Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus type 1 (HIV-1) exclusively selects tRNA3Lys as the primer for the initiation of reverse transcription, even though both tRNA3Lys and tRNA1,2Lys are found in HIV-1 virions. Alteration of the HIV-1 primer-binding site (PBS) to be complementary to alternate tRNAs results in the use of those tRNAs for replication, indicating that primer complementarity with the PBS is an important determinant of primer selection. In previous studies, we have exploited this fact to develop a system in which yeast (Saccharomyces cerevisiae) tRNAPhe is provided in trans to complement the replication of HIV-1 with a PBS complementary to yeast tRNAPhe. Recent studies have demonstrated that the presence of lysyl-tRNA synthetase in HIV-1 virions might account for the preference for the selection of tRNA3Lys in HIV-1 replication. To establish a complementation system more reflective of HIV-1 primer selection, we have altered the HIV-1 PBS to be complementary to the Escherichia coli tRNA3Lys, which shares near identity with mammalian tRNA3Lys except in the 3'-terminal 18-nucleotide sequence that binds to the PBS. E. coli tRNA3Lys expressed from a plasmid was aminoacylated in mammalian cells. Cotransfection of cells with a plasmid that encodes E. coli tRNA3Lys and a plasmid encoding an HIV-1 provirus with a PBS complementary to E. coli tRNA3Lys resulted in the production of infectious virus. A comparison of the two complementation systems revealed that higher levels of intracellular E. coli tRNA3Lys than of yeast tRNAPhe were needed to achieve equal levels of infectious virus, indicating that there was no preferential selection of E. coli tRNA3Lys. To examine the specificity of tRNALys selection, E. coli tRNA3Lys was modified to tRNA1,2Lys. This tRNA was also aminoacylated when expressed in mammalian cells and complemented the infectivity of HIV-1 at levels similar to those seen for E. coli tRNA3Lys. Additional mutations in the anticodon of E. coli tRNA3Lys were constructed; these mutations did not significantly correlate with the capacity of the tRNA primer to complement infectivity of HIV-1, even though they had a drastic effect on the aminoacylation of the tRNAs. The results of these studies demonstrate that E. coli tRNA3Lys provided in trans can complement HIV-1 genomes with the PBS altered to E. coli tRNA3Lys. However, the capacity of tRNA3Lys to interact with lysyl-tRNA synthetase does not entirely explain the enhanced preference for selection of tRNA3Lys for the replication of HIV-1.
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PMID:Complementation of human immunodeficiency virus type 1 replication by intracellular selection of Escherichia coli formula supplied in trans. 1697 68

The primer for reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome is tRNA3(Lys). During assembly of HIV-1 particles, tRNA3(Lys) is taken up from the host cell along with lysyl-tRNA synthetase (LysRS), the tRNA binding protein that specifically aminoacylates the different tRNA(Lys) isoacceptors. In humans, the cytoplasmic and mitochondrial species of LysRS are encoded by a single gene by means of alternative splicing. Here, we show that polyclonal antibodies directed to the full-length cytoplasmic enzyme equally recognized the two enzyme species. We raised antibodies against synthetic peptides that allowed discrimination between the two enzymes and found that mitochondrial LysRS is the only cellular source of LysRS detected in the virions. These results open new routes for understanding the molecular mechanisms involved in the specific packaging of tRNA3(Lys) into viral particles.
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PMID:Viral hijacking of mitochondrial lysyl-tRNA synthetase. 1705 Jun 5

Mitochondrial lysyl-tRNA synthetase (LysRS) is thought to be involved in the specific packaging of tRNA(3)(Lys) into HIV-1 viral particles. The HIV-1 auxiliary viral protein Vpr is an apoptogenic protein that affects the integrity of the mitochondrial membrane and has also been reported to interact with LysRS. In the present study, we show that HIV-1 Vpr expressed in E. coli and purified to homogeneity does not interact specifically with LysRS and does not impact its aminoacylation activity. However, we also show that the mitochondrial localization of LysRS in HeLa cells is altered after addition of Vpr in the culture medium. These results suggest that HIV-1 Vpr fulfills an essential role in the process of packaging of mitochondrial LysRS.
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PMID:Role of HIV-1 Vpr-induced apoptosis on the release of mitochondrial lysyl-tRNA synthetase. 1756 Sep 97

Human tRNALys3 is used as the primer for human immunodeficiency virus type 1 (HIV-1) reverse transcription. HIV-1 Gag and GagPol, as well as host cell factor lysyl-tRNA synthetase (LysRS), are required for specific packaging of tRNALys into virions. Gag alone is sufficient for packaging of LysRS, and these two proteins have been shown to interact in vitro with an equilibrium binding constant of approximately 310 nM. The capsid (CA) domain of Gag binds to LysRS with a similar affinity as full-length Gag. In this work, we report further characterization of the interaction between HIV-1 CA and human LysRS using truncation constructs and point mutations in the putative interaction helices. Fluorescence anisotropy binding measurements reveal that a LysRS variant lacking the N-terminal 219 residues still displays high affinity binding to CA. The CA C-terminal domain (CTD) is also sufficient for binding to LysRS. Nuclear magnetic resonance spectroscopy studies using 15N-labeled CA-CTD reveal chemical shift perturbations of residues in and proximal to helix 4 of CA-CTD upon LysRS binding. A synthetic peptide that includes helix 4 binds to LysRS with high affinity, whereas peptides derived from the other three helical domains of CA-CTD do not. Alanine-scanning mutagenesis studies targeting residues in the helix 4 region support a direct interaction between this domain of CA-CTD and LysRS. The high resolution mapping studies reported here will facilitate future work aimed at disrupting the Gag-LysRS interaction, which represents a novel anti-viral strategy.
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PMID:Critical role of helix 4 of HIV-1 capsid C-terminal domain in interactions with human lysyl-tRNA synthetase. 1772 17

