Annealing Of tRNA Research Articles

In virio SHAPE analysis of tRNA(Lys3) annealing to HIV-1 genomic RNA in wild type and protease-deficient virus.

BackgroundtRNALys3 annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys3 annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNALys3 to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNALys3, resulting in a more tightly bound and efficient primer for reverse transcription.ResultsIn this report, we have used in virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNALys3 to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNALys3 annealed by Gag in Pr- virions reflects both missing interactions of tRNALys3 with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA.ConclusionsWe propose secondary structure models for the tRNALys3/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNALys3 to HIV-1 viral RNA, followed by a more complete annealing by NCp7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0171-7) contains supplementary material, which is available to authorized users.

Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism

RNA helicase A (RHA) promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNALys3 to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs) that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNALys3 annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNALys3 annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5′-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.

TRNA Annealing onto the HIV-1 Genome Studied by FRET Spectroscopy and Microscopy

In the HIV life cycle, reverse transcription allows conversion of a single stranded genomic RNA into double stranded DNA, capable of integration. A prerequisite for the reverse transcription process is the formation of the initiation complex between the RNA genome and a host-cell tRNA that is used as a primer by reverse transcriptase. The nucleocapsid protein (NC) is involved in this process, which requires hybridization of the tRNA to the complementary primer binding site (PBS) sequence on the RNA genome. Besides the tRNA-PBS interaction, other interactions were proposed to play a role such as an 8-nucleotide motif in the U5 region of the untranslated leader RNA, named the primer activation signal (PAS). We investigate the tRNA annealing process and the presence of a tRNA-PAS interaction using fluorescence resonance energy transfer (FRET) spectroscopy and single molecule FRET microscopy. In our assay, fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the region of the HIV-1 genome in which the PBS sequence is located. We observe better folding of the RNA molecules in the presence of NC than with heat annealing and a large change in conformation of the RNA molecule upon tRNA annealing. Our results give further evidence that tRNA also interacts with the viral RNA PAS motif.

Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA(3)(Lys) serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA(3)(Lys) placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA(3)(Lys) annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

Coordinate Roles of Gag and RNA Helicase A in Promoting the Annealing of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} to HIV-1 RNA

RNA helicase A (RHA) has been shown to promote HIV-1 replication at both the translation and reverse transcription stages. A prerequisite step for reverse transcription involves the annealing of tRNA(3)(Lys), the primer for reverse transcription, to HIV-1 RNA. tRNA(3)(Lys) annealing is a multistep process that is initially facilitated by Gag prior to viral protein processing. Herein, we report that RHA promotes this annealing through increasing both the quantity of tRNA(3)(Lys) annealed by Gag and the ability of tRNA(3)(Lys) to prime the initiation of reverse transcription. This improved annealing is the result of an altered viral RNA conformation produced by the coordinate action of Gag and RHA. Since RHA has been reported to promote the translation of unspliced viral RNA to Gag protein, our observations suggest that the conformational change in viral RNA induced by RHA and newly produced Gag may help facilitate the switch in viral RNA from a translational mode to one facilitating tRNA(3)(Lys) annealing.

Nucleocapsid protein function in early infection processes

The role of nucleocapsid protein (NC) in the early steps of retroviral replication appears largely that of a facilitator for reverse transcription and integration. Using a wide variety of cell-free assay systems, the properties of mature NC proteins (e.g. HIV-1 p7 NC or MLV p10 NC) as nucleic acid chaperones have been extensively investigated. The effect of NC on tRNA annealing, reverse transcription initiation, minus-strand-transfer, processivity of reverse transcription, plus-strand-transfer, strand-displacement synthesis, 3′ processing of viral DNA by integrase, and integrase-mediated strand-transfer has been determined by a large number of laboratories. Interestingly, these reactions can all be accomplished to varying degrees in the absence of NC; some are facilitated by both viral and non-viral proteins and peptides that may or may not be involved in vivo. What is one to conclude from the observation that NC is not strictly required for these necessary reactions to occur? NC likely enhances the efficiency of each of these steps, thereby vastly improving the productivity of infection. In other words, one of the major roles of NC is to enhance the effectiveness of early infection, thereby increasing the probability of productive replication and ultimately of retrovirus survival.

New insights into the formation of HIV-1 reverse transcription initiation complex

HIV-1 reverse transcriptase uses the host tRNA 3 Lys as a primer for the synthesis of the minus DNA strand. The first event in viral replication is thus the annealing of tRNA to the primer binding site (PBS) in the 5′ UTR of the viral RNA. This event requires a major RNA rearrangement which is chaperoned by the viral NC protein. The binding of NC to nucleic acids is essentially non-specific, however, NC is known to bind selectively to hairpins located in the 5′ region of the viral RNA. In a previous study, using an NMR approach in which the reaction is slowed down by controlling temperature, we were able to follow details in this RNA unfolding/refolding process and to uncover an intermediate state. We showed that annealing initiates at the junction between the acceptor and the TΨC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TΨC/D loops. In the present work, we have refined our model of the formation of the tRNA 3 Lys/PBS duplex. First, we show that annealing can initiate both from the single-stranded CCA 3′-end bases of the acceptor stem and from the bases in the TΨC stem. Secondly, by NMR and fluorescence spectroscopy, we have studied the complex between the NC protein and RNA hairpins that mimic the D and T arms of the tRNA 3 Lys. Interestingly, the NC protein shows strong and specific binding to the D arm of tRNA 3 Lys, which could explain the overall annealing mechanism.

