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Graphic description of reverse transcription reaction. (Part 1.) The RNA template consists of R, U5, PBS, and a 282-nt 3-flanking sequence. The position and size of each region are indicated. The numbers in parentheses designate nucleotide positions within the viral RNA template. (Part 2.) RNA template and tRNA 3 Lys
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Retroviral reverse transcription starts near the 5' end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA(Lys3) to the PBS and in reverse transcriptase (RT) activity, using a...
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... . To investigate the effect of NCp7 on ()ssDNA synthesis, we reconstituted a reverse transcription reaction mixture consisting of HIV-1 template, human tRNA 3 Lys , RT, and NCp7. As shown in Fig. 1, the template and tRNA were preincubated with various concentrations of NCp7 prior to the addition of enzyme. The final product is 259 nt long and con- sists of ()DNA (183 nt) and the attached primer tRNA 3 Lys (76 nt) at the 5 end. Figure 2A shows that ()ssDNA was clearly detected when NCp7 was present at concentrations of 14, 9.3, ...
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... also blocked the terminal nucleoside 3 hydroxyl of the RNA template by treatment with excess cytidine 3,5-biphos- phate (pCP), prior to the addition of template to the reverse transcription mixture (29). This resulted in a disappearance of nonspecific products (Fig. 2B, lanes 1 to 3). In the absence of NCp7, no nonspecific DNA products were detected and syn- thesis of ()ssDNA was less efficient (lane 1) than when NCp was present (lane 3). Denaturation of the RNA template (92C for 2 min followed by quick chilling on ice) (4, 37) before addition to the reaction mixture resulted in synthesis of more were preincubated ...
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... of NCp7 led to decreased accumulation of nonspecific reverse-transcribed DNA products and increased generation of specific ()ssDNA (Fig. 2A, lanes 1 to 8). Inhibition effect of NCp7 on reverse transcription primed by either viral template or by ribooligonucleotide (rPR) comple- mentary to the PBS. We next investigated reverse transcription using a 18-mer ribooligonucleotide (rPR) complementary to the PBS, as a primer in the place of tRNA 3 Lys . In this instance, NCp7 displayed ...
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... template or by ribooligonucleotide (rPR) comple- mentary to the PBS. We next investigated reverse transcription using a 18-mer ribooligonucleotide (rPR) complementary to the PBS, as a primer in the place of tRNA 3 Lys . In this instance, NCp7 displayed dose-dependent inhibitory rather than stimu- latory effects on rPR-primed synthesis of ()ssDNA (Fig. 3, lanes 1 to 8). Indeed, the strongest ()ssDNA signal was obtained in the absence of NCp7 (Fig. 3, lane 8). Figure 3 also shows that NCp7 inhibited formation of nonspecific DNA products of reverse transcription (lanes 1 to 8), as described above. No specific ()ssDNA products were detected when PBS() template was used in place of the PBS-containing ...
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... PBS() template was used in place of the PBS-containing PBS/WT template in the reverse transcription reaction (not shown). Treatment of the reaction products with NaOH to digest the RNA portion of the reaction products resulted in a smaller size of ()ssDNA (approximately 18 nt shorter), con- firming that these products were indeed initiated by rPR (Fig. 3, lanes 9 to 16). The smaller size of nonspecific DNA products after NaOH treatment also confirmed that these products were caused by self-priming of RNA templates (Fig. 3, lanes 9 to 16). Thus, NCp7 appears to inhibit formation of reverse-tran- scribed DNA products initiated either by rPR or by RNA ...
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... the RNA portion of the reaction products resulted in a smaller size of ()ssDNA (approximately 18 nt shorter), con- firming that these products were indeed initiated by rPR (Fig. 3, lanes 9 to 16). The smaller size of nonspecific DNA products after NaOH treatment also confirmed that these products were caused by self-priming of RNA templates (Fig. 3, lanes 9 to 16). Thus, NCp7 appears to inhibit formation of reverse-tran- scribed DNA products initiated either by rPR or by RNA ...
