Abstract
From retroviral initiation to eukaryotic genome replication, priming cDNA is a challenging task. An underlying problem is that the template lacks a 3′-OH substrate required for faithful initiation of RNA or DNA synthesis by the replicative enzyme, whether it is HIV-1 reverse transcriptase (RT) or eukaryotic pol α. The solutions to this problem are various – in the case of hepadnaviruses, the replicase itself uses a tyrosine residue for the substrate to mimic a primer’s 3′-OH (reviewed in (Salas 1991)), and in the case of eukaryotic replication, a second enzyme, the pol α-primase domain, serves the role of synthesizing a primer for use by the replicase pol α. Retroviruses best exemplify genomic brevity by their ability to accomplish so many activities in so few nucleotides (nt). Thus, retroviruses and retrotransposons have solved the priming problem by co-opting an abundant highly conserved cellular factor to serve as the primer for reverse transcriptase and adapting their genomic viral RNA (vRNA) sequences to be complementary to their primers (Dahlberg et al. 1974; Harada et al. 1975; Kikuchi et al. 1986; Peters and Glover 1980). Although the primer is always a tRNA for retroviruses, the specific primer used by a retrovirus subgroup is unique, with all lentiviruses including HIV-1 using solely tRNALys3 in vivo (reviewed in (Mak and Kleiman 1997; Marquet et al. 1995)).
Conflict of interest statement: None declared.
Access provided by Autonomous University of Puebla. Download chapter PDF
Similar content being viewed by others
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
1 Introduction
From retroviral initiation to eukaryotic genome replication, priming cDNA is a challenging task. An underlying problem is that the template lacks a 3′-OH substrate required for faithful initiation of RNA or DNA synthesis by the replicative enzyme, whether it is HIV-1 reverse transcriptase (RT) or eukaryotic pol α. The solutions to this problem are various – in the case of hepadnaviruses, the replicase itself uses a tyrosine residue for the substrate to mimic a primer’s 3′-OH (reviewed in (Salas 1991)), and in the case of eukaryotic replication, a second enzyme, the pol α-primase domain, serves the role of synthesizing a primer for use by the replicase pol α. Retroviruses best exemplify genomic brevity by their ability to accomplish so many activities in so few nucleotides (nt). Thus, retroviruses and retrotransposons have solved the priming problem by co-opting an abundant highly conserved cellular factor to serve as the primer for reverse transcriptase and adapting their genomic viral RNA (vRNA) sequences to be complementary to their primers (Dahlberg et al. 1974; Harada et al. 1975; Kikuchi et al. 1986; Peters and Glover 1980). Although the primer is always a tRNA for retroviruses, the specific primer used by a retrovirus subgroup is unique, with all lentiviruses including HIV-1 using solely tRNALys3 in vivo (reviewed in (Mak and Kleiman 1997; Marquet et al. 1995)).
Upon infection of a host cell and fusion with the plasma membrane, the HIV-1 particle releases its capsid into the cell’s cytoplasm (Fig. 10.1). Contained within the capsid are components necessary for viral replication, including reverse transcriptase, the annealed tRNALys3/vRNA primer/template duplex, the replication chaperone, nucleocapsid protein (NC), and other factors. Reverse transcription begins from the 3′-OH of the CCA end of tRNALys3, which is annealed to the highly conserved primer-binding site (PBS) region on the vRNA. NC facilitates reverse transcription, in part, by destabilizing structured regions of the vRNA to allow RT read through (Ji et al. 1996; Wu et al. 1996). Once reverse transcription is complete, the cDNA is imported into the nucleus and integrated into cellular genomic DNA. Newly synthesized vRNA serves as the template for both viral protein synthesis (in both spliced and unspliced forms) and as the genomic material for incorporation into newly assembled virions (unspliced form only). During assembly, the Gag protein packages the unspliced genomic vRNA together with other necessary viral proteins and cellular components, such as the tRNA primer, and altogether the ~100 nm virus particle buds off from the infected cell. The tRNA content of virions is critical for viral infectivity due to the essential role the primer plays in initiating cDNA synthesis – overexpression of tRNALys3 yields more infectious virions with higher tRNALys3 content, and preventing tRNALys3 incorporation produces less infectious virions (Guo et al. 2003). Thus, preventing tRNALys packaging and inhibiting priming are viable strategies for reducing virus infectivity (Fig. 10.1).
In this chapter, we discuss investigations into the mechanism of priming of HIV-1 reverse transcription with emphasis on how findings may impact the development of antiretroviral drugs targeting tRNALys3 priming. First, we review what is known about the mechanism of tRNA annealing and the structure of the primer/template complex. As virion tRNA content is modulated by the reverse transcriptase (RT) domain of GagPol and the cellular factor lysyl-tRNA synthetase (LysRS), the contribution of these accessory proteins to tRNA packaging and annealing is also discussed. Finally, we review efforts to date to directly prevent priming and to inhibit primer tRNALys3 packaging, noting recent developments in understanding the complex interactions between the protein and RNA components in the initiation complex. Reverse transcription initiation (tRNALys3 priming) is not the only priming event in the HIV-1 life cycle, and the subsequent polypurine tract priming events for positive strand synthesis will not be discussed here.
2 Mechanism of tRNALys3 Annealing
Duplex formation between the 3′ 18 nt of tRNALys3 and a complementary and absolutely conserved 18-nt PBS on the HIV-1 genome is the primary interaction that allows for initiation of DNA synthesis by RT. This annealing reaction is catalyzed by the NC domain of Gag (Feng et al. 1999; Jones et al. 2011; Roldan et al. 2005; Wu et al. 2010), which acts as a nucleic acid chaperone (Darlix et al. 2011; Levin et al. 2005, 2010). Deletion of the PBS produces noninfectious virions despite expression of Gag and Env gene products (Rhim et al. 1991). Additional tRNA-vRNA interactions outside of the PBS have also been reported, including an interaction between the anticodon stem/variable loop of tRNALys3 and a sequence upstream of the PBS (Iwatani et al. 2003). An A-rich loop or bulge in the genomes of some isolates has also been reported to interact with the U-rich anticodon of tRNALys3 (Isel et al. 1993). An alternative interaction between the TΨC stem and nt 123–130 of the HIV-1 genome termed the primer activation signal (PAS) has been proposed to regulate reverse transcription initiation (Beerens and Berkhout 2002; Beerens et al. 2001). The PAS appears to contribute to the virus’s preference for tRNALys3, as mutating both the PBS and PAS is required to switch the preference of HIV-1 from tRNALys3 to tRNALys1,2 (Abbink et al. 2004).
