Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase

Ilina, Tatiana V.; Slack, Ryan L.; Elder, John H.; Sarafianos, Stefan G.; Parniak, Michael A.; Ishima, Rieko

doi:10.1016/j.jmb.2018.02.027

Cited by 8 publications

(42 citation statements)

References 51 publications

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“…Production of p51 subunit from p66 was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following incubation of purified p66 with HIV-1 PR for varying amounts of time. 18 Generation of equivalent amounts of p66 and p51 is indicative of p66/p51 heterodimer production. As shown previously, 18 incubation of p66 alone with PR did not generate significant p66/p51 heterodimer ( Figure 2 A) while incubation in the presence of tRNA Lys3 did ( Figure 2 B).…”

Section: Resultsmentioning

confidence: 99%

“… 18 Generation of equivalent amounts of p66 and p51 is indicative of p66/p51 heterodimer production. As shown previously, 18 incubation of p66 alone with PR did not generate significant p66/p51 heterodimer ( Figure 2 A) while incubation in the presence of tRNA Lys3 did ( Figure 2 B). A similar result was obtained in the presence of LysRS ( Figure 2 C,D): p51 band generation was more significant in the presence of tRNA Lys3 than in its absence.…”

Section: Resultsmentioning

confidence: 99%

“… 26 In the tRNA Lys3 –p66 interaction study, the well-defined free tRNA Lys3 band was present at low p66 concentrations and shifted to a diffuse band in the presence of higher p66 concentrations (>2 μM p66), reflecting generation of p66–tRNA Lys3 and p66/p66–tRNA Lys3 complexes ( Figure 4 B), which is in agreement with our previous observation. 18 …”

Section: Resultsmentioning

confidence: 99%

“…We previously found that the interaction of tRNA Lys3 with p66/p66 enhances specific cleavage by PR. 18 , 19 However, tRNA Lys3 is not recruited to the virus by itself and is known to be packaged in a complex with LysRS. 20 , 21 , 40 , 41 Thus, we tested whether LysRS affects RT maturation in vitro .…”

Section: Discussionmentioning

confidence: 99%

“…HIV-1 p66 protein was prepared, as described previously. 18 tRNA Lys3 was prepared by in vitro transcription of its DNA using NTPs and T7 RNA polymerase, as described previously. 19 Note, tRNA Lys3 does not have amino-acyl modification.…”

Section: Methodsmentioning

confidence: 99%

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Effect of Lysyl–tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase

et al. 2020

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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 tRNA Lys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNA Lys3 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 tRNA Lys3 from LysRS. Thus, we conclude that a free fraction of tRNA Lys3 , which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNA Lys3 from LysRS. Given that only transient tRNA Lys3 –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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Effect of Lysyl–tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase

et al. 2020

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Interplay between protease and reverse transcriptase dimerization in a model HIV‐1 polyprotein

Chagas,

Zhou,

Guerrero

et al. 2024

Protein Science

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The Gag‐Pol polyprotein in human immunodeficiency virus type I (HIV‐1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR‐RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO‐tag, at the N‐terminus of the PR region. We also prepared two mutants of PR‐RT containing a dimer dissociation mutation either in the PR region, PR(T26A)‐RT, or in the RT region, PR‐RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR‐RT and PR(T26A)‐RT, but only monomer in PR‐RT(W401A). SEC experiments of PR‐RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR‐RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer‐interface on these functions. Our results consistently indicate that the RT dimer‐interface plays a crucial role in the dimerization in PR‐RT, whereas the PR dimer‐interface has a lesser role.

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Evolving understanding of HIV-1 reverse transcriptase structure, function, inhibition, and resistance

Ruiz

Arnold

2020

Current Opinion in Structural Biology

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Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase

Cited by 8 publications

References 51 publications

Effect of Lysyl–tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase

Effect of Lysyl–tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase

Interplay between protease and reverse transcriptase dimerization in a model HIV‐1 polyprotein

Evolving understanding of HIV-1 reverse transcriptase structure, function, inhibition, and resistance

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