tRNA Lys Research Articles

Reverse transcription of plasma-derived HIV-1 RNA generates multiple artifacts through tRNA(Lys-3)-priming.

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.IMPORTANCEThe 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.

Complete mitochondrial genomes of three Oxya grasshoppers (Orthoptera) and their implications for phylogenetic reconstruction

Oxya is a genus of grasshoppers (Orthoptera: Acridoidea) attacking rice and other gramineous plants in Africa and Asia. In the present study, we characterized complete mitochondrial genomes (mitogenomes) of three species, Oxya japonica japonica (15,427 bp), Oxya hainanensis (15,443 bp) and Oxya agavisa robusta (15,552 bp) collected from China. The three mitogenomes contained a typical gene set of metazoan mitogenomes and shared the same gene order with other Acridid grasshoppers, including the rearrangement of tRNAAsp and tRNALys. Analyses of pairwise genetic distances showed that ATP8 was the least conserved gene, while COI the most conserved. To determine the position of Oxya grasshoppers in the phylogeny of Acrididae, we reconstructed phylogenetic trees among 64 species from across 11 subfamilies using nucleotide sequences of mitogenomes. While the tree confirms traditional classifications of Acrididae at major higher-levels, it suggests a few modifications for classifications at lower-levels.

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

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

Designing peptide sequences in flexible chain conformations to bind RNA: a search algorithm combining Monte Carlo, self-consistent mean field and concerted rotation techniques.

A search algorithm combining Monte Carlo, self-consistent mean field, and concerted rotation techniques was developed to discover peptide sequences that are reasonable HIV drug candidates due to their exceptional binding to human tRNAUUU(Lys3), the primer of HIV replication. The search algorithm allows for iteration between sequence mutations and conformation changes during sequence evolution. Searches conducted for different classes of peptides identified several potential peptide candidates. Analysis of the energy revealed that the asparagine and cysteine at residues 11 and 12 play important roles in "recognizing" tRNA(Lys3) via van der Waals interactions, contributing to binding specificity. Arginines preferentially attract the phosphate linkage via charge-charge interaction, contributing to binding affinity. Evaluation of the RNA/peptide complex's structure revealed that adding conformation changes to the search algorithm yields peptides with better binding affinity and specificity to tRNA(Lys3) than a previous mutation-only algorithm.

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Amino Acid Signature Enables Proteins to Recognize Modified tRNA

Human tRNALys3UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNALys3UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNALys3UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N6-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNALys3UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA’s fully modified anticodon stem and loop domain, hASLLys3UUU. Peptides of this sequence specifically recognized and bound modified htRNALys3UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

RNA Structural Rearrangements during Reverse Transcription Initiation in HIV

Reverse transcription is the first step in the replication of the Human Immunodeficiency Virus (HIV) and is a target of multiple therapies. The initiation phase of reverse transcription in human immunodeficiency virus (HIV) is the slowest and least processive step of the reverse transcription process; however, premature initiation of reverse transcription leads to failed infection. During this phase, several structures between the viral genomic RNA and tRNA(Lys3) primer are formed and broken as reverse transcriptase transcribes viral genomic RNA into negative sense DNA. Using NMR spectroscopy, X-Ray crystallography, single molecule FRET spectroscopy, and biochemical methods, we are characterizing these RNA structural rearrangements in order to understand their role in regulating the initiation of reverse transcription in HIV.

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

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

Mapping the Small RNA Content of Simian Immunodeficiency Virions (SIV)

Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV), which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs) that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNALys3 and tRNALys isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging.