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNALys,2 to two distinct primer binding sites (PBS) at the genome 5′ and 3′ ends. Only the 5′ PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.

The selective packaging and annealing of primer tRNALys3 in HIV-1.

In HIV-1, tRNA(Lys3) serves as the primer for reverse transcriptase, and during viral assembly, the tRNA(Lys) isoacceptors, tRNA(Lys1,2) and tRNA(Lys3), are selectively packaged into the virion. In this review, we shall first discuss the evidence for the formation of a tRNA(Lys) packaging complex, whose components include Gag, GagPol, genomic RNA, lysyl-tRNA synthetase (LysRS), and tRNA(Lys). Evidence suggests that the formation of this complex involves a Gag/GagPol/viral genomic RNA complex interacting with a tRNA(Lys)/ LysRS complex, with Gag interacting with both GagPol and LysRS, and GagPol interacting with the tRNA(Lys). The interaction of Gag with LysRS is quite specific, does not require the presence of tRNA(Lys), and LysRS is believed to be the target that allows the specific packaging of tRNA(Lys) into the virion. The parameters influencing these interactions, and the molecular sites of interaction, will be discussed. The selective packaging of tRNA(Lys3) into HIV-1 facilitates annealing of tRNA(Lys) to the 5' region of viral RNA genome. This region contains a series of stem loops, and there exists several regions in both the viral RNA and the tRNA(Lys) that are believed to be important for tRNA(Lys) annealing. The annealing is facilitated by viral proteins such as unprocessed Gag, nucleocapsid, and reverse transcriptase.

Mechanistic Insights into the Kinetics of HIV-1 Nucleocapsid Protein-facilitated tRNA Annealing to the Primer Binding Site

HIV-1 reverse transcriptase uses human tRNALys,3 as a primer to initiate reverse transcription. Prior to initiation, the 3′ 18 nucleotides of this tRNA are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). Here, we show that the HIV-1 nucleocapsid protein (NC) enhances this annealing by approximately five orders of magnitude in vitro, decreasing the transition state enthalpy from approximately 20kcalmol−1 for the uncatalyzed reaction to 13kcalmol−1 for the NC-catalyzed process. Moreover, the annealing follows second-order kinetics, consistent with the nucleation of the intermolecular duplex being the rate-limiting step. This nucleation is preceded by melting of a small duplex region within the original structure, and is followed by much faster zipping of the rest of the 18 base-pair (bp) duplex. A tRNA mutational analysis shows that destabilization of the tRNA acceptor stem has only a minor effect on the annealing rate. In contrast, addition of bases to the 5′ end of tRNA that are complementary to its single-stranded 3′ end interferes with duplex nucleation and therefore has a much larger effect on the net reaction rate. Assuming that the apparent transition free energy of the annealing reaction, ΔG‡, is a sum of the melting (ΔGm) and nucleation (ΔGnuc) free energies, we show that NC affects both ΔGm and ΔGnuc. We estimate that ten to 100-fold of the overall rate enhancement is due to NC-induced destabilization of a 4 bp helix in the PBS, while the additional factor of 103–104 rate enhancement is a result of NC-facilitated duplex nucleation. The apparently similar effectiveness of wild-type and SSHS NC, a mutant that lacks the zinc finger structures, in facilitating the tRNA annealing reaction is most likely the result of the mutual cancellation of two factors: SSHS NC is less effective than wild-type NC as a duplex destabilizer, but more effective as a duplex nucleating agent.

In vitro characterization of a base pairing interaction between the primer binding site and the minimal packaging signal of avian leukosis virus genomic RNA.

The 5' leader region of avian sarcoma-leukosis viruses (ASLVs) folds into a series of RNA secondary structures which are involved in key steps in the viral replication cycle such as reverse transcription, dimerization and packaging of genomic RNA. The O3 stem and three stem-loops (O3SLa, O3SLb and O3SLc) form the minimal packaging signal that is located downstream of the primer binding site (PBS). The U5-PBS region contributes to packaging via a mechanism that remains unknown. In this in vitro study, we have investigated the possibility of interactions between the R-U5-PBS region and the minimal packaging signal using chemical and enzymatic probing, antisense oligonucleotides and site-directed mutagenesis. We have identified a base pairing interaction between the PBS sequence and the terminal loop of O3SLa. It was found that the PBS/O3SLa interaction was intramolecular since it occurred not only in dimeric RNA but also in monomeric RNA. This interaction probably corresponds to a pseudoknot interaction. The PBS/O3SLa interaction may be formed in vivo since the sequences are highly conserved in ASLV strains. The PBS/O3SLa interaction may regulate the processes of primer tRNA annealing, packaging and initiation of Gag translation through its involvement in leader tertiary structure. Interestingly, we found that in other retroviruses the PBS sequence can also base pair with a terminal loop of the stem-loops involved in RNA packaging.