Reversion from an altered PBS sequence back to wild type (WT) can occur in a single virus replication cycle, as the PBS sequence in the viral cDNA is copied directly from the tRNA primer. Efforts to engineer HIV-1 to use alternative primers by mutation of the PBS have shown varied success – either immediate reversion to tRNALys3 or replication failure is observed in most cases. However, use of tRNALys1,2, tRNAHis, tRNAMet, and tRNAGlu produces infectious virions although the replication characteristics of these viruses are altered (Ni and Morrow 2007; Wei et al. 2005). A similar observation has been made in avian leukosis virus where the use of other tRNA primers is possible although with observed growth defects (Whitcomb et al. 1995). Aberrant annealing has also been observed – either due to the use of the incorrect tRNA to misprime reverse transcription (Colicelli and Goff 1986a, b; Wakefield et al. 1995) or due to the priming of cellular mRNAs with short “tRNA-like” primers (Taylor and Cywinski 1984). HIV-1 RT can cope with misaligned annealing, and minor insertions and deletions to the PBS can still be extended at low levels, eventually leading to the recovery of WT PBS upon virus replication (Das and Berkhout 1995).
The precise timing of tRNALys3 annealing in the HIV-1 life cycle is not known. However, protease-negative virions contain annealed tRNALys3 (Fu et al. 1997; Huang et al. 1997; Stewart et al. 1990) and mature virions contain a greater amount of tRNA stably annealed to vRNA (Guo et al. 2009). Thus, annealing has been described as a multistep process that is initially catalyzed by Gag in the cell and later remodeled by the Gag cleavage product NC in the mature virion. A corresponding maturation step in genome dimerization has also been proposed (Jalalirad and Laughrea 2010), suggesting that liberation of NC from Gag leads to changes in chaperone activity as well. Highlighting a newly discovered role for NC in the HIV-1 life cycle, recent studies have observed a loss in virus infectivity in NC zinc-finger mutant viruses in which reverse transcription occurs prematurely (i.e., priming and extension in the cytoplasm prior to budding) (Houzet et al. 2008; Thomas et al. 2008). However, the loss in infectivity is not due to early reverse transcription (Thomas et al. 2011). These studies demonstrate that WT virus suppresses reverse transcription until after assembly and budding.
In vitro, tRNA annealing can be observed by mixing tRNALys3 and vRNA in the presence of NC (Chan et al. 1999; De Rocquigny et al. 1992) or Gag (Jones et al. 2011; Roldan et al. 2005; Wu et al. 2010). The tRNA may also be hybridized to the template by thermal denaturation and still be competent for reverse transcription (Waters et al. 1975). Previous reports found no difference in the structure of the template between heat-annealed and NC-annealed RNAs (Brule et al. 2002; Kim et al. 2012). Alternatively, vRNA and tRNALys3 gently extracted from virions can serve as substrates for in vitro extension assays in which extension via radiolabel incorporation is a measure of the amount of annealed complex (Cen et al. 2009; Huang et al. 1997). A detailed kinetic analysis of tRNA annealing in the presence and absence of NC found that NC accelerates annealing by up to ~105-fold and that the uncatalyzed reaction would proceed with a t1/2 of ~4.5 days (Hargittai et al. 2004). Ten- to 100-fold of this rate enhancement is due to NC’s destabilization of secondary structure within the PBS template, and a further 103- to 104-fold enhancement is achieved by NC-facilitated duplex nucleation (i.e., bringing the strands together).
In vivo, the initial annealing step is performed by Gag (Feng et al. 1999), which facilitates the annealing reaction at least tenfold more slowly in vitro when compared to mature NC (Jones et al. 2011; Wu et al. 2010). The reduced chaperone activity of Gag relative to NC is partly attributed to the matrix (MA) domain, which also interacts with nucleic acids. Inositol phosphates can stimulate Gag’s tRNA annealing activity by competing with MA to prevent it from interacting with nucleic acids (Jones et al. 2011). In addition, deletion of the MA domain stimulates Gag’s chaperone activity.
3 Structure of the Annealed tRNA/vRNA Complex
The structure of the HIV-1 genome has been probed using chemical and enzymatic methods (reviewed in (Lu et al. 2011b)), NMR spectroscopy (Lu et al. 2011a; Puglisi and Puglisi 2011; Tisné et al. 2004), and mass spectrometry-based approaches (Turner et al. 2009; Yu et al. 2008). Recently, the secondary structure of the entire genome has been investigated using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) (Watts et al. 2009). The three-dimensional structure of the complete ~340-nt 5′ UTR, a highly structured region of the genome, is unknown, and the differences in the reported secondary structures likely reflect the dynamics of the vRNA, as well as sequence differences between different isolates (Goldschmidt et al. 2004). Recent work has suggested that the variety of secondary structure models is an accurate depiction of conformational heterogeneity, and it is likely that the HIV-1 genome adopts alternate conformations depending on its involvement in translation or packaging (Lu et al. 2011a) or its monomeric/dimeric state. Examination of long constructs has proven difficult; however, shorter regions of the genome have been examined via NMR, and high-resolution structural models are available for various stem loops, including SL1 (Lawrence et al. 2003), SL2 (Amarasinghe et al. 2000), SL3 (De Guzman et al. 1998), and SL4 (Kerwood et al. 2001) of the packaging signal; the end of TAR (Aboul-ela et al. 1996); and an A-rich stemloop upstream of the PBS in the MAL isolate (Puglisi and Puglisi 1998).
Understanding how the 5′ UTR structure changes as a consequence of tRNALys3 primer annealing poses an even greater challenge. The crystal structure of the fully modified primer tRNA alone has been solved to 3.3 Å and adopts a canonical L-shaped tRNA fold (Benas et al. 2000). Studies to understand the mechanism of tRNA annealing have been performed to probe how NC affects tRNA structure and to determine which parts of NC mediate changes within tRNALys3 and vRNA (Barraud et al. 2007; Tisné et al. 2001, 2003, 2004). NC’s interactions with the primer tRNA appear to be primarily within the acceptor stem, on the inside of the L shape, and on the D stem at the point of tertiary interactions in the loop (Tisné et al. 2001). Using either NC or heat, tRNALys3 was annealed to an 18-nt PBS sequence or a 65-nt RNA containing the PBS and examined by NMR. These studies found that melting initiated at the four-way junction and continued at the base of the acceptor and TΨC stems (Tisné et al. 2004). These findings are consistent with terbium probing, which also revealed that NC first disrupts the metal binding pockets of the tRNA, including those that stabilize the tRNA tertiary fold at the D/TΨC junction (Hargittai et al. 2001). An important finding from these studies and FRET-based studies (Chan et al. 1999) is that NC weakly disrupts the overall structure of tRNALys3 and that complete unwinding of the acceptor stem is not observed in the absence of vRNA.
