Tristan Piolot

Close but Distinct Regions of Human Herpesvirus 8 Latency-Associated Nuclear Antigen 1 Are Responsible for Nuclear Targeting and Binding to Human Mitotic Chromosomes

ABSTRACT Human herpesvirus 8 is associated with all forms of Kaposis sarcoma, AIDS-associated body cavity-based lymphomas, and some forms of multicentric Castlemans disease. Herpesvirus 8, like other gammaherpesviruses, can establish a latent infection in which viral genomes are stably maintained as multiple episomes. The latent nuclear antigen (LANA or LNAI) may play an essential role in the stable maintenance of latent episomes, notably by interacting concomitantly with the viral genomes and the metaphase chromosomes, thus ensuring an efficient transmission of the neoduplicated episomes to the daughter cells. To identify the regions responsible for its nuclear and subnuclear localization in interphase and mitotic cells, LNAI and various truncated forms were fused to a variant of green fluorescent protein. This enabled their localization and chromosome binding activity to be studied by low-light-level fluorescence microscopy in living HeLa cells. The results demonstrate that nuclear localization of LNAI is due to a unique signal, which maps between amino acids 24 and 30. Interestingly, this nuclear localization signal closely resembles those identified in EBNA1 from Epstein-Barr virus and herpesvirus papio. A region encompassing amino acids 5 to 22 was further proved to mediate the specific interaction of LNA1 with chromatin during interphase and the chromosomes during mitosis. The presence of putative phosphorylation sites in the chromosome binding sites of LNA1 and EBNA1 suggests that their activity may be regulated by specific cellular kinases.

Picosecond-Hetero-FRET Microscopy to Probe Protein-Protein Interactions in Live Cells

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.

Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

Homo- and Hetero-oligomerization of β-Arrestins in Living Cells

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous β-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of β-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged β-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, β-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of β-arrestin2 and β-arrestin1 prevented β-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that β-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.

Homo-FRET versus hetero-FRET to probe homodimers in living cells.

Publisher Summary The purpose of this chapter is to provide information on the homo-fluorescence resonance energy transfer (FRET) versus hetero-FRET to probe homodimers in living cells. FRET is a nonradiative phenomenon in which energy is transferred from a donor fluorophore to an acceptor chromophore with an efficiency that depends on the distance between the two chromophores, the extent of overlap between the donor emission and acceptor excitation spectra, the quantum yield of the donor, and the relative orientation of the donor and acceptor. For homo-FRET, because the photophysical properties of the two donor molecules are the same, the excitation energy is reversibly transferred between the fluorescent tags. Time-resolved fluorescence anisotropy monitors any process that changes the polarization of the emitted fluorescence during the excited state. Consequently, the fluorescence anisotropy decay depends on (1) rotational movements of the fluorescent molecules and (2) energy transfer taking place within the fluorescence time scale. In addition, according to the type of interaction, hetero-or homodimer, the methodology, hetero- or homo-FRET, must be judiciously chosen to obtain the best information about structural data within the macromolecular complex.

Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

ABSTRACT Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.

Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements

Verrier & Soling1 and Thaler et al.2 did not observe yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) photoconversion during YFP photobleaching, questioning our previous results3. To exclude the possibility that photoconversion was specific to our experiments, we tested different microscopes, constructs and cells, including the sample used in Supplementary Figure 2b of the Verrier & Soling report. We detected photoconversion in all cases (Supplementary Fig. 1 online), indicating that it occurred independently of the cell lines, cell preparation and microscope used.