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IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions
Journal of Biotechnology 155 (2011) 284–286
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions ˜ 1,2 , Iria Otero-Romero 1 , Javier Benavente, Jose M. Martinez-Costas ∗ Alberto Brandariz-Nunez Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia y Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), na), Spain Universidad de Santiago de Compostela, Campus Vida S/N, 15782-Santiago de Compostela (A Coru˜
a r t i c l e
i n f o
Article history: Received 18 May 2011 Received in revised form 5 July 2011 Accepted 11 July 2011 Available online 22 July 2011 Keywords: Protein–protein interactions IC-tagging Particulate protein inclusions Reovirus
a b s t r a c t We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore. © 2011 Elsevier B.V. All rights reserved.
1. Results and discussion Avian reovirus (ARV) viral factories are dense globular cytoplasmic structures where the different viral components required for viral replication and morphogenesis are sequentially and specifically attracted to increase the overall efficiency of the replication process (Benavente, 2006a,b; Touris-Otero et al., 2004a). The viral protein muNS is believed to form the matrix of such factories because of its ability to form globular inclusions in the cytoplasm of eukaryotic cells in absence of any other viral protein (TourisOtero et al., 2004b). We have recently developed a versatile tagging system (IC-tagging) that uses the IC domain of the viral protein muNS. As the IC domain is an interaction domain involved in muNS homo-oligomer formation, it causes relocation of the IC-tagged proteins to ARV muNS-derived intracellular globular inclusions ˜ (Brandariz-Nunez et al., 2010a,b,c). We have also demonstrated the utility of our method for purifying foreign proteins (Brandariz˜ et al., 2010c). Proteins purified with the IC-tagging system Nunez can be obtained in soluble form or integrated into particulate pellets formed by protein muNS or its derivatives. Pellet-integrated enzymes have the additional advantage that they can be re-used or eliminated from solution by simple centrifugation or filtration.
∗ Corresponding author. Tel.: +34 981599157; fax: +34 981599157. E-mail address: [email protected] (J.M. Martinez-Costas). 1 These authors contributed equally to this work. 2 Present address: Microbiology and Immunology Department, Albert Einstein College of Medicine, New York, USA. 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.07.010
Additionally, muNS purified inclusions can also be used as T-cell response-inducer vaccination vectors, when decorated with appropriate foreign IC-tagged epitopes. The IC-tagging method can also be used for detecting or validating protein-protein interactions in the cytoplasm or nucleus of living cells because IC-tagged proteins are able to attract their untagged binding partners to the muNS˜ inclusions (Brandariz-Nunez et al., 2010b). However, we do not know if the IC-tagged molecules are able to interact between them while integrated in the inclusions as they are directed to specific places inside the inclusions guided by the IC-tag. We have proposed that the IC-tagging system could be used for recruiting and concentrating the components of supra-molecular complexes in the inclusions to increase the efficiency of complex formation, mimicking the natural activity of muNS in the viral factories but, as said before, we showed no proof yet of the interaction between IC-tagged proteins. To test this hypothesis we explored the ability of DsRed protein to fluoresce when integrated into muNSderived inclusions. We used DsRed, because it displays strong red fluorescence emission (Matz et al., 1999), and also because it is an obligate tetramer whose oligomerization is essential for the formation of a functional red fluorescent chromophore (Baird et al., 2000; Sacchetti et al., 2002). The crystal structure for DsRed revealed a tetrameric quaternary structure where the N-terminus of each protomer points towards the surface of the tetramer, while the Cterminus is directly involved in dimer to dimer contacts (Fig. 1A). Such contacts seemed essential for tetramerization, which lead the authors to predict the non-functionality of any construct that would involve the fusion of the tagged protein to the carboxyl-terminus of DsRed (Yarbrough et al., 2001). In fact, successful DsRed tagging
A. Brandariz-Nu˜ nez et al. / Journal of Biotechnology 155 (2011) 284–286
Fig. 1. DsRed tagging and intracellular distribution. (A) Ribbon representation of the DsRed tetramer produced with Chimera (http://www.cgl.ucsf.edu/chimera) using the PDB file 1G7K (Yarbrough et al., 2001). Monomers are labelled in capital letters and their C-termini with lower case letters. (B) Intracellular distribution and fluorescence ability of untagged and IC-tagged DsRed proteins. Vero cells were transfected with plasmids expressing the proteins indicated at the right of the figure. DsRed was visualized without antibodies (red), and the IC-tag with antibodies against muNS (muNS). Nuclei were counterstained blue with DAPI.