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... an additional control, we asked what the effect would be on the formation of the tRNA-RNA complex if we replaced NCp7 by other proteins. In comparison with high-level com- plex formation obtained in the presence of NCp7 (Fig. 4B, lane 1), only minimal placement of tRNA onto RNA was obtained in the presence of a basic protein, e.g., histone (lane 2), an acidic protein, e.g., BSA (lane 3), or RT (lane 4). No proteins were present in the experiment of lane 5, while only 32 P-5- end-labelled tRNA 3 Lys was present in lane 6. This shows that the effects of NCp7 on formation ...
Similar publications
The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNA(Lys) 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-ene...
Citations
... Annealing between the tRNA and vRNA of the reverse transcription initiation complex is proposed to be achieved by the mature NC. [11][12][13][14][15] NMR studies showed that the NC protein may not be required for the formation of the primertemplate complex; instead, it may help in accelerating the process. The unwinding process could be initiated by the unpaired 3 ′ CCA end of the tRNA 3 Lys and the unpaired bases from the acceptor and TJC stem junction. ...
RNA interference is an upcoming methodology being designed to specifically target viral infections. The current study suggests a strategy to design probable small interfering RNAs (siRNA) for targeting the viral genome of SARS-CoV-2, as a case study. siRNAs were designed against the targets from a highly conserved region of the spike gene of SARS-CoV-2 having no significant matches within the human genome. Four targets/viral RNAs (vRNA) with high predicted inhibition values were selected for further evaluation. The predicted siRNAs were examined for their properties and stability using molecular dynamics (MD) simulations. Further, to understand the RNA-Induced Silencing Complex (RISC) mechanism of the predicted siRNA targets of SARS-CoV-2, the human argonaute (Ago2) protein in complex with the four siRNA-vRNA duplexes was built. MD simulations of apo-Ago2, four selected siRNA-vRNA duplexes and four Ago2 bound to these siRNA-vRNA duplexes were carried out for 1 μs each. Amongst the four duplex-bound Ago2 simulation systems, the siRNA-vRNA3 duplex showed stable base pairing in the seed region, favourable and strong interactions with functionally important residues of Ago2 protein through the simulation length. Therefore, the designed siRNA3 molecule may act as an effective therapeutic agent against the SARS-CoV-2. The reported in-silico strategy may be beneficial for the identification and designing of probable siRNAs against any viral genome in RNAi therapeutics. However, the experimental validation of these molecules would be required for proving their use as therapeutics.
... Annealing between the tRNA and vRNA of the reverse transcription initiation complex is proposed to be achieved by the mature NC. [11][12][13][14][15] NMR studies showed that the NC protein may not be required for the formation of the primertemplate complex; instead, it may help in accelerating the process. The unwinding process could be initiated by the unpaired 3 ′ CCA end of the tRNA 3 Lys and the unpaired bases from the acceptor and TJC stem junction. ...
The tRNA3Lys, which acts as a primer for human immunodeficiency virus type 1 (HIV-1) reverse transcription, undergoes structural changes required for the formation of a primer–template complex. Small molecules have been targeted against tRNA3Lys to inhibit the primer–template complex formation. The present study aims to understand the kinetics of the conformational landscape spanned by tRNA3Lys in apo form using molecular dynamics simulations and Markov state modeling. The study is taken further to investigate the effect of small molecules like 1,4T and 1,5T on structural conformations and kinetics of tRNA3Lys, and comparative analysis is presented. Markov state modeling of tRNA3Lys apo resulted in three metastable states where the conformations have shown the non-canonical structures of the anticodon loop. Based on analyses of ligand–tRNA3Lys interactions, crucial ion and water mediated H-bonds and free energy calculations, it was observed that the 1,4-triazole more strongly binds to the tRNA3Lys compared to 1,5-triazole. However, the MSM analysis suggest that the 1,5-triazole binding to tRNA3Lys has brought rigidity not only in the binding pocket (TΨC arm, D–TΨC loop) but also in the whole structure of tRNA3Lys. This may affect the easy opening of primer tRNA3Lys required for HIV-1 reverse transcription.