Effect of altering the tRNA(Lys)(3) concentration in human immunodeficiency virus type 1 upon its annealing to viral RNA, GagPol incorporation, and viral infectivity.

Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys)(3) as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNA(Lys), tRNA(Lys)(3). Increasing the cytoplasmic concentration of tRNA(Lys)(3) through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA(Lys)(3) gene results in a greater incorporation of tRNA(Lys)(3) into virions, which is accompanied by increased annealing of tRNA(Lys)(3) to the viral genome and increased infectivity of the viral population. Increased viral tRNA(Lys)(3) is accompanied by decreased viral tRNA(Lys)(3), with the total tRNA(Lys)/virion and the GagPol/Gag ratios remaining unchanged. Viral tRNA(Lys) can be doubled, with increases in both tRNA(Lys)(3) and tRNA(Lys)(1,2) concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA(Lys)(3) annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNA(Lys) incorporation into HIV-1, whereas LysRS is.

TRNA(Lys3): the primer tRNA for reverse transcription in HIV-1.

During the assembly of HIV-1, tRNA(Lys) isoacceptors are selectively packaged into the virion, and one of these, tRNA(Lys3), is annealed to the viral RNA genome where it acts to prime the reverse transcriptase (RT)-catalyzed synthesis of viral DNA. We review herein what is known about the selective packaging and annealing of primer tRNA(Lys3). Current evidence suggests that a complex of two major precursor viral proteins, Pr55gag and Pr160(gag-pol), interact with a tRNA(Lys)/lysyl-tRNA synthetase (LysRS) complex during viral assembly, with Pr55gag interacting with both LysRS and Pr160(gag-pol), and RT sequences within Pr160(gag-pol) binding to tRNA(Lys). LysRS appears to target the tRNA(Lys) isoacceptors for incorporation into HIV-1. In the virion, the 3' terminal 18 nucleotides of tRNA(Lys3) anneals to an 18-nucleotide sequence at the 5' terminal region of viral RNA termed the primer binding site (PBS). Evidence is presented that other regions on the tRNA(Lys3) also anneal with other regions in viral RNA upstream of the PBS, resulting in a destabilized tRNA(Lys3) structure. Both viral and tRNA(Lys3) regions need to be denatured to establish annealing, and the roles of both viral and cellular proteins in this process are discussed.

Identification of the in Vitro HIV-2/SIV RNA Dimerization Site Reveals Striking Differences with HIV-1

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.

The Gag-like Protein of the Yeast Ty1 Retrotransposon Contains a Nucleic Acid Chaperone Domain Analogous to Retroviral Nucleocapsid Proteins

The reverse transcription process for retroviruses and retrotransposons takes place in a nucleocore structure in the virus or virus-like particle. In retroviruses the major protein of the nucleocore is the nucleocapsid protein (NC protein), which derives from the C-terminal region of GAG. Retroviral NC proteins are formed of either one or two CCHC zinc finger(s) flanked by basic residues and have nucleic acid chaperone and match-maker properties essential for virus replication. Interestingly, the GAG protein of a number of retroelements including Spumaviruses does not possess the hallmarks of retroviral GAGs and in particular lacks a canonical NC protein. In an attempt to search for a nucleic acid chaperone activity in this class of retroelements we used the yeast Ty1 retrotransposon as a model system. Results shows that the C-terminal region of Ty1 GAG contains a nucleic acid chaperone domain capable of promoting the annealing of primer tRNA(i)(Met) to the multipartite primer binding site, Ty1 RNA dimerization and initiation of reverse transcription. Moreover Ty1 RNA dimerization, in a manner similar to Ty3 but unlike retroviral RNAs, appears to be mediated by tRNA(i)(Met). These findings suggest that nucleic acid chaperone proteins probably are general co-factors for reverse transcriptases.

In Vitro Studies on tRNA Annealing and Reverse Transcription with Mutant HIV-1 RNA Templates

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semistable stem-loop structure, the U5-PBS hairpin, which occludes part of the tRNA primer binding site (PBS). In previous studies, we demonstrated that mutations that alter the stability of the U5-PBS hairpin inhibit virus replication. A reverse transcription defect was measured in assays with the virion-extracted RNA-tRNA complexes. We now extend these studies with in vitro synthesized wild-type and mutant RNA templates that were tested in primer annealing and reverse transcription assays. The effect of annealing temperature and the presence of the viral nucleocapsid protein on reverse transcription was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to stable template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA primed reactions. The inhibitory effect of template structure on tRNA-primed reverse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex differs from the virion-extracted viral RNA-tRNA complex.

Characterization of Active Reverse Transcriptase and Nucleoprotein Complexes of the Yeast Retrotransposon Ty3 in Vitro

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

The role of Pr55(gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA.

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or Pr160(gag-pol). In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55(gag) or mutant Pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA3Lys placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA3Lys to genomic RNA.

The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site.

The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.