Escherichia coli LysU is a potential surrogate for human lysyl tRNA synthetase in interactions with the C-terminal domain of HIV-1 capsid protein

Human lysyl-tRNA synthetase (hLysRS) is known to interact directly with human immunodeficiency virus type-1 (HIV-1) GagPol polyproteins, and both hLysRS with tRNA(Lys3) are selectively packaged into emerging HIV-1 viral particles. This packaging process appears to be mediated by contact between the motif 1 helix h7 of hLysRS and the C-terminal dimerization domain of the HIV-1 capsid protein (CA) segment of Gag or GagPol. Given similarities between hLysRS and Escherichia coli (E. coli) heat shock protein LysU, we investigate if LysU might be an hLysRS surrogate for interactions with Gag or GagPol proteins. We report on a series of studies involving three CA C-domains: CA(146) (intact domain), CA(151) (truncated domain), and CA(146)-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA(146) are dimeric whilst CA(151) and M185A remain monomeric, we use glutathione S-transferase (GST) pull-down assays to demonstrate the existence of specific interactions between LysU and all three CA-C domains. By means of (1)H-NMR titration experiments, we estimate K(d) values of 50 μM for the interaction between LysU and CA(146) or >500 μM for interactions between LysU and CA(151) or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA(146) take place through dimer-dimer interactions resulting in a α(2)β(2) heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA(146) proteins, and hLysRS/Gag binding interactions have also been suggested to involve only αβ heterodimer formation. Nevertheless, we propose that LysU could act as a surrogate for hLysRS with respect to Gag and GagPol polyprotein interactions although arguably not sufficiently for LysU to act as an inhibitor of the HIV-1 life cycle without further adaptation or mutation. Potentially, LysU and/or LysU mutants could represent a new class of anti-HIV-1 therapeutic agent.

MD simulations of HIV-1 RT primer–template complex: effect of modified nucleosides and antisense PNA oligomer

Human immunodeficiency virus type 1 (HIV-1) requires the human tRNA3 Lys as a reverse transcriptase (RT) primer. The annealing of 3’ terminal 18 nucleotides of tRNA3 Lys with the primer binding site (PBS) of viral RNA (vRNA) is crucial for reverse transcription. Additional contacts between the A rich (A-loop) region of vRNA and the anticodon domain of tRNA3 Lys are necessary, which show the specific requirement of tRNA3 Lys. The importance of modified nucleosides, present in tRNA3 Lys, in giving stability to the primer–template complex has been determined in earlier experiments. It has been observed that the PNA oligomer targeted to PBS of vRNA destabilized the crucial interactions between primer and template due to which the reverse transcription is inhibited. Molecular dynamics simulations have been carried out to study the effect of modified nucleosides on the vRNA–tRNA3 Lys complex stability and the destabilization effect of PNA oligomer on the vRNA–tRNA3 Lys–PNA complex. The root-mean-square deviation, hydrogen bonding, tertiary interactions, and free energy calculations of the simulation data support the experimental results. The analyses have revealed the structural changes in PBS region of vRNA which might be another strong reason for the inability of RT binding to 7F helix for its normal functioning of reverse transcription.

Analysis of the complete mitochondrial genome sequence of Pielomastax zhengi

The complete mitochondrial genome sequence of Pielomastax zhengi was determined by using long PCR and conserved primer walking approaches. The results showed that the entire mitochondrial genome of Pielomastax zhengi is 15 602 bp long with A+T content of 71.8%. All 37 genes are conserved in the positions observed in those of Locusta migratoria. All the genes are closely assembled by leaving 10 intergenic spacers in between. Those intergenic spacers are 47 bp in total (excluding the A+T rich region), with individual size ranges from 1 bp to 20 bp. In addition, there are a total of 52 bp overlapping sequences among 14 genes, ranging from 1-8 bp. All protein-coding genes start with a typical initiation codon in insects, ATN. Twelve protein-coding genes use the usual TAA and TAG termination codons, whereas, the ND5 genes have an incomplete termination codon (T). Except the tRNASer (AGN), whose DHU arm is absent; all the other 21 tRNA genes have typical clover-leaf secondary structures. But in Pielomastax zhengi, the secondary structures of five tRNA (tRNA(Cys), tRNA(Lys), tRNA(Phe), tRNA(Pro), tRNA(Arg)) genes can not be predicted by the conventional methods as they have only 3-4, rather than 5 base pairs in the TψC arm, while, the tRNA(Lys) and tRNA(Arg) have only 4 base pairs in the anticode arm. The predicted secondary structures of lrRNA and srRNA have 6 domains with 44 helices and 3 domains with 30 helices, respectively. The results of the rRNA secondary structure comparison showed that Pielomastax zhengi is more closely related with Locusta migratoria tibetensis than Ruspolia dubia. Like most insects, the mitochondrial genome of Pielomastax zhengi has a non-coding A+T rich region containing a polythymidine stretch, which may be involved in the replication and/or translation initiation of other genes.