Chemical and enzymatic probing of the annealed complex (MAL isolate) has been performed (Isel et al. 1995), and SHAPE was also used to determine the secondary structural models of the free vRNA and annealed tRNALys3/vRNA complex (NL4-3 isolate) (Wilkinson et al. 2008). These models are consistent in predicting both the 18-nt duplex formed between vRNA and tRNALys3, as well as the previously proposed additional interactions (Isel et al. 1993; Iwatani et al. 2003) despite sequence differences between isolates. Additional footprinting assays with the tRNALys3/vRNA complex (MAL isolate) in the presence of RT led to a proposed model for the initiation complex, which predicted that RT could change orientations relative to its tRNALys3/vRNA substrate (Isel et al. 1999). More recently, the initiation complex of the MAL isolate has been investigated using NMR (Puglisi and Puglisi 2011), and substantial secondary structural changes were observed in both the template and primer tRNALys3 in the annealed duplex compared to the free RNAs. One of these changes occurring upstream from the 18-nt PBS duplex involves the formation of a long stable hairpin, which is hypothesized to be a block to RT initiation (further discussed below) (Liu et al. 2010). Similar to the study with the NL4-3-derived PBS (Tisné et al. 2004), the interaction between the U-rich tRNALys3 anticodon and an A-rich loop in the MAL vRNA was not observed by NMR even though DMS probing/foot printing showed protection of the A-rich loop in the presence of either transcribed or authentic tRNALys3 (Puglisi and Puglisi 2011). Taken together, these results suggest that the structure of the annealed template is significantly different from the free vRNA template, but the precise interactions present remain to be determined.
A recent single molecule Förster resonance energy transfer (FRET) study (Liu et al. 2010) was able to observe the dynamic interactions occurring between RT and the tRNALys3/vRNA duplex and demonstrate that a stemloop within the template prevents RT from extending the tRNALys3 primer past 3 nt. Using RT labeled with a fluorescent donor and annealed tRNALys3/PBS template labeled with a fluorescent acceptor, two FRET states were observable, corresponding to RT bound in a polymerase orientation (productive for extension) or an RNase H orientation (nonproductive for extension). In the presence of extended tRNALys3 + 3, which mimics the tRNALys3 primer after 3 nt have been added to the CCA end, RT remained primarily in the nonproductive orientation. However, in the presence of tRNALys3 + 6, which mimics the primer after 6 nt of extension, RT was found predominantly in the productive orientation, which was attributed to destabilization of a hairpin upstream of the PBS (Liu et al. 2010). The presence of NC was also sufficient to destabilize this hairpin, and mutation of the stemloop to destabilize its secondary structure also allowed for productive RT binding. These findings support a mechanism in which RT prevents premature initiation by remaining in a nonproductive orientation on the template but binds productively and extends the tRNA primer once NC is present.
4 Accessory Factors to tRNA Packaging
Retroviruses are ribonucleoprotein complexes that package various levels of host tRNAs. In some cases, the total tRNA content of virions reflects the cellular milieu in which assembling particles coalesce (Kelly et al. 2003); however, many studies have noted that the tRNA in cells differs considerably from the tRNA in retroviruses (Jiang et al. 1993; Pavon-Eternod et al. 2010; Waters 1975; Waters et al. 1975, 1980). Since the vRNA serves as both template for Gag translation and future packaging material, it has been suggested that newly translated Gag interacts with nearby vRNA, thereby shutting off translation for that particular vRNA and eventually leading to its incorporation into assembling virions. Even though Gag binds relatively tightly to all RNAs, Gag preferentially binds the packaging signal or ψ region of the genome in the 5′ UTR and beginning of the Gag open reading frame (ORF), which allows the precise selection of the full-length vRNA instead of spliced versions of vRNA or other cellular mRNAs (reviewed in (Lu et al. 2011b)). Thus, preference for Ψ excludes other cellular RNAs from being packaged. tRNAs and other noncoding relatively short RNAs are exceptions: many tRNAs are packaged into each HIV-1 virion along with copies of 7SL RNA (Onafuwa-Nuga et al. 2006). MLV also packages 7SL RNA and a variety of other cellular RNAs, including Ro RNP RNAs, 5S rRNA, and U6 snRNA (Onafuwa-Nuga et al. 2005). Of the tRNAs packaged in HIV-1, tRNALys (~35 %) and tRNAAsn(GUU) (11 %) are the most abundant and are enriched in virions, as their relevant percentages in the cytoplasm are 4.5 % and 3.4 % of total tRNA, respectively (Pavon-Eternod et al. 2010).
In avian retroviruses, it was suggested that RT within GagPol is the major determinant for tRNA packaging and that RT binds specifically to its primer tRNATrp (Cordell et al. 1979; Garret et al. 1984; Panet et al. 1975; Peters and Hu 1980). Studies of HIV-1 also initially suggested RT to be crucial for tRNA packaging via an interaction between RT and the anticodon domain of its tRNALys3 primer (Baratet al. 1989; Mak et al. 1994). However, later reports excluded the anticodon sequence to be required for RT-tRNA interaction and attributed the overall tRNA fold to be the recognition motif for RT, which binds preferentially to tRNAs but otherwise shows no specificity (Arion et al. 1996; Oude Essink et al. 1995; Thrall et al. 1996). Moreover, the contribution of GagPol is essential for tRNA packaging as Gag-only virus-like particles contain >80 % less tRNALys than WT particles (Pavon-Eternod et al. 2010). Nevertheless, the specificity must be derived from another factor. The interaction between RT and the tRNA/vRNA complex is not unlike the interaction between ribosomes and internal ribosome entry sites – both occur at the 5′ UTR of the RNA in question, recognize tRNA or tRNA-like structural motifs, and signal the starting points for cDNA and protein synthesis, respectively.
The observation that all tRNALys isoacceptors, tRNALys3 and tRNALys1,2, are selectively packaged into HIV-1 virions and that HIV-1 RT only generally recognizes the tRNA fold implies that a common interacting factor specific for tRNALys is involved in selective primer packaging. A likely candidate, lysyl-tRNA synthetase (LysRS), is a translation factor and natural binding partner of all tRNALys isoacceptors, which are aminoacylated with lysine by LysRS for use by the ribosome in protein synthesis. Indeed, it was discovered that LysRS is packaged into HIV-1 virions in an ~1:1 correspondence with tRNALys (Cen et al. 2001, 2002) while other aminoacyl-tRNA synthetases and associated factors are undetectable in particles (Halwani et al. 2004). In RSV, which primes reverse transcription with tRNATrp, tryptophanyl-tRNA synthetase (TrpRS) is packaged whereas other aminoacyl-tRNA synthetases are not, suggesting that other retroviruses may also package tRNA/synthetase complexes (Cen et al. 2002). The ability of LysRS to bind its cognate tRNA influences packaging efficiency, as tRNALys3 anticodon mutations reduce their incorporation (Javanbakht et al. 2002). In vitro studies and cell-based assays suggest that LysRS is packaged via an interaction between the motif 1 dimerization interface of the synthetase and the CA domain of Gag (Javanbakht et al. 2003). A recent mass spectrometry-based proteomics analysis also identified the multisynthetase complex (MSC) as an interacting partner of Gag’s MA domain, although the significance of this interaction is unclear (Jager et al. 2012). LysRS is present in cells in premitochondrial, mitochondrial, and cytoplasmic forms (Cen et al. 2001, 2002; Kaminska et al. 2007). These forms are differentiated by the inclusion of a signal peptide in the premitochondrial LysRS, which is cleaved in the mitochondrial form. Cytoplasmic and mitochondrial LysRS are derived from the same gene, which undergoes alternative splicing, and are identical except for ~20 amino acids at the N-terminus (Tolkunova et al. 2000; Dias 2012).