... We suspected that the 12-NC-annealed complex exposed certain residues that mediate dimerization of the annealed complex (slow-complex 2), but these residues were in a different structure and not available to mediate dimerization when the complex was annealed by 1-NC or by heat. These fast-and slow-migration complex bands were also reported previously in NC-promoted vRNA: tRNA Lys3 complexes [40]. Furthermore, the position of the slow complex formed in the 1-NC-annealed complex (slow-complex 1) was lower (migrated faster) than the slow complex formed in the 12-NCannealed complex (slow-complex 2, Figure 6c), suggesting structural differences in these slow complexes. ...
The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.
... During the extension phase, RT functions in a processive manner, and DNA synthesis is much faster (11,14). Viral nucleocapsid (NC) proteins participate in reverse transcription by promoting tRNA Lys3 annealing and stimulating specific cDNA synthesis primed by tRNA Lys3 (15,16). Several cellular factors found in virus particles are reported to impact RT activity, but their roles remain controversial (17). ...
... A 5-Cy3-labeled tRNA Lys3 was annealed to the 5 (nt 1-344) of the HIV-1 genomic RNA in the presence of NC protein (Fig. 3B). NC was added at a ratio of one NC per 15 nt RNA, which is predicted to be close to the ratio within virions without inhibiting reverse transcription (15,28). The 5-UTR was predimerized to mimic the RNA conformation that directs genome packaging in the late phase of replication (29,30). ...
... In summary, our results demonstrate that RHA positively regulates the processivity during the elongation phase of reverse transcription. RNA secondary/tertiary structures and proteins bound to the RNA template typically hinder RT scanning and lead to dissociation of RT from the template (15,33,39). Hence, RHA most likely removes the physical roadblocks for RT, such as RNA structures, NC, and/or other proteins coating the RNA and thereby improves RT's processivity (Fig. 8). ...
DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3:viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.
... Mature NC results from the protease-mediated cleavage of the Gag polyprotein precursor and plays key roles in HIV-1 replication (2)(3)(4)(5). During the early steps, NC acts as a cofactor of the reverse transcriptase (RT), to promote the initiation of reverse transcription (6)(7)(8), as well as the two obligatory strand transfers (9,10). Moreover, NC also reduces RT pauses at the initiation step (11)(12)(13), increases the overall RT processivity (14,15), promotes synthesis through pause sites (16)(17)(18)(19), helps to remove the RNA fragments resulting from the RT RNase H activity (20), and may contribute to the protection of the viral DNA (vDNA) during its nuclear import (21). ...
Unlabelled:
The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (∼ 10(7) M(-1)) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM.
Importance:
The NC protein is a highly conserved nucleic acid binding protein that plays numerous key roles in HIV-1 replication. While accumulating evidence shows that NC either as a mature protein or as a domain of the Gag precursor also interacts with host proteins, only a few data are available on the possible interaction of NC with lipid membranes. Interestingly, during HIV-1 assembly, the Gag precursor is thought to adopt a bent conformation where the NC domain may interact with the plasma membrane. In this context, we quantitatively characterized the binding of NC, as a free protein or as a complex with nucleic acids, to lipid membranes and showed that the latter constitute a binding platform for NC. Taken together, our data suggest that the NC domain may play a role in the initial binding events of Gag to the plasma membrane during HIV-1 assembly.
... This transfer is achieved by the annealing of the repeat regions (R) (containing the TAR element) at the 3 ′ ends of the (−) ssDNA and the (+) sense viral RNA (Basu et al., 2008). NCp7 chaperones minus-strand DNA transfer by promoting the annealing of the complementary (+) and (−) TAR sequences and preventing non-specific self-priming reactions (Driscoll and Hughes, 2000;Guo et al., 1997;Li et al., 1996). Vif is also able to stimulate (−) ssDNA transfer, although with a reduced efficiency compared to NC proteins (NCp15, NCp9 and NCp7) . ...
