The use of fluorescence microscopy to visualise homotypic interactions of tomato spotted wilt virus nucleocapsid protein in living cells

被引:19
作者
Snippe, M
Borst, JW
Goldbach, R
Kormelink, R
机构
[1] Univ Wageningen & Res Ctr, Virol Lab, NL-6709 PD Wageningen, Netherlands
[2] Univ Wageningen & Res Ctr, Biochem Lab, MicroSpectroscopy Ctr, NL-6703 HA Wageningen, Netherlands
关键词
fluorescence resonance energy transfer (FRET); fluorescence lifetime imaging microscopy (FLIM); protein-protein interaction; tospovirus; bunyaviridae; virus assembly;
D O I
10.1016/j.jviromet.2004.11.028
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) were employed to study homotypic protein-protein interactions in living cells. To this end, the nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) was expressed as a fusion protein with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). Co-expression experiments of the two fusion proteins were carried out in baby hamster kidney (BHK21) cells. Both the wild type and the fusion proteins showed a peri-nuclear localisation pattern and were observed to form aggregates. In sensitised emission experiments, energy transfer was observed to take place from CFP to YFP when the two fluorophores were fused to TSWV N protein, indicating strongly homotypic interaction of the N proteins. This was confirmed by acceptor photobleaching studies as well as by FLIM experiments. All three methods showed interactions taking place, not only in the aggregates in the peri-nuclear region, but also throughout the cytoplasm. These experiments clearly demonstrated the potential of these fluorescence methods for studying the interactions of viral proteins in living cells. (c) 2005 Elsevier B.V. All rights reserved.
引用
收藏
页码:15 / 22
页数:8
相关论文
共 36 条
[21]   Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region [J].
Ravkov, EV ;
Compans, RW .
JOURNAL OF VIROLOGY, 2001, 75 (04) :1808-1815
[22]   Watching proteins fold one molecule at a time [J].
Rhoades, E ;
Gussakovsky, E ;
Haran, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2003, 100 (06) :3197-3202
[23]   Characterization of the nucleic acid binding properties of tomato spotted wilt virus nucleocapsid protein [J].
Richmond, KE ;
Chenault, K ;
Sherwood, JL ;
German, TL .
VIROLOGY, 1998, 248 (01) :6-11
[24]   XENOPUS-EMBRYOS REGULATE THE NUCLEAR-LOCALIZATION OF XMYOD [J].
RUPP, RAW ;
SNIDER, L ;
WEINTRAUB, H .
GENES & DEVELOPMENT, 1994, 8 (11) :1311-1323
[25]   Fluorescent indicators for imaging protein phosphorylation in single living cells [J].
Sato, M ;
Ozawa, T ;
Inukai, K ;
Asano, T ;
Umezawa, Y .
NATURE BIOTECHNOLOGY, 2002, 20 (03) :287-294
[26]   IDENTIFICATION, BY A MONOCLONAL-ANTIBODY, OF A 53-KD PROTEIN ASSOCIATED WITH A TUBULO-VESICULAR COMPARTMENT AT THE CIS-SIDE OF THE GOLGI-APPARATUS [J].
SCHWEIZER, A ;
FRANSEN, JAM ;
BACHI, T ;
GINSEL, L ;
HAURI, HP .
JOURNAL OF CELL BIOLOGY, 1988, 107 (05) :1643-1653
[27]   Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations [J].
Sekar, RB ;
Periasamy, A .
JOURNAL OF CELL BIOLOGY, 2003, 160 (05) :629-633
[28]   Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus [J].
Takeda, A ;
Sugiyama, K ;
Nagano, H ;
Mori, M ;
Kaido, M ;
Mise, K ;
Tsuda, S ;
Okuno, T .
FEBS LETTERS, 2002, 532 (1-2) :75-79
[29]   Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt tospovirus: Identification and characterization of two interacting domains [J].
Uhrig, JF ;
Soellick, TR ;
Minke, CJ ;
Philipp, C ;
Kellmann, JW ;
Schreier, PH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (01) :55-60
[30]   DETECTION OF THE L-PROTEIN OF TOMATO SPOTTED WILT VIRUS [J].
VANPOELWIJK, F ;
BOYE, K ;
OOSTERLING, R ;
PETERS, D ;
GOLDBACH, R .
VIROLOGY, 1993, 197 (01) :468-470