Myristoylation-Enhanced Binding of the HIV-1 Net Protein to T Cell Skeletal Matrix

Niederman, T. M. J.; Hastings, William R.; Ratner, Lee

doi:10.1006/viro.1993.1605

Cited by 75 publications

(63 citation statements)

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“…Cytoplasmic and nuclear fractions were analyzed by Western blot with anti-EBNA1 and anti-Rch1 mAb. To determine the subcellular localization of EBNA1 and Rch1, transfected 293 GP cells were lysed by Dounce homogenization exactly as described by Niedermann et al (41). The nuclear and cytosolic fractions were analyzed with the EBNA1-specific mAb 1H4 and the Rch1-specific mAb 2G7.…”

Section: Leumentioning

confidence: 99%

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

Fischer

Kremmer²,

Lautscham

et al. 1997

Journal of Biological Chemistry

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The Epstein-Barr virus (EBV) is implicated in the induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the viral episome in the infected cell and exhibits oncogenic activity in transgenic mice. Here we report the identification of the nuclear transporter karyopherin ␣2 as a cellular partner of EBNA1 using the yeast "two-hybrid system." Karyopherin ␣2 is also called importin ␣ or Rch1. The binding to karyopherin ␣2 was mediated through a C-terminal region of EBNA1 encompassing the nuclear localization signal, whereas clones of EBNA1 devoid of the nuclear localization signal failed to bind to karyopherin ␣2. The interaction was biochemically confirmed by far-Western analysis using bacterially expressed karyopherin ␣2 and karyopherin ␣2-specific monoclonal antibodies. The nuclear transport of EBNA1 was impaired by expression of N-terminally truncated karyopherin ␣2. Zone velocity sedimentation in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize; and (ii) the association of karyopherin ␣2 with high molecular weight protein complexes might be impeded by the presence of EBNA1.The Epstein-Barr virus (EBV) 1 is the etiological agent of infectious mononucleosis and is implicated in the induction of several malignancies including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, T-cell lymphoma, and polyclonal B-cell lymphoma that arise under immunosuppression (reviewed in Ref. 1). The expression of EBV-encoded proteins is most stringently restricted in primary Burkitt's lymphoma in that only EBNA1 and the nontranslated EpsteinBarr virus-encoded small nuclear RNAs are transcribed. EBNA1 binds to the viral origin of replication (oriP)(2, 3) and is the only virus protein necessary and sufficient to replicate and maintain the EBV genome in the infected cell (4). In addition, experiments using transgenic animals show that EBNA1 by itself is able to induce tumors in vivo (5, 6). EBNA1 is a multifunctional phosphoprotein (7) with a variety of properties, including sequence-specific binding to DNA (8, 9), formation of homodimers (10), nuclear localization (11), and stimulation of cellular gene expression (12).Import of proteins into the nucleus is an active process that can be divided into at least two steps: 1) binding of proteins with a nuclear localization signal (NLS) to a cytosolic NLSreceptor and translocation to the nuclear pore complex; and 2) transport of NLS-bearing proteins into the nucleus. The NLSreceptor consists of two subunits, a 60-kDa protein that is called karypherin ␣2 (alternatively, it is called importin ␣ in vertebrates and Srp1 in yeast) and a 95-kDa protein, called karyopherin ␤ (also known as importin ␤ in vertebrates and Kap95p in yeast). Karyopherin ␣2 serves as the NLS-receptor, whereas karyopherin ␤ functions as an adapter that mediates binding to nucleoporins. Two additional proteins, GTPase Ran/ TC4 and p15 (NTF2...

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Section: Leumentioning

confidence: 99%

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

Fischer

Kremmer²,

Lautscham

et al. 1997

Journal of Biological Chemistry

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“…Compared to expression in lower-order organisms, recombinant protein expression in mammalian cells offers significant advantages in protein quality and post-translational modification 15 . Moreover the protein of interest 27 kDa nef is N-terminally myristoylated at the second glycine of the highly conserved sequences MGG 16 . Though transient transfection of mammalian cells has been employed for low recombinant protein expression since the invention of transfection reagents, the ability to express milligram-to-gram amounts of recombinant protein has relied mainly on the creation of stable cell lines 17 .…”

Section: Purification Of Nef Protein By Hispur Cobalt Columnmentioning

confidence: 99%

Stable Transfection Study for Cloning and Expression of Hiv-1 Nef Protein in Hek 293 Cells

2017

Rasayan J Chem

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Human immunodeficiency virus continues to be a major global populace health issue, there were approximately 34 million people living with HIV in 2011. However, the development of anti -viral has blunted the AIDS epidemic in the Western world but globally the epidemic has not been curtailed. Nef is one of these accessory genes that are only present within HIV and SIV genome and thought to play a in the progression to AIDS. The given its central purpose in HIV Pathogenesis, Nef considered as a potential anti-viral target for preventing or at least delaying Pathogenesis. The biologically active 27 kDa myristoylated Nef protein expressed from HEK 293 is a protein model to be used for significantly specific antibody production to lower the pathogenicity of HIV infection. Cultivated HEK 293 was transfected by 4ug/cubic centimetre of successful clone of pQBI-Nef-6His by stable transfection selected in 0.7 mg/cc of G418 antibiotic. It display that targeted 27 kDa HIV-1 Nef was not successfully expressed in HEK 293 cells either in stable transfection when transfected. However, non-targeted HIV-1 Nef was detected in western blot by anti-Nef (anti manufactured by Thermo scientific. It suggesting that, the Nef protein that was detected not identically synthesis through post-translation modification though it was expressed in the cytoplasm of HEK 293 cells. The ability of not expressing the targeted myristoylated 27 kDa nef protein was to various unpreventable factors due to time limitation and lacking of skills in the filed cloning and cell culture.

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“…Cytoskeletal association of Nef was significantly enhanced by myristoylation (Niederman et al, 1993). It is therefore desirable to express Nef protein in mammalian system to produce high quality protein.…”

Section: Nef Protein/mammalian Systemmentioning

confidence: 99%

A Review on Biologically Active N-Myristoylated HIV-1 Nef Protein

Balakrishn¹,

Abdullah²,

Huri³

et al. 2015

Asian J. of Biochemistry

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Myristoylation-Enhanced Binding of the HIV-1 Net Protein to T Cell Skeletal Matrix

Cited by 75 publications

References 0 publications

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

Epstein-Barr Virus Nuclear Antigen 1 Forms a Complex with the Nuclear Transporter Karyopherin α2

Stable Transfection Study for Cloning and Expression of Hiv-1 Nef Protein in Hek 293 Cells

A Review on Biologically Active N-Myristoylated HIV-1 Nef Protein

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