Cellular Distribution of Lysyl-tRNA Synthetase and Its Interaction with Gag during Human Immunodeficiency Virus Type 1 Assembly

ABSTRACT Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA3 Lys, the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i) in the nucleus, (ii) in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii) in mitochondria, and (iv) associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [35S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.

Download Full-text

Inability of Human Immunodeficiency Virus Type 1 Produced in Murine Cells To Selectively Incorporate Primer \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}

Journal of Virology ◽

10.1128/jvi.01744-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12049-12059 ◽

Cited By ~ 4

Author(s):

Min Wei ◽

Yiliang Yang ◽

Meijuan Niu ◽

Laurie Desfosse ◽

Robert Kennedy ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Leukemia Virus ◽

Trna Synthetase ◽

Primer Binding Site ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into HIV-1 produced in murine cells. HIV-1 and murine leukemia virus (MuLV) use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} and tRNAPro, respectively, as primers for reverse transcription. Selective packaging of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into HIV-1 produced in human cells is much stronger than that for tRNAPro incorporation into MuLV produced in murine cells, and different packaging mechanisms are used. Thus, both lysyl-tRNA synthetase and GagPol are required for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} packaging into HIV-1, but neither prolyl-tRNA synthetase nor GagPol is required for tRNAPro 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} to an MuLV-like packaging of tRNAPro. The primer binding site in viral RNA remains complementary to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , resulting in a significant decrease in reverse transcription and infectivity. Reduction in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} into another retrovirus that uses \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} as a primer, the mouse mammary tumor virus.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.78.3.1595-1601.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1595-1601 ◽

Cited By ~ 39

Author(s):

Shan Cen ◽

Hassan Javanbakht ◽

Meijuan Niu ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Trna Synthetase ◽

Wild Type ◽

Viral Packaging ◽

Immunodeficiency Virus ◽

Strong Candidate ◽

Hiv 1

ABSTRACT The major human tRNALys 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 tRNALys, via its interaction with Gag, and it is a strong candidate for being the signal that specifically targets tRNALys 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 tRNALys. Herein, we show that this increase in tRNALys incorporation into virions is dependent upon the ability of LysRS to bind to tRNALys but not upon its ability to aminoacylate the tRNALys. 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 tRNALys, does not increase tRNALys packaging into viruses, while C-terminally truncated LysRS, which binds to but does not aminoacylate tRNALys, still facilitates an increase in tRNALys packaging into virions.

Download Full-text

Human Immunodeficiency Virus Type 1 Matrix Protein Interacts with Cellular Protein HO3

Journal of Virology ◽

10.1128/jvi.72.2.1671-1676.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1671-1676 ◽

Cited By ~ 23

Author(s):

Juan Lama ◽

Didier Trono

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Matrix Protein ◽

Critical Role ◽

Trna Synthetase ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) plays a critical role in virion morphogenesis and fulfills important functions during the early steps of infection. In an effort to identify cellular partners of MA, a Saccharomyces cerevisiae two-hybrid screen was utilized. A specific interaction between MA and HO3, a putative histidyl-tRNA synthetase, was demonstrated in this system. HO3-specific mRNA was detected in several tissues relevant for HIV infection, such as spleen, thymus, and peripheral blood lymphocytes, as well as in a number of T-lymphoid-cell lines. The binding of MA to HO3 was confirmed in transfected cells by coimmunoprecipitation. This interaction was abrogated by replacing two lysine residues at positions 26 and 27 of MA by threonine (MAKK27TT ). HO3 localized both to the cytoplasm and to the nucleus of acutely transfected 293T cells. When overexpressed in HIV-1-producing cells, HO3 was incorporated into wild-type virions but not in ones containing the dilysine-mutated variant of MA. Correspondingly, overexpression of HO3 in virus producer cells enhanced the infectivity of wild-type but not MAKK27AA HIV-1 particles. The stimulating effect of HO3 was independent from the presence of Envelope, Vpr, or Vpu. Taken together, these results suggest that HO3, through its recognition of MA, plays a role in the life cycle of HIV-1.

