop or (-)ssDNA, is transferred from the donor RNA onto the acceptor RNA. This strand transfer reaction is a rather inefficient process in vitro. We found that this is in part due to the presence of an excess donor RNA, and highly efficient strand transfer was achieved by reducing the amount of donor RNA. We suggest that annealing of the (-)ssDNA to the excess donor RNA is preferred over productive binding to the acceptor RNA because of a higher basepair complementarity. ActD remains a potent inhibitor of strand transfer in this optimized assay system. We measured no effect of ActD on the elongation of reverse transcription or the RNase H action of the RT enzyme. Instead, we provide evidence that ActD acts through direct interaction with the (-)ssDNA, thereby blocking the basepairing capacity of this molecule. The possible use of single-stranded DNA binding molecules as antiretroviral agents is discussed.

In VitroCharacterization of Late Steps of RNA Recombination in Turnip Crinkle Virus: II. The Role of the Priming Stem and Flanking Sequences

Turnip crinkle carmovirus (TCV) has a uniquely high recombination frequency and nonrandom cross-over site distribution among the recombining TCV-associated satellite RNAs. Anin vitrosystem has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putativein vivorecombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E.,EMBO J.17, 2392–2403, 1998). This system mimics the strand transfer and primer extension steps of recombination events. We characterize in detail three RNA factors that, in addition to the previously characterized motif1-hairpin, can influence the efficient generation of 3′-terminal extension products: (i) a primer binding region, termed the priming stem; (ii) a spacer region; and (iii) a U-rich sequence located 5′ of the motif1-hairpin. The priming stem is formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template in the recombinants. The stability and location of the priming stem relative to the motif1-hairpin can influence both the efficiency and initiation site of 3′-terminal extension. A short flexible spacer region between the motif1-hairpin and the priming stem can increase the efficiency of 3′-terminal extensions. A U-rich sequence 5′ of the motif1-hairpin facilitates 3′-terminal extensions and its function partly overlaps with that of the spacer region. These RNA factors may also affect the late steps of RNA recombination in TCV.

Diverse RNA substrates for aminoacylation: clues to origins?

The aminoacylation of RNA structures generally is considered the starting point for the emergence of the theater of proteins from the RNA world. Once an aminoacyl ester linkage is established with an RNA acceptor, peptide bond formation is thermodynamically favored. In contemporary protein biosynthesis, aminoacylation is directed to the 3′-end of the tRNA structure, which, in turn, brings the activated amino acid to the ribosomes for addition to the growing polypeptide chain. But during the last 9 years a variety of artificial RNA substrates for aminoacylation has been reported (1–8). And in this issue of the Proceedings, the list of examples is expanded in a significant way. Felden and Giege (9) describe for the first time an aminoacylation system based on a circular RNA substrate. The results obtained with this novel substrate add further support to the idea that the contemporary system of tRNA aminoacylation could have grown out of early aminoacylation systems that used diverse RNA oligonucleotide substrates. Interactions between some of these aminoacylated substrates could have led to a primitive system of peptide synthesis.

Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays.

In this report we demonstrate that human immunodeficiency virus type 1 (HIV-1) minus-strand transfer, assayed in vitro and in endogenous reactions, is greatly inhibited by actinomycin D. Previously we showed that HIV-1 nucleocapsid (NC) protein (a nucleic acid chaperone catalyzing nucleic acid rearrangements which lead to more thermodynamically stable conformations) dramatically stimulates HIV-1 minus-strand transfer by preventing TAR-dependent self-priming from minus-strand strong-stop DNA [(-) SSDNA]. Despite this potent activity, the addition of NC to in vitro reactions with actinomycin D results in only a modest increase in the 50% inhibitory concentration (IC50) for the drug. PCR analysis of HIV-1 endogenous reactions indicates that minus-strand transfer is inhibited by the drug with an IC50 similar to that observed when NC is present in the in vitro system. Taken together, these results demonstrate that NC cannot overcome the inhibitory effect of actinomycin D on minus-strand transfer. Other experiments reveal that at actinomycin D concentrations which severely curtail minus-strand transfer, neither the synthesis of (-) SSDNA nor RNase H degradation of donor RNA is affected; however, the annealing of (-) SSDNA to acceptor RNA is significantly reduced. Thus, inhibition of the annealing reaction is responsible for actinomycin D-mediated inhibition of strand transfer. Since NC (but not reverse transcriptase) is required for efficient annealing, we conclude that actinomycin D inhibits minus-strand transfer by blocking the nucleic acid chaperone activity of NC. Our findings also suggest that actinomycin D, already approved for treatment of certain tumors, might be useful in combination therapy for AIDS.

Dissecting RNA recombination in vitro: role of RNA sequences and the viral replicase.

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.

High Fidelity of Internal Strand Transfer Catalyzed by Human Immunodeficiency Virus Reverse Transcriptase

A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in the absence of NCp had essentially the same amount of errors as donor-directed products while those produced with NCp showed a slight increase in error frequency. Overall, strand transfer events on this template were highly accurate. Since experiments with other templates have suggested that transfer is error prone, the fidelity of strand transfer may be highly sequence dependent.

A Novel Mutation in Intron K of the PROS1 Gene Causes Aberrant RNA Splicing and Is a Common Cause of Protein S Deficiency in a UK Thrombophilia Cohort

In the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.

The presence of RNA splicing signals in the cDNA construct of the E2 gene of classical swine fever virus affected its expression

E2 is the major neutralizing antigen for classical swine fever virus (CSFV) infection. Previously, we have cloned and sequenced the E2 cDNA of Taiwan strain p97 by the reverse transcription-polymerase chain reaction (RT-PCR) method from CSFV-infected tissue. The presence of RNA splicing donor and acceptor sites were found in the cDNA sequence. In this study, transfection of E2 cDNA into mammalian cells resulted in the production of a spliced RNA. Site-directed mutagenesis of the donor and acceptor sites prevented the RNA splicing event and generated a full length transcript in COS7 cells. Although the spliced E2 transcript has not been reported in natural infection of CSFV, this study suggested that the potential splicing sites affected the E2 gene expression when the plasmid-based E2 gene was introduced into mammalian cells.

Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA.

During the first strand transfer in reverse transcription, minus-strand strong-stop DNA [(-) SSDNA] is annealed to the 3' end of the acceptor RNA in a reaction mediated by base-pairing between terminal repeat sequences in the RNA and their complement in the DNA. The large stem-loop structure in the repeat region known as TAR could interfere with this annealing reaction. We have developed an in vitro human immunodeficiency virus type 1 (HIV-1) system to investigate the effect of TAR on strand transfer. Mutational analysis demonstrates that the presence of TAR in the donor and acceptor templates inhibits strand transfer and is correlated with extensive synthesis of heterogeneous DNAs formed by self-priming from (-) SSDNA. These DNAs are not precursors to the transfer product. Interestingly, products of self-priming are not detected in HIV-1 endogenous reactions; this suggests that virions contain a component which prevents self-priming. Our results show that the viral nucleocapsid protein (NC), which can destabilize secondary structures, drastically reduces self-priming and dramatically increases the efficiency of strand transfer. In addition, the data suggest that the ability to eliminate self-priming is a general property of NC which is manifested during reverse transcriptase pausing at sites of secondary structure in the template. We conclude that this activity of NC is critical for achieving highly efficient and specific viral DNA synthesis. Our findings raise the possibility that inactivation of NC could provide a new approach for targeting reverse transcription in anti-HIV therapy.