Secondary Structure of the HIV Reverse Transcription Initiation Complex by NMR

Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA–RNA complex formed between a host transfer RNA (tRNA Lys 3) and a region at the 5′ end of genomic RNA; the 3′ end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA–human tRNA Lys 3 initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3′ end of tRNA Lys 3 drives large conformational rearrangements of the tRNA at the 5′ end while maintaining the anticodon loop for potential loop–loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.

Functional Recognition of the Modified Human tRNA Lys3UUU Anticodon Domain by HIV's Nucleocapsid Protein and a Peptide Mimic

The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA Lys3 UUU as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA Lys3 UUU, the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL Lys3) were studied. NCp7 bound the hASL Lys3 UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm 5s 2U 34) and 2-methylthio- N 6 -threonylcarbamoyladenosine at position-37 (ms 2t 6A 37) with a considerably higher affinity than the unmodified hASL Lys3 UUU ( K d = 0.28 ± 0.03 and 2.30 ± 0.62 μM, respectively). NCp7 denatured the structure of the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 more effectively than that of the unmodified hASL Lys3 UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K d = 0.55 ± 0.10 μM) and specificity for the ASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37 comparable to that of NCp7. The peptides recognized a t 6A 37-modified ASL with an affinity ( K d = 0.60 ± 0.09 μM) comparable to that for hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37, indicating a preference for the t 6A 37 modification. Significantly, one of the peptides was capable of relaxing the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 37;Ψ 39 structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA Lys3 UUU have been found to be important determinants of NCp7's recognition prior to the tRNA Lys3 UUU being annealed to the viral genome as the primer of reverse transcription.

Prediction of folding nuclei in tRNA molecules

Prediction of folding nuclei in RNA molecules allows one to look in a new way at the problem of possible RNA base sequence folding and at problems associated with incorrect RNA folding, as well as at RNA structure stability. We have chosen a model and energy parameters for description of RNA structure. The algorithm for studying processes including protein folding/unfolding was successfully applied to calculations on tRNA. Four tRNA molecules were considered whose structures were obtained in the free state (tRNA(Phe), tRNA(Asp), tRNA(fMet), and tRNA(Lys)). The calculated Φ-values for tRNA molecules correlate with experimental data showing that nucleotide residues in the D and T hairpin regions are involved in tRNA structure last, or more exactly, they are not included in the tRNA folding nucleus. High Φ-values in the anticodon hairpin region show that the nucleus of tRNA folding is localized just in that place.

Packaging HIV Virion Components through Dynamic Equilibria of a Human tRNA Synthetase

Aminoacyl tRNA synthetases, components of the translation apparatus, have alternative functions outside of translation. The structural and mechanistic basis of these alternative functions is of great interest. As an example, reverse transcription of the HIV genome is primed by a human lysine-specific tRNA (tRNA(Lys3)) that is packaged (into the virion) by the HIV Gag protein with lysyl-tRNA synthetase (LysRS). Not understood is the structural basis for simultaneous packaging of tRNA(Lys3), LysRS, and Gag. Here, ab initio computational methods, together with our recent high-resolution 3-D structure of human LysRS, produced an energy-minimized model where Gag, tRNA(Lys), and LysRS form a ternary complex. Interestingly, the model requires normally homodimeric LysRS to dissociate into a monomer that bridges between Gag and tRNA(Lys3). Earlier experiments of others and new experiments presented here, which tested an engineered dissociated form of LysRS, were consistent with the ab initio "bridging monomer" model. The results support an emerging theme that alterative functions of tRNA synthetases may come, in part, from protein surfaces exposed by dynamic equilibria.

HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses.

