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![Schematic diagram illustrating possible mechanisms for removal of 5′ terminal RNA fragments in the context of minus-strand transfer. Step 1 shows small 5′ genomic RNA fragments (red) annealed to the 3′ end of (−) SSDNA (purple). These fragments, derived by RNase H cleavage of viral RNA, must be removed to allow annealing of (−) SSDNA to acceptor RNA and successful minus-strand transfer (steps 2–4). In our assay, the 5′ RNA fragment (either RNA 20 or RNA 14 [Table 1]) is heat-annealed to DNA 128 (Table 1) and then acceptor RNA 148 (green) is added (step 2). Subsequent addition of RT and NC results in productive strand transfer, i.e., elongation of (−) SSDNA using RNA 148 as the template, as long as the 5′ fragment is removed (step 3). The fragment could be removed by NC-mediated destabilization of the RNA–DNA hybrid (left side), RNase H cleavage of the short RNA (right side), or both (step 3). The full-length transfer product is shown in step 4. The acceptor RNA is ultimately degraded by RNase H (not shown). RNA 148 contains R plus a portion of U3 (Guo et al., 1997) and DNA 128 contains the complementary sequences of R and a portion of U5 (shown here in lower case) (Heilman-Miller et al., 2004; Wu et al., 2010). The first 59 nt of R at the 3′ ends of RNA 148 and DNA 128 form the TAR RNA and TAR DNA stem-loop structures, respectively (Levin et al., 2010). The diagram is not drawn to scale.](profile/Tiyun-Wu/publication/233410635/figure/fig3/AS:269784168267779@1441332966960/Schematic-diagram-illustrating-possible-mechanisms-for-removal-of-5-terminal-RNA.png)
Schematic diagram illustrating possible mechanisms for removal of 5′ terminal RNA fragments in the context of minus-strand transfer. Step 1 shows small 5′ genomic RNA fragments (red) annealed to the 3′ end of (−) SSDNA (purple). These fragments, derived by RNase H cleavage of viral RNA, must be removed to allow annealing of (−) SSDNA to acceptor RNA and successful minus-strand transfer (steps 2–4). In our assay, the 5′ RNA fragment (either RNA 20 or RNA 14 [Table 1]) is heat-annealed to DNA 128 (Table 1) and then acceptor RNA 148 (green) is added (step 2). Subsequent addition of RT and NC results in productive strand transfer, i.e., elongation of (−) SSDNA using RNA 148 as the template, as long as the 5′ fragment is removed (step 3). The fragment could be removed by NC-mediated destabilization of the RNA–DNA hybrid (left side), RNase H cleavage of the short RNA (right side), or both (step 3). The full-length transfer product is shown in step 4. The acceptor RNA is ultimately degraded by RNase H (not shown). RNA 148 contains R plus a portion of U3 (Guo et al., 1997) and DNA 128 contains the complementary sequences of R and a portion of U5 (shown here in lower case) (Heilman-Miller et al., 2004; Wu et al., 2010). The first 59 nt of R at the 3′ ends of RNA 148 and DNA 128 form the TAR RNA and TAR DNA stem-loop structures, respectively (Levin et al., 2010). The diagram is not drawn to scale.
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During (-) strong-stop DNA [(-) SSDNA] synthesis, RNase H cleavage of genomic viral RNA generates small 5'-terminal RNA fragments (14 to 18 nt) that remain annealed to the DNA. Unless these fragments are removed, the minus-strand transfer reaction, required for (-) SSDNA elongation, cannot occur. Here, we describe the mechanism of 5'-terminal RNA r...
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Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and co...
Citations
... NC proteins also have a significance in terms of destabilizing RNA and DNA hairpins. Unfolding these hairpins is essential for the formation of the DNA-RNA heteroduplex in strand transfer (38)(39)(40). Furthermore, it is supposed that the close proximity of the two co-packaged RNAs in the viral particles is essential for proper template switching (41,42). ...