During HIV-1 assembly, tRNA(Lys3), the primer for reverse transcriptase (RT) in HIV-1, is selectively packaged into the virus due to a specific interaction between Gag and lysyl-tRNA synthetase (LysRS). However, while Gag alone will incorporate LysRS, tRNA(Lys3) packaging also requires the presence of RT thumb domain sequences in GagPol. The formation of a tRNA(Lys3) packaging/annealing complex involves an interaction between Gag/GagPol/viral RNA and LysRS/tRNA(Lys), and herein, we have investigated whether the transfer of tRNA(Lys3) from LysRS to RT sequences in Pol by a currently unknown mechanism is facilitated by an interaction between LysRS and Pol. We demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. However, cytoplasmic Gag/Pol interactions, detected by either coimmunoprecipitation or incorporation of Pol into Gag viral-like particles, were found to be insensitive to the overexpression or underexpression of LysRS, indicating that a Gag/LysRS/RT interaction is not essential for Gag/Pol interactions. Based on this and previous work, including the observation that the RT connection domain is not required for tRNA(Lys3) packaging, but is required for tRNA(Lys3) annealing, a model is proposed for a tRNA(Lys3) packaging/annealing complex in which the interaction of Gag with Pol sequences during early viral assembly facilitates the retention in budding viruses of both tRNA(Lys3) and early Pol processing intermediates, with tRNA(Lys3) annealing to viral RNA further facilitated by the LysRS/RT interaction.
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PMID:Interactions of reverse transcriptase sequences in Pol with Gag and LysRS in the HIV-1 tRNALys3 packaging/annealing complex. 1870 37

Attempts to use the mouse as a model system for studying AIDS are stymied by the multiple blocks to human immunodeficiency virus type 1 (HIV-1) replication that exist in mouse cells at the levels of viral entry, transcription, and Gag assembly and processing. In this report, we describe an additional block in the selective packaging of tRNA(3Lys) into HIV-1 produced in murine cells. HIV-1 and murine leukemia virus (MuLV) use tRNA(3Lys) and tRNA(Pro), respectively, as primers for reverse transcription. Selective packaging of tRNA(3Lys) into HIV-1 produced in human cells is much stronger than that for tRNA(Pro) incorporation into MuLV produced in murine cells, and different packaging mechanisms are used. Thus, both lysyl-tRNA synthetase and GagPol are required for tRNA(3Lys) packaging into HIV-1, but neither prolyl-tRNA synthetase nor GagPol is required for tRNA(Pro) packaging into MuLV. In this report, we show that when HIV-1 is produced in murine cells, the virus switches from an HIV-1-like incorporation of tRNA(3Lys) to an MuLV-like packaging of tRNA(Pro). The primer binding site in viral RNA remains complementary to tRNA(3Lys), resulting in a significant decrease in reverse transcription and infectivity. Reduction in tRNA(3Lys) incorporation occurs even though both murine lysyl-tRNA synthetase and HIV-1 GagPol are packaged into the HIV-1 produced in murine cells. Nevertheless, the murine cell is able to support the select incorporation of tRNA(3Lys) into another retrovirus that uses tRNA(3Lys) as a primer, the mouse mammary tumor virus.
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PMID:Inability of human immunodeficiency virus type 1 produced in murine cells to selectively incorporate primer formula. 1884 18

Human immunodeficiency virus 1 (HIV-1) uses a host cell tRNA(Lys,3) molecule to prime reverse transcription of the viral RNA genome into double-stranded DNA prior to integration into the host genome. All three human tRNA(Lys) isoacceptors along with human lysyl-tRNA synthetase (LysRS) are selectively packaged into HIV-1. Packaging of LysRS requires the viral Gag polyprotein and incorporation of tRNA(Lys) additionally requires the Gag-Pol precursor. A model that incorporates the known interactions between components of the putative packaging complex is presented. The molecular interactions that direct assembly of the tRNA(Lys)/LysRS packaging complex hold promise for the development of new anti-viral agents.
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PMID:Formation of the tRNALys packaging complex in HIV-1. 1991 38

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.
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PMID:Packaging HIV virion components through dynamic equilibria of a human tRNA synthetase. 2105 83

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.
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PMID:Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor. 2223 46

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNA(Lys,3), which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents.
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PMID:Cyclic peptide inhibitors of HIV-1 capsid-human lysyl-tRNA synthetase interaction. 2227 94


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