The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting cellular cytosine deaminases APOBEC3G (A3G) and A3F by different mechanisms and thus preventing its incorporation into viral particles. The Vif-induced degradation of A3G through the proteasome pathway has been extensively studied, but little is known about the translational repression of A3G mRNA by Vif. After cellular co-transfection of A3G mRNA constructs mutated in their untranslated regions (UTRs) in presence or absence of Vif, and in conditions where the proteasome-induced degradation of A3G was inhibited, we show that the 5’-UTR of A3G mRNA is crucial for the translational inhibition by Vif. The core binding factor, CBF-, required to stabilize the Vif/A3G complex is dispensable for this specific repression. According to our previous secondary structural model of the 5’-UTR, the two distal stem-loop structures are sufficient for a complete translational inhibition of A3G. We show that residue K26 of Vif is critical for A3G neutralization, both for its proteasome-induced degradation and translation inhibition of itsmRNA. Interestingly, we observe a strict correlation between the cellular reduction of A3G through translation inhibition and the quantity of A3G incorporated into viral particles. Both mechanisms account for about 50% decrease of A3G in cell. Thus, we showed for the first time that A3G mRNA translational inhibition by Vif is a 5’-UTR mRNA-dependent mechanism, and that any of these two mechanisms, degradation or translation, is sufficient to restore viral infectivity. Regulating the translation of A3G could thus be considered as a new target to restore a functional expression of A3G and viral restriction.
... RTn takes place in a complex organization called Reverse transcription complex (RTC), a nucleoprotein complex comprising viral RNA, a tRNA primer and newly synthesized DNA. Along with these nucleic acids, viral factors, including Nef [2], Vif [3][4][5], matrix protein (MA) [6], nucleocapsid protein (NCp7) [7,8], Integrase (IN) [9] and Tat [10] are known to be present alongside of reverse transcriptase (RT). In addition to viral factors, very little information is known about the role of host factors in the viral life cycle. ...
HIV-1 reverse transcription (RTn) involves synthesis of double strand DNA (dsDNA) from viral genomic RNA. Topoisomerase II (Topo II) alpha and beta maintains topological reorganization of dsDNA regions and catalytic inhibition of these isoforms repressed viral replicative cycle. Present study is aimed to understand the role of Topo II isoforms in HIV-1 early replication. Topo IIα and β showed differential expression in SupT1 cells and PBMCs during early hours of HIV-1 infection where Topo IIα expression increased after 4h, while Topo IIβ showed relatively higher expression at 1 and 4h. In Topo IIα and/or β down regulated cells, transcription of viral genes gag, pol and env as well as proviral DNA synthesis was abolished. In Topo IIα and/or β down regulated cells, strong stop DNA synthesis was unaffected while other downstream events of reverse transcription such as first strand transfer, full length minus strand synthesis, and second strand transfer were completely inhibited, which affects HIV-1 replication. Further, co-localization of Topo II isoforms with HIV-1 reverse transcriptase was observed in SupT1 cells and PBMCs by immunofluorescence. These results collectively suggest a role of Topo II isoforms during HIV-1 RTn probably by promoting the alignment of viral RNA-DNA hybrids.
... The chaperone properties of the NC domain in Pr55gag are thought to facilitate the specific placement of the tRNA Lys,3 primer on the primer binding site (PBS) of the viral RNA (Cen et al., 1999;Feng et al., 1999;Cruceanu et al., 2006b;Guo et al., 2009;Wu et al., 2010). In vitro, NCp7 directs the annealing of the tRNA Lys,3 primer to the PBS (Li et al., 1996), by facilitating the strand exchange at the level of the tRNA acceptor stem and by unlocking in the presence of the complementary genomic RNA sequence, the highly stable interactions at the level of the T C loop (Chan and Musier-Forsyth, 1997;Tisné et al., 2003Tisné et al., , 2004Hargittai et al., 2004;Tisné, 2005;Barraud et al., 2007). The kinetics of the tRNA Lys,3 annealing on the PBS sequence follow a nucleation-limited bimolecular reaction. ...
