Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During the assembly of HIV-1, tRNA(Lys) isoacceptors are selectively packaged into the virion, and one of these, tRNA(Lys3), is annealed to the viral RNA genome where it acts to prime the reverse transcriptase (RT)-catalyzed synthesis of viral DNA. We review herein what is known about the selective packaging and annealing of primer tRNA(Lys3). Current evidence suggests that a complex of two major precursor viral proteins, Pr55gag and Pr160(gag-pol), interact with a tRNA(Lys)/lysyl-tRNA synthetase (LysRS) complex during viral assembly, with Pr55gag interacting with both LysRS and Pr160(gag-pol), and RT sequences within Pr160(gag-pol) binding to tRNA(Lys). LysRS appears to target the tRNA(Lys) isoacceptors for incorporation into HIV-1. In the virion, the 3' terminal 18 nucleotides of tRNA(Lys3) anneals to an 18-nucleotide sequence at the 5' terminal region of viral RNA termed the primer binding site (PBS). Evidence is presented that other regions on the tRNA(Lys3) also anneal with other regions in viral RNA upstream of the PBS, resulting in a destabilized tRNA(Lys3) structure. Both viral and tRNA(Lys3) regions need to be denatured to establish annealing, and the roles of both viral and cellular proteins in this process are discussed.
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PMID:tRNA(Lys3): the primer tRNA for reverse transcription in HIV-1. 1204 92

HIV-1 utilizes cellular tRNA(3)(Lys) to prime the initiation of reverse transcription. The selective incorporation of cytoplasmic tRNA(3)(Lys) into HIV-1 particles was recently shown to involve the lysyl-tRNA synthetase, and hence, the encapsidated tRNA(3)(Lys) is likely to be aminoacylated. Here, we tested the effect of aminoacylation on the initiation of reverse transcription. We show that HIV-1 reverse transcriptase is unable to extend lysyl-tRNA(3)(Lys). In addition, the viral polymerase does not significantly enhance the rate of tRNA deacylation, in contrast with previous studies on avian retroviruses. Thus, aminoacylation of the primer tRNA might prevent the initiation of HIV-1 reverse transcription from taking place before viral budding and maturation.
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PMID:Effects of tRNA 3 Lys aminoacylation on the initiation of HIV-1 reverse transcription. 1276 11

In HIV-1, tRNA(Lys3) serves as the primer for reverse transcriptase, and during viral assembly, the tRNA(Lys) isoacceptors, tRNA(Lys1,2) and tRNA(Lys3), are selectively packaged into the virion. In this review, we shall first discuss the evidence for the formation of a tRNA(Lys) packaging complex, whose components include Gag, GagPol, genomic RNA, lysyl-tRNA synthetase (LysRS), and tRNA(Lys). Evidence suggests that the formation of this complex involves a Gag/GagPol/viral genomic RNA complex interacting with a tRNA(Lys)/ LysRS complex, with Gag interacting with both GagPol and LysRS, and GagPol interacting with the tRNA(Lys). The interaction of Gag with LysRS is quite specific, does not require the presence of tRNA(Lys), and LysRS is believed to be the target that allows the specific packaging of tRNA(Lys) into the virion. The parameters influencing these interactions, and the molecular sites of interaction, will be discussed. The selective packaging of tRNA(Lys3) into HIV-1 facilitates annealing of tRNA(Lys) to the 5' region of viral RNA genome. This region contains a series of stem loops, and there exists several regions in both the viral RNA and the tRNA(Lys) that are believed to be important for tRNA(Lys) annealing. The annealing is facilitated by viral proteins such as unprocessed Gag, nucleocapsid, and reverse transcriptase.
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PMID:The selective packaging and annealing of primer tRNALys3 in HIV-1. 1507 80

The primer tRNA for reverse transcription in HIV-1, tRNALys3, is selectively packaged into the virus during its assembly, and annealed to the viral genomic RNA. The ribonucleoprotein complex that is involved in the packaging and annealing of tRNALys into HIV-1 consists of Gag, GagPol, tRNALys, lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNALys for viral packaging through Gag's interaction with LysRS, a tRNALys-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNALys. The further annealing of tRNALys3 to viral RNA requires nucleocapsid (NC) sequences in Gag, but not the NC sequences GagPol. In this report, we further show that while the RT connection domain in GagPol is not required for tRNALys3 packaging into the virus, it is required for tRNALys3 annealing to the viral RNA genome.
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PMID:The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNALys3 to HIV-1 genomic RNA. 1549 76

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

The major cellular tRNA(Lys) isoacceptors are tRNA(Lys1,2) and tRNA(Lys3). During the replication of human immunodeficiency virus 1 (HIV-1), tRNA(Lys3) is used to prime reverse transcription of the viral RNA genome into double-stranded DNA, which is then integrated into the host genome. The annealing of tRNA(Lys3) to 5'-terminal sequences of viral RNA is multi-staged, with an initial poor quality, cytoplasmic annealing promoted by the Gag precursor protein, followed by a more effective annealing imposed upon the Gag-annealed tRNA(Lys3) that occurs after viral protein processing, and that is facilitated by mature nucleocapsid (NCp7). The initial annealing by Gag is assisted by the architecture of an early viral assembly intermediate we term the "tRNA(Lys3) annealing complex" whose composition includes Gag, GagPol, viral RNA, lysyl-tRNA synthetase (LysRS), and the tRNA(Lys) isoacceptors. Our model proposes that the reverse transcriptase sequences in GagPol bind all tRNAs non-specifically, and that the cytoplasmic tRNA population to which GagPol is exposed is enriched in tRNA(Lys) isoacceptors due to a specific interaction between Gag and LysRS. We further predict a protein conformation within the annealing complex that not only promotes this tRNA(Lys) enrichment, but that also facilitates the transfer of tRNA(Lys3) from GagPol to the viral RNA where annealing is carried out by nucleocapsid sequences within Gag.
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PMID:Aspects of HIV-1 assembly that promote primer tRNA(Lys3) annealing to viral RNA. 2269 76

A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNALys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNALys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication.IMPORTANCE Human tRNALys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNALys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNALys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNALys Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.
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PMID:HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication. 2881 26

In human immunodeficiency virus-1 (HIV-1), reverse transcriptase (RT) is encoded as a 66 kDa protein, p66, in the Gag-Pol polyprotein. This protein is proteolytically cleaved by HIV-1 protease (PR) to finally generate a mature RT that is a heterodimer, composed of a p66 subunit and a p66-derived 51 kDa subunit, p51. In our prior work, we demonstrated that tRNALys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNALys3 is known to be recruited to the virus by forming a complex with lysyl-tRNA synthetase (LysRS). Herein, we tested whether LysRS can have an effect on RT maturation in vitro. Importantly, our data show no significant differences in RT maturation in the presence of LysRS. Furthermore, no apparent p66/66 interaction with LysRS was observed. Although PR cleaved LysRS, it did not immediately release tRNALys3 from LysRS. Thus, we conclude that a free fraction of tRNALys3, which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNALys3 from LysRS. Given that only transient tRNALys3-p66/p66 interaction is needed for efficient RT maturation, a small amount of free tRNA may be sufficient for this process. These studies reveal molecular level insights into RT maturation and will be useful for the design of cellular/viral experiments to better understand the role of tRNA in HIV-1 replication.
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PMID:Effect of Lysyl-tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase. 3268 28