Download Full-text

Human Immunodeficiency Virus Type 1 (HIV-1) Viral Protein R (Vpr) Interacts with Lys-tRNA Synthetase: Implications for Priming of HIV-1 Reverse Transcription

Journal of Virology ◽

10.1128/jvi.72.4.3037-3044.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 3037-3044 ◽

Cited By ~ 46

Author(s):

Lesley A. Stark ◽

Ronald T. Hay

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Reverse Transcription ◽

Viral Protein ◽

Trna Synthetase ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 96-amino-acid 14-kDa protein (viral protein R [Vpr]), which is produced late in the viral life cycle and is incorporated into the virion. Although Vpr is not required for viral replication in transformed cell lines and primary T lymphocytes, it is essential for productive infection of macrophages and monocytes and appears to be important for pathogenesis in vivo. To establish the role of Vpr in HIV-1 replication and pathogenesis, we have isolated cellular proteins with which Vpr interacts. By using the yeast two-hybrid system, Lys-tRNA synthetase (LysRS) was identified as a Vpr-interacting protein. The interaction between Vpr and LysRS was characterized both in vitro and in vivo, and the domains of Vpr required for the interaction were defined. In the presence of Vpr, LysRS-mediated aminoacylation of tRNALys is inhibited. Since tRNALys is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription.

Download Full-text

Specific Inhibition of the Synthesis of Human Lysyl-tRNA Synthetase Results in Decreases in tRNALys Incorporation, tRNA3LysAnnealing to Viral RNA, and Viral Infectivity in Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.77.18.9817-9822.2003 ◽

2003 ◽

Vol 77 (18) ◽

pp. 9817-9822 ◽

Cited By ~ 43

Author(s):

Fei Guo ◽

Shan Cen ◽

Meijuan Niu ◽

Hassan Javanbakht ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Early Time ◽

Trna Synthetase ◽

Viral Rna ◽

Viral Infectivity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The major human tRNALys isoacceptors, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{1,2}^{Lys}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} , are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNALys, via its interaction with Gag, and is a strong candidate for being the signal that specifically targets tRNALys for viral incorporation. We have transfected 293T cells with HIV-1 proviral DNA and short interfering RNA (siRNA) specific for LysRS to study the effect of diminished cellular LysRS upon tRNALys packaging, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document} annealing to viral genomic RNA, and viral production and infectivity. At early time points after siRNA transfection, an 80% inhibition of LysRS incorporation into viruses reflects an 80% reduction of newly synthesized LysRS, rather than a more limited 20 to 25% decrease in the concentration of total cell LysRS, indicating that newly synthesized LysRS in the cell may be the main source of viral LysRS. Viruses produced from cells transfected with siRNA show reduced tRNALys packaging, reduced \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}B\) \end{document} annealing to viral RNA, and reduced viral infectivity.

Download Full-text

Effect of Altering the tRNA\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document}Concentration in Human Immunodeficiency Virus Type 1 upon Its Annealing to Viral RNA, GagPol Incorporation, and Viral Infectivity

Journal of Virology ◽

10.1128/jvi.76.18.9096-9102.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9096-9102 ◽

Cited By ~ 50

Author(s):

Juliana Gabor ◽

Shan Cen ◽

Hassan Javanbakht ◽

Meijuan Niu ◽

Lawrence Kleiman

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Trna Synthetase ◽

Viral Rna ◽

Viral Population ◽

Viral Infectivity ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) uses tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNALys, tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{1,2}^{Lys}\) \end{document} . Increasing the cytoplasmic concentration of tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} gene results in a greater incorporation of tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} into virions, which is accompanied by increased annealing of tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} to the viral genome and increased infectivity of the viral population. Increased viral tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} is accompanied by decreased viral tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{1,2}^{Lys}\) \end{document} , with the total tRNALys/virion and the GagPol/Gag ratios remaining unchanged. Viral tRNALys can be doubled, with increases in both tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} and tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{1,2}^{Lys}\) \end{document} concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{3}^{Lys}\) \end{document} annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNALys incorporation into HIV-1, whereas LysRS is.

Download Full-text