In addition to its canonical function in tRNA aminoacylation, LysRS also has additional “moonlighting” jobs in the cell (reviewed in (Nechushtan et al. 2009)). LysRS is believed to be stored in the MSC for translation and mobilized for its other cellular roles. For example, in mast cells LysRS functions as an Ap4A-producing transcription factor in complex with microphthalmia transcription factor (MITF) and is released from the MSC via a conformational change induced by phosphorylation (Fang et al. 2011; Yannay-Cohen et al. 2009). tRNAs are channeled for translation and are rarely free in the cytoplasm in their non-aminoacylated form; however, separate pools of active synthetase/tRNA have been observed that do not participate in translation (Kyriacou and Deutscher 2008). Thus, the source of primer tRNALys and LysRS in HIV-1 is either non-MSC bound or mobilized from the MSC via a viral signal. Based upon siRNA knockdown data, in early time points, a 20–25 % decrease in total cellular LysRS correlated with an 80 % decrease in packaged LysRS, suggesting that newly synthesized LysRS might be the cellular source of LysRS in virions. LysRS is presumably bound to tRNALys3 when it encounters the assembly complex, and the RT domain of GagPol may simultaneously interact with the primer tRNA as RT recognizes the features of tRNAs that are not identity elements for LysRS. Mitochondrial LysRS has also been proposed to be the source of LysRS in HIV-1 (Kaminska et al. 2007).
The state of tRNALys is critical for priming because the site of lysine attachment in aminoacylated tRNA is the CCA-3′-OH, which is also the substrate for RT (Shimada et al. 1994). In avian myeloblastosis virus, which uses tRNATrp as its primer, RT was reported to have deacylation activity in the presence of Trp-tRNATrp and viral RNA but not in the presence of tRNATrp alone (Litvak et al. 1983; Sarih et al. 1982), suggesting that primer/template annealing facilitates tRNATrp deacylation by RT. In HIV-1, aminoacylated Lys-tRNALys3 cannot be extended by HIV-1 RT, and RT does not accelerate the rate of deacylation of Lys-tRNALys3 (Rigourd et al. 2003). Spontaneous deacylation of charged tRNAs and the synthetase-catalyzed reverse reaction also occurs to varying extents (Bonnet and Ebel 1972). Alternatively, tRNALys3 charging may be prevented by an unknown mechanism such that tRNALys3 is packaged in an uncharged state.
5 Inhibitors of tRNA Priming
The most desirable drug targets in the HIV-1 genome are highly conserved sites, and many of the most highly conserved RNA sequences are found within the 5′ UTR, such as the PBS. Strategies for inhibiting tRNA priming include (1) preventing packaging of the tRNALys3 primer by disruption of the tRNALys/LysRS/Gag/GagPol packaging complex, (2) blocking NC’s chaperone activity and thus preventing tRNA annealing, and (3) inhibiting tRNALys3 extension by RT (Fig. 10.1). Disrupting the packaging complex could involve inhibition of the interaction between Gag and LysRS. Cyclic peptides selected for binding to Gag’s CA domain have been recently developed and shown to effectively compete for the Gag/LysRS interaction in vitro (Dewan et al. 2012). Inhibition of NC’s chaperone activity is a promising strategy, as multiple steps in reverse transcription would be inhibited using this approach.
More recent research in antiretroviral therapies has been progressing toward the goal of disrupting NC/tRNALys3 interactions (Graham et al. 2011). The anticodon stemloop of tRNALys3 is highly posttranscriptionally modified, containing 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34 or S34), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37), and pseudouridine (Ψ39) modifications. According to a recent report, NC’s affinity to the tRNALys3 anticodon is enhanced by these modifications, which allow NC to better melt the anticodon stemloop and facilitate tRNA annealing (Graham et al. 2011). A more general series of NC inhibitors, which would potentially prevent priming as well as other RNA refolding activities chaperoned by NC, are the zinc-ejecting class of drugs, such as S-acyl-2-mercaptobenzamide thioesters and others (de Rocquigny et al. 2008; Miller Jenkins et al. 2007, 2010; Morcock et al. 2008; Pannecouque et al. 2010; Rice et al. 1993; Wallace et al. 2009). These inhibitors irreversibly react with the cysteines of the CCHC zinc fingers, thus preventing zinc binding and eliminating NC’s duplex destabilization activity (reviewed in (Supuran et al. 2004)). As the zinc fingers also play a well established role in ψ recognition, this class of drugs could also disrupt virus assembly provided that they penetrate cell membranes. A unique feature of some compounds in this drug class is inactivation of NC followed by regeneration of the active drug by cellular enzymes (Miller Jenkins et al. 2010). One recent screen for inhibitors of NC interactions with SL2, a stemloop in ψ region of the vRNA, identified compounds with modest antiviral activity (Breuer et al. 2012). Another class of NC-inhibiting compounds, methylated oligonucleotides, compete for NC/vRNA interactions and inhibit viral replication (Avilov et al. 2012; Grigorov et al. 2011).
For inhibitors of RT initiation, one strategy is to use a mimic of the primer tRNALys3 to block annealing such that RT initiation is prevented or leads to inert cDNA. For example, one paradigm is to provide a tRNALys3 mimic that would inactivate RT. Alternatively, the annealed primer/template complex could be targeted and cleaved. Antisense phosphorothioate oligonucleotides to the Gag, Tat, and Rev ORFs and siRNAs to the 5′ UTR, Vif, Tat, Rev, and Nef have been shown to be inhibitors of HIV replication (Coburn and Cullen 2002; Jacque et al. 2002; Lisziewicz et al. 1993), and phosphorothioate oligonucleotides derived from the sequence of tRNALys3 have been shown to inhibit RT (El Dirani-Diab et al. 1997). A tRNALys3 mispriming mimic designed to be complementary to a region in TAR was found to modestly inhibit HIV-1 replication (10- to 50-fold) due to its ability to prime reverse transcription from both the PBS and the TAR sequence (Lu et al. 1997). This tRNA contained a modified acceptor stem that allowed for TAR priming, and complementary mutations in the acceptor stem modestly impaired PBS priming. A second tRNALys3 mimicry strategy used targeted ribozyme cleavage with a tRNALys3 mimic fused to a ribozyme and was shown to be active in vitro (Westaway et al. 1995). Aside from the challenges posed by drug delivery, a difficulty with these strategies is that any primer mimic that resembles the authentic tRNALys3 might compete for tRNALys3 in translation or disrupt charging and thus be toxic. Indirect methods targeting tRNALys3 priming or the PBS have also been developed. For example, targeting the integrated PBS cDNA with an engineered zinc-finger transcription factor designed to repress HIV-1 transcription from the 5′ LTR leads to escape mutations in the PBS (e.g., single or double point mutations), which in turn reduce the rate of viral replication (Eberhardy et al. 2006).