In vitro reverse transcription of full-length HIV-1 RNA extracted from the blood plasma of people living with HIV-1 remains challenging. Here, we describe the initiation of reverse transcription of plasma-derived viral RNA in the absence of an exogenous primer. Real-time PCR and Sanger sequencing were applied to identify the source and to monitor the outcome of this reaction. Results demonstrated that during purification of viral RNA from plasma, tRNA(Lys-3) is co-extracted in a complex with the viral RNA. In the presence of a reverse transcription enzyme, this tRNA(Lys-3) can induce reverse transcription, a reaction that is not confined to transcription of the 5’ end of the viral RNA. A range of cDNA products is generated, most of them indicative for the occurrence of in vitro strand transfer events that involve translocation of cDNA from the 5’ end to random positions on the viral RNA. This process results in the formation of cDNAs with large internal deletions. However, near full-length cDNA and cDNA with sequence patterns resembling multiple spliced HIV-1 RNA were also detected. Despite its potential to introduce significant bias in the interpretation of results across various applications, tRNA(Lys-3)-driven reverse transcription has been overlooked thus far. A more in-depth study of this tRNA-driven in vitro reaction may provide new insight into the complex process of in vivo HIV-1 replication.
IMPORTANCE
The use of silica-based extraction methods for purifying HIV-1 RNA from viral particles is a common practice, but it involves co-extraction of human tRNA(Lys-3) due to the strong interactions between these molecules. This co-extraction becomes particularly significant when the extracted RNA is used in reverse transcription reactions, as the tRNA(Lys-3) then serves as a primer. Reverse transcription from tRNA(Lys-3) is not confined to cDNA synthesis of the 5’ end of the viral RNA but extends across various regions of the viral genome through in vitro strand transfer events. Co-extraction of tRNA(Lys-3) has been overlooked thus far, despite its potential to introduce bias in downstream, reverse transcription-related applications. The observed events in the tRNA(Lys-3)-induced in vitro reverse transcription resemble in vivo replication processes. Therefore, these reactions may offer a unique model to better understand the replication dynamics of HIV-1.
... More recently, Hergott et al. [87] showed that NC is required for removal of 5 0 -terminal RNA fragments (14e18 nucleotides) in the presence of an acceptor RNA template. In fact, NC's destabilizing activity promotes the displacement of short RNAs by the longer acceptor RNA, which forms a thermodynamically more stable duplex with (À) ssDNA (Fig. 4D). ...
An infectious retroviral particle contains 1000–1500 molecules of the nucleocapsid protein (NC) that cover the diploid RNA genome. NC is a small zinc finger protein that possesses nucleic acid chaperone activity that enables NC to rearrange DNA and RNA molecules into the most thermodynamically stable structures usually those containing the maximum number of base pairs. Thanks to the chaperone activity, NC plays an essential role in reverse transcription of the retroviral genome by facilitating the strand transfer reactions of this process. In addition, these reactions are involved in recombination events that can generate multiple drug resistance mutations in the presence of anti-HIV-1 drugs. The strand transfer reactions rely on base pairing of folded DNA/RNA structures. The molecular mechanisms responsible for NC-mediated strand transfer reactions are presented and discussed in this review. Antiretroviral strategies targeting the NC-mediated strand transfer events are also discussed.
... Polyacrylamide gel electrophoresis in 6% denaturing gels and PhosphorImager analysis were performed as described previously [107]. The % strand transfer product formed was calculated by dividing the amount of transfer product by the total signal in the gel lane and multiplying by 100 [108]. ...
Background
The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC. Non-human primates are frequently used as a platform to study retroviral infection in vivo; thus, it is important to understand underlying mechanistic differences between HIV-1 and SIV NC. ResultsHere, we characterize SIV NC chaperone activity for the first time. Only modest differences are observed in the ability of SIV NC to facilitate reactions that mimic the minus-strand annealing and transfer steps of reverse transcription relative to HIV-1 NC, with the latter displaying slightly higher strand transfer and annealing rates. Quantitative single molecule DNA stretching studies and dynamic light scattering experiments reveal that these differences are due to significantly increased DNA compaction energy and higher aggregation capability of HIV-1 NC relative to the SIV protein. Using salt-titration binding assays, we find that both proteins are strikingly similar in their ability to specifically interact with HIV-1 Psi RNA. In contrast, they do not demonstrate specific binding to an RNA derived from the putative SIV packaging signal. Conclusions
Based on these studies, we conclude that (1) HIV-1 NC is a slightly more efficient NA chaperone protein than SIV NC, (2) mechanistic differences between the NA interactions of highly similar retroviral NC proteins are revealed by quantitative single molecule DNA stretching, and (3) SIV NC demonstrates cross-species recognition of the HIV-1 Psi RNA packaging signal.
