Amino Acid Substitution Analyses of the DNA Contact Region, Two Amphipathic α-Helices and a Recognition-Helix-Like Helix outside the Dimeric β-Barrel of Epstein-Barr Virus Nuclear Antigen 1

Intervirology ◽  
2001 ◽  
Vol 44 (5) ◽  
pp. 271-282 ◽  
Author(s):  
Tomomichi Fujita ◽  
Masato Ikeda ◽  
Shuichi Kusano ◽  
Makoto Yamazaki ◽  
Sayuri Ito ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Epstein Barr Virus ◽  
Contact Region ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Recognition Helix ◽  
Epstein Barr

scholarly journals Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association

2002 ◽  
Vol 76 (3) ◽  
pp. 1025-1032 ◽  
Author(s):  
Michiko Tanaka ◽  
Akihiko Yokoyama ◽  
Mie Igarashi ◽  
Go Matsuda ◽  
Kentaro Kato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Epstein Barr Virus ◽  
Cellular Proteins ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr ◽  
Self Association

ABSTRACT Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate γ-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401–413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

scholarly journals Epstein-Barr Virus Nuclear Protein 3A Domains Essential for Growth of Lymphoblasts: Transcriptional Regulation through RBP-Jκ/CBF1 Is Critical

2005 ◽  
Vol 79 (16) ◽  
pp. 10171-10179 ◽  
Author(s):  
Seiji Maruo ◽  
Eric Johannsen ◽  
Diego Illanes ◽  
Andrew Cooper ◽  
Bo Zhao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epstein Barr Virus ◽  
Genetic System ◽  
Nuclear Antigen ◽  
Regulation Of Transcription ◽  
Amino Acid Residues ◽  
Barr Virus ◽  
Biochemical Assays ◽  
Conditional Mutant ◽  
Epstein Barr

ABSTRACT Experimental reverse genetic replacement of Epstein-Barr virus nuclear antigen 3A (EBNA3A) with a conditional mutant EBNA3A revealed that EBNA3A is critical for continued lymphoblastoid cell (LCL) growth. Wild-type (wt) EBNA3A expressed in the LCLs specifically sustained growth under nonpermissive conditions, whereas EBNA3B or EBNA3C expression had no effect (S. Mauro, E. Johannsen, D. Illanes, A. Cooper, and E. Kieff, J. Virol. 77:10437-10447, 2003). This genetic system and related biochemical assays have now been used to discover that EBNA3A lacking amino acid residues 170 to 240 (Δ170-240), TLGC202 to AAGA202, or Δ300-386, which are deficient in repression of EBNA2 activation of an RBP-Jκ/CBF1-dependent EBV Cp enhancer, are null mutations for LCL growth, whereas EBNA3A Δ2-124, Δ410-439, Δ440-470, Δ470-500, Δ500-523, Δ523-612, and Δ620-820, which are wt in repression are wt for LCL growth. Thus, EBNA3A regulation of transcription through RBP-Jκ/CBF1 is critical for LCL growth. EBNA3A mutants deleted of amino acid residues 240 to 300, 386 to 410, or 827 to 944 were intermediate, null, or intermediate, respectively, for LCL growth despite being wt for RBP-Jκ association and repression. Amino acid residues 240 to 300, 386 to 410, and, particularly, C-terminal residues 827 to 944 are therefore likely to contribute to LCL growth through RBP-Jκ-independent mechanisms.

scholarly journals mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

2008 ◽  
Vol 83 (3) ◽  
pp. 1289-1298 ◽  
Author(s):  
Sebastien Apcher ◽  
Anastassia Komarova ◽  
Chrysoula Daskalogianni ◽  
Yili Yin ◽  
Laurence Malbert-Colas ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epstein Barr Virus ◽  
Mrna Translation ◽  
Underlying Mechanism ◽  
Nuclear Antigen ◽  
Translation Regulation ◽  
Antigenic Peptides ◽  
Barr Virus ◽  
Histocompatibility Complex ◽  
Epstein Barr

ABSTRACT The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.

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