Cellular defenses against retroviruses like HIV-1 include strategies that are often thwarted by HIV-1 accessory proteins. APOBEC3G, a cellular cytidine deaminase, has potent anti-HIV-1 activity in viruses lacking the Vif accessory protein. In addition to hypermutation of viral cDNA, APOBEC3G also appears to function in blocking RT elongation (Bishop et al. 2008; Iwatani et al. 2007) and has been reported to inhibit tRNALys3 annealing (Guo et al. 2009). Moreover, the presence of cleaved primer/template complexes in HIV-1-infected cells suggests that cellular countermeasures against retroviruses by targeting annealed complexes may have already evolved (Yeung et al. 2009). RNA interference (RNAi) is utilized by the cell to silence retrotransposons (Yang and Kazazian 2006), which also have complex 5′ UTRs and prime reverse transcription using cellular tRNAs. Future anti-priming strategies derived from known host defense mechanisms may also provide new approaches to combat HIV-1.
Abbreviations
- FRET:
-
Förster resonance energy transfer
- HIV-1:
-
Human immunodeficiency virus type 1
- LysRS:
-
Lysyl-tRNA synthetase
- MA:
-
Matrix
- NC:
-
Nucleocapsid
- NMR:
-
Nuclear magnetic resonance
- nt:
-
Nucleotide(s)
- ORF:
-
Open reading frame
- PBS:
-
Primer-binding site
- RSV:
-
Rous sarcoma virus
- RT:
-
Reverse transcriptase
- TrpRS:
-
Tryptophanyl-tRNA synthetase
- UTR:
-
Untranslated region
- vRNA:
-
Viral RNA
- WT:
-
Wild type
References
Abbink TE, Beerens N, Berkhout B (2004) Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme. J Virol 78(19):10706–10714
Aboul-ela F, Karn J, Varani G (1996) Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge. Nucleic Acids Res 24(20):3974–3981
Amarasinghe GK, De Guzman RN, Turner RB, Summers MF (2000) NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform. J Mol Biol 299(1):145–156
Arion D, Harada R, Li X, Wainberg MA, Parniak MA (1996) HIV-1 reverse transcriptase shows no specificity for the binding of primer tRNALys3. Biochem Biophys Res Commun 225(3):839–843
Avilov SV, Boudier C, Gottikh M, Darlix JL, Mély Y (2012) Characterization of the inhibition mechanism of HIV-1 nucleocapsid protein chaperone activities by methylated oligoribonucleotides. Antimicrob Agents Chemother 56(2):1010–1018
Barat C, Lullien V, Schatz O, Keith G, Nugeyre MT, Gruninger-Leitch F et al (1989) HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J 8(11):3279–3285
Barraud P, Gaudin C, Dardel F, Tisné C (2007) New insights into the formation of HIV-1 reverse transcription initiation complex. Biochimie 89(10):1204–1210
Beerens N, Berkhout B (2002) The tRNA primer activation signal in the human immunodeficiency virus type 1 genome is important for initiation and processive elongation of reverse transcription. J Virol 76(5):2329–2339
Beerens N, Groot F, Berkhout B (2001) Initiation of HIV-1 reverse transcription is regulated by a primer activation signal. J Biol Chem 276(33):31247–31256
Benas P, Bec G, Keith G, Marquet R, Ehresmann C, Ehresmann B et al (2000) The crystal structure of HIV reverse-transcription primer tRNALys3 shows a canonical anticodon loop. RNA 6(10):1347–1355
Bishop KN, Verma M, Kim EY, Wolinsky SM, Malim MH (2008) APOBEC3G inhibits elongation of HIV-1 reverse transcripts. PLoS Pathog 4(12):e1000231
Bonnet J, Ebel JP (1972) Interpretation of incomplete reactions in tRNA aminoacylation. Aminoacylation of yeast tRNAVal II with yeast valyl-tRNA synthetase. Eur J Biochem 31(2):335–344
Breuer S, Chang MW, Yuan J, Torbett BE (2012) Identification of HIV-1 inhibitors targeting the nucleocapsid protein. J Med Chem 55(11):4968–4977
Brule F, Marquet R, Rong L, Wainberg MA, Roques BP, Le Grice SF et al (2002) Structural and functional properties of the HIV-1 RNA-tRNALys3 primer complex annealed by the nucleocapsid protein: comparison with the heat-annealed complex. RNA 8(1):8–15
Cen S, Khorchid A, Javanbakht H, Gabor J, Stello T, Shiba K et al (2001) Incorporation of lysyl-tRNA synthetase into human immunodeficiency virus type 1. J Virol 75(11):5043–5048
Cen S, Javanbakht H, Kim S, Shiba K, Craven R, Rein A et al (2002) Retrovirus-specific packaging of aminoacyl-tRNA synthetases with cognate primer tRNAs. J Virol 76(24):13111–13115
Cen S, Guo F, Kleiman L (2009) Methods for analysis of incorporation and annealing methods for analysis of tRNALys in HIV-1. Methods Mol Biol 485:223–232
Chan B, Weidemaier K, Yip WT, Barbara PF, Musier-Forsyth K (1999) Intra-tRNA distance measurements for nucleocapsid protein dependent tRNA unwinding during priming of HIV reverse transcription. Proc Natl Acad Sci 96(2):459–464
Coburn GA, Cullen BR (2002) Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 76(18):9225–9231
Colicelli J, Goff SP (1986a) Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J Virol 57(1):37–45
Colicelli J, Goff SP (1986b) Structure of a cloned circular retroviral DNA containing a tRNA sequence between the terminal repeats. J Virol 57(2):674–677
Cordell B, Swanstrom R, Goodman HM, Bishop JM (1979) tRNATrp as primer for RNA-directed DNA polymerase: structural determinants of function. J Biol Chem 254(6):1866–1874
Dahlberg JE, Sawyer RC, Taylor JM, Faras AJ, Levinson WE, Goodman HM et al (1974) Transcription of DNA from the 70S RNA of Rous sarcoma virus. I. Identification of a specific 4S RNA which serves as primer. J Virol 13(5):1126–1133
Darlix JL, Godet J, Ivanyi-Nagy R, Fosse P, Mauffret O, Mély Y (2011) Flexible nature and specific functions of the HIV-1 nucleocapsid protein. J Mol Biol 410(4):565–581
Das AT, Berkhout B (1995) Efficient extension of a misaligned tRNA-primer during replication of the HIV-1 retrovirus. Nucleic Acids Res 23(8):1319–1326
De Guzman RN, Wu ZR, Stalling CC, Pappalardo L, Borer PN, Summers MF (1998) Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element. Science 279(5349):384–388
De Rocquigny H, Gabus C, Vincent A, Fournie-Zaluski MC, Roques B, Darlix JL (1992) Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. Proc Natl Acad Sci 89(14):6472–6476