... The relationship between binding polarity and the polarized destabilization of stem-loops may result from the lack of equivalence of the two zinc fingers, ZF1 and ZF2, with ZF1 more involved in the destabilization than ZF2. In contrast, ZF2 is involved in the specific recognition of guanine residues (Guo et al. 2002;Heath et al. 2003;Beltz et al. 2005;Narayanan et al. 2006;Hergott et al. 2013;Mitra et al. 2013). Binding polarity may therefore be responsible for the localization of ZF1 close to a double-stranded region. ...
The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.
... In addition, NCp7 chaperones the first strand transfer by annealing cTAR DNA with TAR RNA allowing RT to resume the minus-strand DNA elongation step . Moreover, NCp7 ensures the fidelity of plus-strand DNA priming at the two polypurine tracts (PPT) by blocking mispriming by non-PPT RNAs and by removing the 5′-terminal fragments annealed to minus-strand DNA (Hergott et al. 2013). In order for RT to perform the plus-strand synthesis after its pausing, NCp7 must chaperone the second strand transfer (i) by facilitating the RT-RNaseH removal of primer tRNALys3 from the 5´-end of minus-strand DNA, and (ii) by promoting the annealing of the PBS DNA copy at the 3´-end of plus-strand DNA Fig. ...
... These NA chaperone properties rely on the ability of NCp7 to transiently destabilize the NA secondary structure Beltz et al. 2003Beltz et al. , 2004Bernacchi et al. 2002;Cosa et al. 2006;Egele et al. 2004;Godet et al. 2011Godet et al. , 2013Liu et al. 2005;Williams et al. 2001). This destabilization is mainly mediated by the hydrophobic region located on the top of the folded ZFs and strongly depends on the NA stability and structure, suggesting a coevolutionary relationship between NCp7 and its NA targets (Beltz et al. 2003(Beltz et al. , 2005Godet et al. 2011Godet et al. , 2013Hergott et al. 2013). Guanosine is the pivotal nucleoside to be trapped (Grohman et al. 2013). ...
The currently available anti-HIV-1 therapeutics is highly beneficial to infected patients. However, clinical failures occur as a result of the ability of HIV-1 to rapidly mutate. One approach to overcome drug resistance is to target HIV-1 proteins that are highly conserved among phylogenetically distant viral strains and currently not targeted by available therapies. In this respect, the nucleocapsid (NC) protein, a zinc finger protein, is particularly attractive, as it is highly conserved and plays a central role in virus replication, mainly by interacting with nucleic acids. The compelling rationale for considering NC as a viable drug target is illustrated by the fact that point mutants of this protein lead to noninfectious viruses and by the inability to select viruses resistant to a first generation of anti-NC drugs. In our review, we discuss the most relevant properties and functions of NC, as well as recent developments of small molecules targeting NC. Zinc ejectors show strong antiviral activity, but are endowed with a low therapeutic index due to their lack of specificity, which has resulted in toxicity. Currently, they are mainly being investigated for use as topical microbicides. Greater specificity may be achieved by using non-covalent NC inhibitors (NCIs) targeting the hydrophobic platform at the top of the zinc fingers or key nucleic acid partners of NC. Within the last few years, innovative methodologies have been developed to identify NCIs. Though the antiviral activity of the identified NCIs needs still to be improved, these compounds strongly support the druggability of NC and pave the way for future structure-based design and optimization of efficient NCIs.
... conformations (Tsuchihashi and Brown, 1994) (reviewed in Darlix et al., 2011;Godet and Mély, 2010;Levin et al., 2005Levin et al., , 2010Rein et al., 1998) (also see more recent Refs. Hergott et al., 2013;Mitra et al., 2013;Wu et al., 2013Wu et al., , 2014. Effective chaperone activity depends on three properties: (i) aggregation of nucleic acids, which is important for annealing (associated with the basic residues); (ii) moderate duplex destabilizing activity (associated with the ZFs); and (iii) rapid on-off nucleic acid binding kinetics (Cruceanu et al., 2006a) (reviewed in Levin et al., 2005Levin et al., , 2010Mirambeau et al., 2010;Wu et al., 2010a). ...
... To assay the kinetics of strand transfer, reaction mixtures were scaled up as needed and 10-l aliquots were removed at the indicated times. The percentage of strand transfer product formation was calculated by dividing the amount of transfer product by the total signal present in the gel lane and multiplying by 100 (Hergott et al., 2013). ...