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tRMDtRL, or one of two truncated tRNAs (Lys-3), tRMDΔARMtRL or tRMD3′-endtRL. Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.

A miRNA-tRNA mix-up: tRNA origin of proposed miRNA.

The rapid release of new data from DNA genome sequencing projects has led to a variety of misannotations in public databases. Our results suggest that next generation sequencing approaches are particularly prone to such misannotations. Two related miRNA candidates did recently enter the miRBase database, miR-1274b and miR-1274a, but they share identical 18-nucleotide stretches with tRNA (Lys3) and tRNA (Lys5) , respectively. The possibility that the small RNA fragments that led to the description of these two miRNAs originated from the two tRNAs was examined. The ratio of the miR-1274b:miR-1274a fragments does closely resemble the known tRNA lys3:lys5 ratio in the cell. Furthermore, the proposed miRNA hairpins have a very low prediction score and the proposed miRNA genes are in fact endogenous retroviral elements. We searched for other miRNA-mimics in the human genome and found more examples of tRNA-miRNA mimicry. We propose that the corresponding miRNAs should be validated in more detail, as the small RNA fragments that led to their description are likely derived from tRNA processing.

TRNA Lys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs

Using exogenous sequences to express RNA interference (RNAi) activators has potential for the treatment of chronic viral infections. However, availability of a variety of suitable of promoter elements is important to optimize transcription control of silencing sequences and facilitate multitargeting. Recent demonstration that tRNA miR genes occur naturally has prompted investigating the incorporation these tRNA Pol III promoters into exogenous RNAi-activating cassettes. We have assessed efficacy of Pol III tRNA Lys3 short hairpin RNA (shRNA) sequences that target hepatitis B virus (HBV). These cassettes achieved good silencing at low concentrations, and efficacy compared favorably to that of equivalent U6, H1 and CMV expression cassettes. HBV replication in cell culture was inhibited and northern blot hybridization analysis confirmed processing of the tRNA Lys3 transcripts to form intended antiviral guide sequences. Importantly effects were observed without evidence of disruption of endogenous miR function. Analysis in a murine hydrodynamic injection model of HBV replication confirmed that the tRNA Lys3 expression cassettes are also effective in vivo. Usefulness of tRNA Lys3 antiviral expression cassettes expands the repertoire of promoters available for RNAi-mediated HBV silencing and advances the application of expressed sequences for therapeutic gene silencing.

The contribution of the primer activation signal to differences between Gag- and NCp7-facilitated tRNALys3 annealing in HIV-1

During tRNA(Lys3) annealing in HIV-1, tRNA(Lys3) binds to both the primer binding site (PBS) and to an 8 nucleotide base-paired sequence upstream of the PBS known as the primer activation signal (PAS). In protease-negative (Pr(-)) HIV-1, the amount of tRNA(Lys3) annealed by Gag is 35% less than that annealed by mature nucleocapsid (NCp7) in protease-positive (Pr(+)) virions. Gag-annealed tRNA(Lys3) also has a reduced ability to initiate reverse transcription, and binds less tightly to viral RNA than NCp7-annealed tRNA(Lys3). Pr(-) virions containing a constitutively single-stranded PAS (2R mutant), show a significant increase in the ability to initiate reverse transcription with little change in the amount of tRNA(Lys3) annealed. However, the 2R mutant does not achieve levels of RT initiation achieved in Pr(+) virions, and tRNA(Lys3) binding to viral RNA remains weak. Wild type levels of initiation and tRNA(Lys3) binding to viral RNA can only be recovered by transient exposure of Pr(-) or Pr(-)2R viral RNA to NCp7. This suggests that in addition to facilitating annealing of tRNA(Lys3) to the PBS and possible denaturation of the PAS, other functions of NCp7 involved in annealing are required. The effect of an inactive protease and/or the 2R mutation upon tRNA(Lys3) annealing and initiation are also observed when the tRNA(Lys3) is annealed in vitro to wild type or mutant viral RNA using either NCp7 or GagDeltap6, indicating a direct effect of the 2R mutation upon tRNA(Lys3) annealing.