de Rocquigny H, Shvadchak V, Avilov S, Dong CZ, Dietrich U, Darlix JL et al (2008) Targeting the viral nucleocapsid protein in anti-HIV-1 therapy. Mini Rev Med Chem 8(1):24–35
Dewan V, Liu T, Chen KM, Qian Z, Xiao Y, Kleiman L et al (2012) Cyclic peptide inhibitors of HIV-1 capsid-human lysyl-tRNA synthetase interaction. ACS Chem Biol 7(4):761–769
Dias J, Octobre G, Kobbi L, Comisso M, Flisiak S, Mirande M (2012) Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor. Biochemistry 51(4):909–916
Eberhardy SR, Goncalves J, Coelho S, Segal DJ, Berkhout B, Barbas CF 3rd (2006) Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site. J Virol 80(6):2873–2883
El Dirani-Diab R, Sarih-Cottin L, Delord B, Dumon B, Moreau S, Toulme JJ et al (1997) Phosphorothioate oligonucleotides derived from human immunodeficiency virus type 1 (HIV-1) primer tRNALys3 are strong inhibitors of HIV-1 reverse transcriptase and arrest viral replication in infected cells. Antimicrob Agents Chemother 41(10):2141–2148
Fang P, Zhang HM, Shapiro R, Marshall AG, Schimmel P, Yang XL et al (2011) Structural context for mobilization of a human tRNA synthetase from its cytoplasmic complex. Proc Natl Acad Sci 108(20):8239–8244
Feng YX, Campbell S, Harvin D, Ehresmann B, Ehresmann C, Rein A (1999) 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. J Virol 73(5):4251–4256
Fu W, Ortiz-Conde BA, Gorelick RJ, Hughes SH, Rein A (1997) Placement of tRNA primer on the primer-binding site requires pol gene expression in avian but not murine retroviruses. J Virol 71(9):6940–6946
Garret M, Romby P, Giegé R, Litvak S (1984) Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases. Nucleic Acids Res 12(5):2259–2271
Goldschmidt V, Paillart JC, Rigourd M, Ehresmann B, Aubertin AM, Ehresmann C et al (2004) Structural variability of the initiation complex of HIV-1 reverse transcription. J Biol Chem 279(34):35923–35931
Graham WD, Barley-Maloney L, Stark CJ, Kaur A, Stolyarchuk K, Sproat B et al (2011) Functional recognition of the modified human tRNALys3(UUU) anticodon domain by HIV’s nucleocapsid protein and a peptide mimic. J Mol Biol 410(4):698–715
Grigorov B, Bocquin A, Gabus C, Avilov S, Mély Y, Agopian A et al (2011) Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex. Nucleic Acids Res 39(13):5586–5596
Guo F, Cen S, Niu M, Javanbakht H, Kleiman L (2003) Specific inhibition of the synthesis of human lysyl-tRNA synthetase results in decreases in tRNALys incorporation, tRNALys3 annealing to viral RNA, and viral infectivity in human immunodeficiency virus type 1. J Virol 77(18):9817–9822
Guo F, Saadatmand J, Niu M, Kleiman L (2009) Roles of Gag and NCp7 in facilitating tRNALys3 annealing to viral RNA in human immunodeficiency virus type 1. J Virol 83(16): 8099–8107
Halwani R, Cen S, Javanbakht H, Saadatmand J, Kim S, Shiba K et al (2004) Cellular distribution of Lysyl-tRNA synthetase and its interaction with Gag during human immunodeficiency virus type 1 assembly. J Virol 78(14):7553–7564
Harada F, Sawyer RC, Dahlberg JE (1975) A primer ribonucleic acid for initiation of in vitro Rous sarcarcoma virus deoxyribonucleic acid synthesis. J Biol Chem 250(9):3487–3497
Hargittai MR, Mangla AT, Gorelick RJ, Musier-Forsyth K (2001) HIV-1 nucleocapsid protein zinc finger structures induce tRNALys3 structural changes but are not critical for primer/template annealing. J Mol Biol 312(5):985–997
Hargittai MR, Gorelick RJ, Rouzina I, Musier-Forsyth K (2004) Mechanistic insights into the kinetics of HIV-1 nucleocapsid protein-facilitated tRNA annealing to the primer binding site. J Mol Biol 337(4):951–968
Houzet L, Morichaud Z, Didierlaurent L, Muriaux D, Darlix JL, Mougel M (2008) Nucleocapsid mutations turn HIV-1 into a DNA-containing virus. Nucleic Acids Res 36(7):2311–2319
Huang Y, Wang J, Shalom A, Li Z, Khorchid A, Wainberg MA et al (1997) Primer tRNA3 Lys on the viral genome exists in unextended and two-base extended forms within mature human immunodeficiency virus type 1. J Virol 71(1):726–728
Hulme AE, Perez O, Hope TJ (2011) Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci 108(24):9975–9980
Isel C, Marquet R, Keith G, Ehresmann C, Ehresmann B (1993) Modified nucleotides of tRNALys3 modulate primer/template loop-loop interaction in the initiation complex of HIV-1 reverse transcription. J Biol Chem 268(34):25269–25272
Isel C, Ehresmann C, Keith G, Ehresmann B, Marquet R (1995) Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNALys3 (template/primer). J Mol Biol 247(2):236–250
Isel C, Westhof E, Massire C, Le Grice SF, Ehresmann B, Ehresmann C et al (1999) Structural basis for the specificity of the initiation of HIV-1 reverse transcription. EMBO J 18(4): 1038–1048
Iwatani Y, Rosen AE, Guo J, Musier-Forsyth K, Levin JG (2003) Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions. J Biol Chem 278(16):14185–14195
Iwatani Y, Chan DS, Wang F, Maynard KS, Sugiura W, Gronenborn AM et al (2007) Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Res 35(21):7096–7108
Jacque JM, Triques K, Stevenson M (2002) Modulation of HIV-1 replication by RNA interference. Nature 418(6896):435–438
Jager S, Cimermancic P, Gulbahce N, Johnson JR, McGovern KE, Clarke SC et al (2012) Global landscape of HIV-human protein complexes. Nature 481(7381):365–370
Jalalirad M, Laughrea M (2010) Formation of immature and mature genomic RNA dimers in wild-type and protease-inactive HIV-1: differential roles of the Gag polyprotein, nucleocapsid proteins NCp15, NCp9, NCp7, and the dimerization initiation site. Virology 407(2):225–236
Javanbakht H, Cen S, Musier-Forsyth K, Kleiman L (2002) Correlation between tRNALys3 aminoacylation and its incorporation into HIV-1. J Biol Chem 277(20):17389–17396
Javanbakht H, Halwani R, Cen S, Saadatmand J, Musier-Forsyth K, Göttlinger H et al (2003) The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly. J Biol Chem 278(30):27644–27651