... Three key steps of reverse transcription, tRNA primer annealing (29), minus-strand transfer and plus-strand transfer (30)(31)(32)(33)(34), require significant rearrangement of NA secondary structure. Previous work demonstrated that HIV-1 NC greatly facilitates these processes through its chaperone activity, which includes NA aggregation, destabilization and rapid protein-NA interaction kinetics (15,16,18,(35)(36)(37)(38)(39). In vitro studies suggest that the ability of NC proteins to aggregate NA is due to their cationic character, and this activity is largely independent of their specific zinc-finger structures (9,36,40). ...
During human immunodeficiency virus type 1 (HIV-1) maturation, three different forms of nucleocapsid (NC) protein—NCp15 (p9
+ p6), NCp9 (p7 + SP2) and NCp7—appear successively. A mutant virus expressing NCp15 shows greatly reduced infectivity. Mature
NCp7 is a chaperone protein that facilitates remodeling of nucleic acids (NAs) during reverse transcription. To understand
the strict requirement for NCp15 processing, we compared the chaperone function of the three forms of NC. NCp15 anneals tRNA
to the primer-binding site at a similar rate as NCp7, whereas NCp9 is the most efficient annealing protein. Assays to measure
NA destabilization show a similar trend. Dynamic light scattering studies reveal that NCp15 forms much smaller aggregates
relative to those formed by NCp7 and NCp9. Nuclear magnetic resonance studies suggest that the acidic p6 domain of HIV-1 NCp15
folds back and interacts with the basic zinc fingers. Neutralizing the acidic residues in p6 improves the annealing and aggregation
activity of NCp15 to the level of NCp9 and increases the protein–NA aggregate size. Slower NCp15 dissociation kinetics is
observed by single-molecule DNA stretching, consistent with the formation of electrostatic inter-protein contacts, which likely
contribute to the distinct aggregate morphology, irregular HIV-1 core formation and non-infectious virus.
... Three key steps of reverse transcription, tRNA primer annealing (29), minus-strand transfer and plus-strand transfer (30)(31)(32)(33)(34), require significant rearrangement of NA secondary structure. Previous work demonstrated that HIV-1 NC greatly facilitates these processes through its chaperone activity, which includes NA aggregation, destabilization and rapid protein-NA interaction kinetics (15,16,18,(35)(36)(37)(38)(39). In vitro studies suggest that the ability of NC proteins to aggregate NA is due to their cationic character, and this activity is largely independent of their specific zinc-finger structures (9,36,40). ...
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC's NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC's aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.
... Previous studies have suggested that NC's duplex destabilization activity is more important for reactions where the rate-limiting step involves significant melting of nucleic acid structure 12 . For example, the zinc finger motifs were shown to be important for annealing of the highly structured transactivation response region (TAR) RNA and TAR DNA stemloops 26 and also for RNA removal reactions during reverse transcription [26][27][28] , but are not needed for human tRNA Lys3 annealing to the primer binding site (PBS) in the viral RNA genome 29,30 . The role of zinc fingers was tested by employing a SSHS NC mutant wherein all the Cys residues of the two zinc fingers are mutated to Ser, thus abolishing the capability to bind zinc 26 . ...
The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is a nucleic acid chaperone that facilitates nucleic acid conformational changes to form the thermodynamically most stable arrangement. The critical role of NC in many steps of the viral life cycle makes it an attractive therapeutic target. The chaperone activity of NC depends on its nucleic acid aggregating ability, duplex destabilizing activity and rapid on/off binding kinetics. During the minus-strand transfer step of reverse transcription, NC chaperones the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nucleotide TAR RNA/DNA annealing was probed by using chemically-synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(1-28), NC(1-35), NC(29-55), NC(36-55) and NC(11-55). Most of these peptides displayed significantly reduced annealing kinetics, even when present at much higher concentrations than wild-type (WT) NC. In addition, these truncated NC constructs generally bind more weakly to single-stranded DNA and are less effective nucleic acid aggregating agents than full-length NC, consistent with the loss of both electrostatic and hydrophobic contacts. However, NC(1-35) displayed annealing kinetics, nucleic acid binding, and aggregation activity that were very similar to that of WT NC. Thus, we conclude that the N-terminal zinc finger, flanked by the N-terminus and linker domains, represents the minimal sequence that is necessary and sufficient for chaperone function in vitro. In addition, covalent continuity of the N-terminal 35 amino acids of NC is critical for full activity. Thus, although the hydrophobic pocket formed by residues proximal to the C-terminal zinc finger has been a major focus of recent anti-NC therapeutic strategies, NC(1-35) represents an alternative target for therapeutics aimed at disrupting NC's chaperone function.
HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing pre-integration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin.