... Specific formation of the initiation complex is mediated by extended interactions between the HIV-1 RNA and tRNA Lys,3 (Goldschmidt et al., 2002). Although variable among HIV strains (Goldschmidt et al., 2004), these extended interactions (Isel et al., 1996(Isel et al., , 1998(Isel et al., , 1999(Isel et al., , 2010Li et al., 1996;Beerens and Berkhout, 2002;Huthoff et al., 2003) are decisive for the efficiency of the initiation of reverse transcription. Formation of the initiation complex requires rearrangements in the 5 UTR vRNA that may be promoted by the fully processed NC (Iwatani et al., 2003;Guo et al., 2009). ...
RNA chaperones are proteins able to rearrange nucleic acid structures towards their most stable conformations. In retroviruses, the reverse transcription of the viral RNA requires multiple and complex nucleic acid rearrangements that need to be chaperoned. HIV-1 has evolved different viral-encoded proteins with chaperone activity, notably Tat and the well described nucleocapsid protein NCp7. We propose here an overview of the recent reports that examine and compare the nucleic acid chaperone properties of Tat and NCp7 during reverse transcription to illustrate the variety of mechanisms of action of the nucleic acid chaperone proteins.
... This transfer is achieved by the annealing of the repeat regions (R) (containing the TAR element) at the 3 ends of the (−) ssDNA and the (+) sense viral RNA (Basu et al., 2008). NCp7 chaperones minus-strand DNA transfer by promoting the annealing of the complementary (+) and (−) TAR sequences and preventing non-specific self-priming reactions (Driscoll and Hughes, 2000;Guo et al., 1997;Li et al., 1996). Vif is also able to stimulate (−) ssDNA transfer, although with a reduced efficiency compared to NC proteins (NCp15, NCp9 and NCp7) (Henriet et al., 2007). ...
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.
... Therefore, the uncoating step in HIV-1 replication is not an immediate post-fusion event, but rather occurs at the nuclear pore upon completion of viral DNA synthesis (Arhel et al., 2007). The efficiency of reverse transcription is affected by a number of proteins encapsidated or not in the virion, including Nef (Aiken and Trono, 1995; Schwartz et al., 1995), Tat (Harrich et al., 1997), Vif (Goncalves et al., 1996; Sova and Volsky, 1993), Vpr (Stark and Hay, 1998), the matrix protein (Kiernan et al., 1998), NCp7 (Li et al., 1996), the integrase (Tsurutani et al., 2000; Wu et al., 1999; Zhu et al., 2004), the cyclophilin A (Thali et al., 1994), the topoisomerase I (Takahashi et al., 1995) and APOBEC3F (Holmes et al., 2007). All retroviruses use a cellular tRNA as a primer to initiate their reverse transcription. ...
HIV-1 reverse transcription is initiated from a tRNA(Lys)(3) molecule annealed to the viral RNA at the primer binding site (PBS). The annealing of tRNA(Lys)(3) requires the opening of its three-dimensional structure and RNA rearrangements to form an efficient initiation complex recognized by the reverse transcriptase. This annealing is mediated by the nucleocapsid protein (NC). In this paper, we first review the actual knowledge about HIV-1 viral RNA and tRNA(Lys)(3) structures. Then, we summarize the studies explaining how NC chaperones the formation of the tRNA(Lys)(3)/PBS binary complex. Additional NMR data that investigated the NC interaction with tRNA(Lys)(3) D-loop are presented. Lastly, we focused on the additional interactions occurring between tRNA(Lys)(3) and the viral RNA and showed that they are dependent on HIV-1 isolates, i.e. the sequence and the structure of the viral RNA.