Ji X, Klarmann GJ, Preston BD (1996) Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro. Biochemistry 35(1):132–143
Jiang M, Mak J, Ladha A, Cohen E, Klein M, Rovinski B et al (1993) Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. J Virol 67(6):3246–3253
Jones CP, Datta SA, Rein A, Rouzina I, Musier-Forsyth K (2011) Matrix domain modulates HIV-1 Gag’s nucleic acid chaperone activity via inositol phosphate binding. J Virol 85(4):1594–1603
Kaminska M, Shalak V, Francin M, Mirande M (2007) Viral hijacking of mitochondrial lysyl-tRNA synthetase. J Virol 81(1):68–73
Kelly NJ, Palmer MT, Morrow CD (2003) Selection of retroviral reverse transcription primer is coordinated with tRNA biogenesis. J Virol 77(16):8695–8701
Kerwood DJ, Cavaluzzi MJ, Borer PN (2001) Structure of SL4 RNA from the HIV-1 packaging signal. Biochemistry 40(48):14518–14529
Kikuchi Y, Ando Y, Shiba T (1986) Unusual priming mechanism of RNA-directed DNA synthesis in copia retrovirus-like particles of Drosophila. Nature 323(6091):824–826
Kim J, Roberts A, Yuan H, Xiong Y, Anderson KS (2012) Nucleocapsid protein annealing of a primer-template enhances (+)-strand DNA synthesis and fidelity by HIV-1 reverse transcriptase. J Mol Biol 415(5):866–880
Kyriacou SV, Deutscher MP (2008) An important role for the multienzyme aminoacyl-tRNA synthetase complex in mammalian translation and cell growth. Mol Cell 29(4):419–427
Lawrence DC, Stover CC, Noznitsky J, Wu Z, Summers MF (2003) Structure of the intact stem and bulge of HIV-1 Psi-RNA stem-loop SL1. J Mol Biol 326(2):529–542
Levin JG, Guo J, Rouzina I, Musier-Forsyth K (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80:217–286
Levin JG, Mitra M, Mascarenhas A, Musier-Forsyth K (2010) Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription. RNA Biol 7(6):754–774
Lisziewicz J, Sun D, Metelev V, Zamecnik P, Gallo RC, Agrawal S (1993) Long-term treatment of human immunodeficiency virus-infected cells with antisense oligonucleotide phosphorothioates. Proc Natl Acad Sci 90(9):3860–3864
Litvak S, Sarih L, Fournier M, von der Haar F, Labouesse B, Araya A (1983) Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions. Recent Results Cancer Res 84:184–190
Liu S, Harada BT, Miller JT, Le Grice SF, Zhuang X (2010) Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription. Nat Struct Mol Biol 17(12):1453–1460
Lu Y, Planelles V, Li X, Palaniappan C, Day B, Challita-Eid P et al (1997) Inhibition of HIV-1 replication using a mutated tRNALys3 primer. J Biol Chem 272(23):14523–14531
Lu K, Heng X, Garyu L, Monti S, Garcia EL, Kharytonchyk S et al (2011a) NMR detection of structures in the HIV-1 5’-leader RNA that regulate genome packaging. Science 334(6053):242–245
Lu K, Heng X, Summers MF (2011b) Structural determinants and mechanism of HIV-1 genome packaging. J Mol Biol 410(4):609–633
Mak J, Kleiman L (1997) Primer tRNAs for reverse transcription. J Virol 71(11):8087–8095
Mak J, Jiang M, Wainberg MA, Hammarskjold ML, Rekosh D, Kleiman L (1994) Role of Pr160gag-pol in mediating the selective incorporation of tRNALys into human immunodeficiency virus type 1 particles. J Virol 68(4):2065–2072
Marquet R, Isel C, Ehresmann C, Ehresmann B (1995) tRNAs as primer of reverse transcriptases. Biochimie 77(1–2):113–124
Miller Jenkins LM, Hara T, Durell SR, Hayashi R, Inman JK, Piquemal JP et al (2007) Specificity of acyl transfer from 2-mercaptobenzamide thioesters to the HIV-1 nucleocapsid protein. J Am Chem Soc 129(36):11067–11078
Miller Jenkins LM, Ott DE, Hayashi R, Coren LV, Wang D, Xu Q et al (2010) Small-molecule inactivation of HIV-1 NCp7 by repetitive intracellular acyl transfer. Nat Chem Biol 6(12):887–889
Morcock DR, Thomas JA, Sowder RC 2nd, Henderson LE, Crise BJ, Gorelick RJ (2008) HIV-1 inactivation by 4-vinylpyridine is enhanced by dissociating Zn2+ from nucleocapsid protein. Virology 375(1):148–158
Nechushtan H, Kim S, Kay G, Razin E (2009) Chapter 1: The physiological role of lysyl tRNA synthetase in the immune system. Adv Immunol 103:1–27
Ni N, Morrow CD (2007) Impact of forced selection of tRNAs on HIV-1 replication and genome stability highlight preferences for selection of certain tRNAs. Virus Res 124(1–2):29–37
Onafuwa-Nuga AA, King SR, Telesnitsky A (2005) Nonrandom packaging of host RNAs in Moloney murine leukemia virus. J Virol 79(21):13528–13537
Onafuwa-Nuga AA, Telesnitsky A, King SR (2006) 7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles. RNA 12(4):542–546
Oude Essink BB, Das AT, Berkhout B (1995) Structural requirements for the binding of tRNALys3 to reverse transcriptase of the human immunodeficiency virus type 1. J Biol Chem 270(40):23867–23874
Panet A, Haseltine WA, Baltimore D, Peters G, Harada F, Dahlberg JE (1975) Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase). Proc Natl Acad Sci USA 72(7):2535–2539
Pannecouque C, Szafarowicz B, Volkova N, Bakulev V, Dehaen W, Mély Y et al (2010) Inhibition of HIV-1 replication by a bis-thiadiazolbenzene-1, 2-diamine that chelates zinc ions from retroviral nucleocapsid zinc fingers. Antimicrob Agents Chemother 54(4):1461–1468
Pavon-Eternod M, Wei M, Pan T, Kleiman L (2010) Profiling non-lysyl tRNAs in HIV-1. RNA 16(2):267–273
Peters G, Glover C (1980) tRNA’s and priming of RNA-directed DNA synthesis in mouse mammary tumor virus. J Virol 35(1):31–40
Peters GG, Hu J (1980) Reverse transcriptase as the major determinant for selective packaging of tRNA’s into Avian sarcoma virus particles. J Virol 36(3):692–700
Puglisi EV, Puglisi JD (1998) HIV-1 A-rich RNA loop mimics the tRNA anticodon structure. Nat Struct Biol 5(12):1033–1036
Puglisi EV, Puglisi JD (2011) Secondary structure of the HIV reverse transcription initiation complex by NMR. J Mol Biol 410(5):863–874
Rhim H, Park J, Morrow CD (1991) Deletions in the tRNALys primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. J Virol 65(9):4555–4564
Rice WG, Schaeffer CA, Harten B, Villinger F, South TL, Summers MF et al (1993) Inhibition of HIV-1 infectivity by zinc-ejecting aromatic C-nitroso compounds. Nature 361(6411):473–475
