The connection domain in reverse transcriptase facilitates the in vivo annealing of tRNA(Lys3 )to HIV-1 genomic RNA

The primer tRNA for reverse transcription in HIV-1, tRNA(Lys3), 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 tRNA(Lys )into HIV-1 consists of Gag, GagPol, tRNA(Lys), lysyl-tRNA synthetase (LysRS), and viral genomic RNA. Gag targets tRNA(Lys )for viral packaging through Gag's interaction with LysRS, a tRNA(Lys)-binding protein, while reverse transcriptase (RT) sequences within GagPol (the thumb domain) bind to tRNA(Lys). The further annealing of tRNA(Lys3 )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 tRNA(Lys3 )packaging into the virus, it is required for tRNA(Lys3 )annealing to the viral RNA genome

Texture evolution induced by twinning and dynamic recrystallization in dilute Mg-1Sn-1Zn-1Al alloy during hot compression

Texture evolution of an extruded dilute Mg-1Sn-1Zn-1Al alloy was thoroughly investigated based on the twinning and dynamic recrystallization (DRX) behavior via hot compression at a strain rate of 10 s−1 and temperature of 225°C. It was found that the types and intensities of the texture are strongly dependent on the fraction of twins and DRX modes as well as regions where sub-grain boundaries (sub-GBs) are intensively accumulated. At the initial stage of deformation, the formation of compression direction (CD)-tilted basal texture was mainly determined by the occurrence of {101¯2} extension twins. As the strain increases, the variation in the texture intensity was greatly dominated by the DRX modes but the type of main texture remained unchanged. These findings are of great importance for texture modification of wrought Mg-Sn-based alloys during post-deformation

Thermoluminescence (TL) analysis for otoliths of the wild carps (cyprinoid) from Baiyangdian Lake and Miyun Reservoir: Some implications for monitoring water environment

Otolith is a typical biomineral carrier growing on insides of fish skull with prominent zoning structure formed by alternating layers of protein and calcium carbonate growing around the nucleus. Even though thermoluminescence (TL) analysis on biomineral has been widely used to measure the radiation exposure in the recent twenty years, the TL characteristics of the fish otolith have not yet been reported in literature. TL characteristics of otoliths from the wild carps (cyprinoid) living in the Baiyangdian Lake, Hebei Province and Miyun Reservoir, Beijing City was first studied, and the differences of energy gap (E) between the fish otoliths in the two waters have also been discussed in this paper. The experimental results indicated that TL curve parameters: peak temperature (Tp), luminous intensity (I), integrated intensity (S) and middle width (Wm) for the glow curves of the cyprinoid otoliths from Baiyangdian Lake are greater than those from Miyun reservoir, and the stability of the formers’ TL curve parameters value and energy gap (E) was weaker than the latter. In comparison to the Miyun Reservoir, the analysis manifested that the electrons and vacancies trapped in the otoliths from Baiyangdian Lake are more likely to escape. According to the investigation, the contaminative degree and eutrophication in the water of Baiyangdian Lake was heavier than that of Miyun Reservoir. Therefore, the characteristics of TL growth curves of the cyprinoid otoliths is quite sensitive to heavier contaminated and less contaminated water, and this could be regarded as an important typomorphic biomineral for monitoring the contaminative degree and environment change of the water.Keywords: Cyprinoid otoliths, thermoluminescence, water environment, typomorphic minera

Quantitative investigation of two metallohydrolases by X-ray absorption spectroscopy near-edge spectroscopy

The last several years have witnessed a tremendous increase in biological applications using X-ray absorption spectroscopy (BioXAS), thanks to continuous advancements in synchrotron radiation (SR) sources and detector technology. However, XAS applications in many biological systems have been limited by the intrinsic limitations of the Extended X-ray Absorption Fine Structure (EXAFS) technique e.g., the lack of sensitivity to bond angles. As a consequence, the application of the X-ray absorption near-edge structure (XANES) spectroscopy changed this scenario that is now continuously changing with the introduction of the first quantitative XANES packages such as Minut XANES (MXAN). Here we present and discuss the XANES code MXAN, a novel XANES-fitting package that allows a quantitative analysis of experimental data applied to Zn K-edge spectra of two metalloproteins: Leptospira interrogans Peptide deformylase (LiPDF) and acutolysin-C, a representative of snake venom metalloproteinases (SVMPs) from Agkistrodon acutus venom. The analysis on these two metallohydrolases reveals that proteolytic activities are correlated to subtle conformation changes around the zinc ion. In particular, this quantitative study clarifies the occurrence of the LiPDF catalytic mechanism via a two-water-molecules model, whereas in the acutolysin-C we have observed a different proteolytic activity correlated to structural changes around the zinc ion induced by pH variations

The connection domain in reverse transcriptase facilitates the <it>in vivo </it>annealing of tRNA^Lys3to HIV-1 genomic RNA

Abstract 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.</p

Ability of Wild-Type and Mutant Lysyl-tRNA Synthetase To Facilitate tRNA(Lys) Incorporation into Human Immunodeficiency Virus Type 1

The major human tRNA(Lys) isoacceptors, tRNA1,2Lys and tRNA3Lys, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where tRNA3Lys acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA(Lys), via its interaction with Gag, and it is a strong candidate for being the signal that specifically targets tRNA(Lys) for viral incorporation. Expression of exogenous wild-type LysRS in cells results in an approximately twofold increase in the viral packaging of both LysRS and tRNA(Lys). Herein, we show that this increase in tRNA(Lys) incorporation into virions is dependent upon the ability of LysRS to bind to tRNA(Lys) but not upon its ability to aminoacylate the tRNA(Lys). COS7 cells were cotransfected with plasmids coding for both HIV-1 and either wild-type or mutant human LysRS, all of which are incorporated into virions with similar efficiency. However, N-terminally truncated LysRS, which binds poorly to tRNA(Lys), does not increase tRNA(Lys) packaging into viruses, while C-terminally truncated LysRS, which binds to but does not aminoacylate tRNA(Lys), still facilitates an increase in tRNA(Lys) packaging into virions

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 tRNA(Lys3) 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 tRNA(Lys3) annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNA(Lys3) 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

Roles of Gag and NCp7 in Facilitating \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathbf{tRNA}}_{3}^{{\mathbf{Lys}}}\end{equation*}\end{document} Annealing to Viral RNA in Human Immunodeficiency Virus Type 1▿

In protease-negative human immunodeficiency virus type 1 (HIV-1) [Pr(-)], the amount of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealed by Gag is modestly reduced (∼25%) compared to that annealed by mature nucleocapsid (NCp7) in protease-positive HIV-1 [Pr(+)]. However, the \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealed by Gag also has a strongly reduced ability to initiate reverse transcription and binds less tightly to viral RNA. Both in vivo and in vitro, APOBEC3G (A3G) inhibits \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealing facilitated by NCp7 but not annealing facilitated by Gag. While transient exposure of Pr(-) viral RNA to NCp7 in vitro returns the quality and quantity of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealing to Pr(+) levels, the presence of A3G both prevents this rescue and creates a further reduction in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealing. Since A3G inhibition of NCp7-facilitated \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealing in vitro requires the presence of A3G during the annealing process, these results suggest that in Pr(+) viruses NCp7 can displace Gag-annealed \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} and re-anneal it to viral RNA, the re-annealing step being subject to A3G inhibition. This supports the possibility that the initial annealing of \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} in wild-type, Pr(+) virus may be by Gag and not by NCp7, perhaps offering the advantage of Gag's preference for binding to RNA stem-loops in the 5′ region of viral RNA near the \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} annealing region