Rigourd M, Bec G, Benas P, Le Grice SF, Ehresmann B, Ehresmann C et al (2003) Effects of tRNALys3 aminoacylation on the initiation of HIV-1 reverse transcription. Biochimie 85(5):521–525
Roldan A, Warren OU, Russell RS, Liang C, Wainberg MA (2005) A HIV-1 minimal Gag protein is superior to nucleocapsid at in vitro annealing and exhibits multimerization-induced inhibition of reverse transcription. J Biol Chem 280(17):17488–17496
Salas M (1991) Protein-priming of DNA replication. Annu Rev Biochem 60:39–71
Sarih L, Fournier M, von der Haar F, Labouesse J, Litvak S (1982) Avian myeloblastosis reverse transcriptase deacylates tryptophanyl-tRNA. Nucleic Acids Res 10(22):7387–7393
Shimada M, Hosaka H, Takaku H, Smith JS, Roth MJ, Inouye S et al (1994) Specificity of priming reaction of HIV-1 reverse transcriptase, 2’-OH or 3’-OH. J Biol Chem 269(6):3925–3927
Stewart L, Schatz G, Vogt VM (1990) Properties of avian retrovirus particles defective in viral protease. J Virol 64(10):5076–5092
Supuran CT, Innocenti A, Mastrolorenzo A, Scozzafava A (2004) Antiviral sulfonamide derivatives. Mini Rev Med Chem 4(2):189–200
Taylor JM, Cywinski A (1984) A defective retrovirus particle (SE21Q1b) packages and reverse transcribes cellular RNA, utilizing tRNA-like primers. J Virol 51(2):267–271
Thomas JA, Bosche WJ, Shatzer TL, Johnson DG, Gorelick RJ (2008) Mutations in human immunodeficiency virus type 1 nucleocapsid protein zinc fingers cause premature reverse transcription. J Virol 82(19):9318–9328
Thomas JA, Shatzer TL, Gorelick RJ (2011) Blocking premature reverse transcription fails to rescue the HIV-1 nucleocapsid-mutant replication defect. Retrovirology 8:46
Thrall SH, Reinstein J, Wöhrl BM, Goody RS (1996) Evaluation of human immunodeficiency virus type 1 reverse transcriptase primer tRNA binding by fluorescence spectroscopy: specificity and comparison to primer/template binding. Biochemistry 35(14):4609–4618
Tisné C, Roques BP, Dardel F (2001) Heteronuclear NMR studies of the interaction of tRNALys3 with HIV-1 nucleocapsid protein. J Mol Biol 306(3):443–454
Tisné C, Roques BP, Dardel F (2003) Specific recognition of primer tRNALys3 by HIV-1 nucleocapsid protein: involvement of the zinc fingers and the N-terminal basic extension. Biochimie 85(5):557–561
Tisné C, Roques BP, Dardel F (2004) The annealing mechanism of HIV-1 reverse transcription primer onto the viral genome. J Biol Chem 279(5):3588–3595
Tolkunova E, Park H, Xia J, King MP, Davidson E (2000) The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript. J Biol Chem 275(45):35063–35069
Turner KB, Yi-Brunozzi HY, Brinson RG, Marino JP, Fabris D, Le Grice SF (2009) SHAMS: combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids. RNA 15(8):1605–1613
Wakefield JK, Wolf AG, Morrow CD (1995) Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNALys3. J Virol 69(10):6021–6029
Wallace GS, Cheng-Mayer C, Schito ML, Fletcher P, Miller Jenkins LM, Hayashi R et al (2009) Human immunodeficiency virus type 1 nucleocapsid inhibitors impede trans infection in cellular and explant models and protect nonhuman primates from infection. J Virol 83(18): 9175–9182
Waters LC (1975) Transfer RNAs associated with the 70S RNA of AKR murine leukemia virus. Biochem Biophys Res Commun 65(3):1130–1136
Waters LC, Mullin BC, Ho T, Yang WK (1975) Ability of tryptophan tRNA to hybridize with 35S RNA of avian myeloblastosis virus and to prime reverse transcription in vitro. Proc Natl Acad Sci 72(6):2155–2159
Waters LC, Mullin BC, Bailiff EG, Popp RA (1980) Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses. Biochim Biophys Acta 608(1):112–126
Watts JM, Dang KK, Gorelick RJ, Leonard CW, Bess JW Jr, Swanstrom R et al (2009) Architecture and secondary structure of an entire HIV-1 RNA genome. Nature 460(7256):711–716
Wei M, Cen S, Niu M, Guo F, Kleiman L (2005) Defective replication in human immunodeficiency virus type 1 when non-primers are used for reverse transcription. J Virol 79(14):9081–9087
Westaway SK, Larson GP, Li S, Zaia JA, Rossi JJ (1995) A chimeric tRNA(Lys3)-ribozyme inhibits HIV replication following virion assembly. Nucleic Acids Symp Ser 33:194–199
Whitcomb JM, Ortiz-Conde BA, Hughes SH (1995) Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers. J Virol 69(10): 6228–6238
Wilkinson KA, Gorelick RJ, Vasa SM, Guex N, Rein A, Mathews DH et al (2008) High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. PLoS Biol 6(4):e96
Wu W, Henderson LE, Copeland TD, Gorelick RJ, Bosche WJ, Rein A et al (1996) Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. J Virol 70(10):7132–7142
Wu T, Datta SA, Mitra M, Gorelick RJ, Rein A, Levin JG (2010) Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: biological implications. Virology 405(2):556–567
Yang N, Kazazian HH Jr (2006) L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells. Nat Struct Mol Biol 13(9):763–771
Yannay-Cohen N, Carmi-Levy I, Kay G, Yang CM, Han JM, Kemeny DM et al (2009) LysRS serves as a key signaling molecule in the immune response by regulating gene expression. Mol Cell 34(5):603–611
Yeung ML, Bennasser Y, Watashi K, Le SY, Houzet L, Jeang KT (2009) Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid. Nucleic Acids Res 37(19):6575–6586
Yu ET, Hawkins A, Eaton J, Fabris D (2008) MS3D structural elucidation of the HIV-1 packaging signal. Proc Natl Acad Sci 105(34):12248–12253
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this chapter
Cite this chapter
Jones, C.P., Musier-Forsyth, K. (2013). tRNA Primer Sequestration as an Antiviral Strategy. In: LeGrice, S., Gotte, M. (eds) Human Immunodeficiency Virus Reverse Transcriptase. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-7291-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4614-7291-9_10
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4614-7290-2
Online ISBN: 978-1-4614-7291-9
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)