was only reported for proteins fused to the N-terminus of the fluorescent protein, and point mutations that abolished the formation of the tetramers, were also non-fluorescent (Campbell et al., 2002; Sacchetti et al., 2002). These reasons prompted us to use DsRed as a suitable model to test whether IC-tagged proteins contact with each other when integrated into muNS-derived inclusions. To this end, we first fused the IC-Tag to either end of the DsRed protein, and checked the ability of the tagged proteins to emit red fluorescence when transiently expressed in mammalian Vero cells. For expressing IC-
285
DsRed, the IC-encoding sequence within plasmid pCDNA3.1/Zeo-IC ˜ et al., 2010c) was amplified and cloned between (Brandariz-Nunez the BglII and EcoRI sites of plasmid pDsRed1-N1 (Clontech, Saint-Germain en Laye, France). For expressing DsRed-IC, the DsRed-encoding sequence within plasmid pDsRed1-N1 was ampli˜ fied and cloned into pCDNA3.1/Zeo-IC (Brandariz-Nunez et al., 2010c). Plasmids expressing untagged and IC-tagged DsRed were transfected into Vero cell monolayers and the cells were analyzed by fluorescence microscopy. As expected, DsRed and IC-DsRed proteins emitted red fluorescence, whereas DsRed-IC did not (Fig. 1B, red panels), confirming that the presence of a C-terminal tag hinders DsRed tetramerization and/or chromophore evolution (Fig. 1B, panel 3). The inability of DsRed-IC to emit red fluorescence was not due to reduced protein expression since the use of antibodies directed against muNS that recognize the IC tag, revealed similar intracellular levels of both tagged proteins (Fig. 1B, ␣-muNS panels). Next, we checked the fluorescence ability of the IC-tagged constructs when integrated into muNS cytoplasmic inclusions. For this, Vero cells were co-transfected with the DsRed-expressing constructs shown in Fig. 1B, and plasmid pCINeo-muNS, which ˜ expresses full-length ARV muNS (Brandariz-Nunez et al., 2010b). The results shown in Fig. 2 revealed that, while untagged DsRed protein does not co-localize with muNS inclusions (lane 1), ICDsRed readily associates with these structures (Fig. 2, lane 2). The strong red fluorescence detected in muNS inclusions not only demonstrates that the IC tag is able to drive fused DsRed to these structures, but also suggests that the IC-DsRed monomers are able to interact with each other to form a functional oligomer when inclusion-associated. However, the possibility still exist that some soluble IC-DsRed monomers are recruited to the inclusions by inclusion-integrated ones. Thus, the observed fluorescence might not really be due to the interaction between monomers that are all associated with the inclusion through their IC-tag. Strikingly, DsRed-IC, which does no emit fluorescence when expressed in the absence of muNS, emitted strong red fluorescence when associated to muNS inclusions (Fig. 2, lane 3). The ability of DsRed-IC proteins to fluoresce only when they are inclusion-integrated, indicates that their recruitment to inclusions by the IC-tag renders the DsRed monomers free to productively interact to each other and evolve a functional chomophore. Thus, our results demonstrate (i) that the IC tag successfully directs re-location to muNS-derived inclusions regardless of its position at the N or C-terminus of the tagged protein, which is very useful for problematic proteins as the DsRed presented here, and (ii) that proteins contained within muNS inclu-
Fig. 2. Association of tagged DsRed with muNS-derived cytoplasmic inclusions. Vero cells were co-transfected with plasmids expressing the proteins indicated on the left of the figure and immunostained with anti-muNS antibodies (muNS). DsRed was visualized without antibodies (red). Nuclei were counterstained blue with DAPI.
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A. Brandariz-Nu˜ nez et al. / Journal of Biotechnology 155 (2011) 284–286
sions are able to interact with each other and catalyze complex reactions as the tetrameric chromophore evolution of DsRed. Competing interests ˜ Javier Benavente Martínez and Jose Alberto Brandariz Nunez, M. Martinez-Costas have a patent application on the IC-tagging system. Acknowledgments This work was supported by grants from the European Commission under contracts ERAS-CT-2003-980409 (as part of the European Science Foundation EUROCORES Programme EuroSCOPE, web – http://www.esf.org/euroscope); the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330, BFU 205-24982-E, web: http://www.mityc.es/) and Xunta de Galicia (08CSA009203PR, web – http://www.conselleriaiei.org/ga/dxidi/index.php). ABN and IOR were recipients of a predoctoral FPI fellowship from the Spanish Ministerio de Ciencia y Tecnología. We thank Rebeca Menaya Vargas and Leticia Barcia Castro for excellent technical assistance. References Baird, G.S., Zacharias, D.A., Tsien, R.Y., 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U. S. A. 97, 11984–11989.
Benavente, J., Martinez-Costas, J., 2006a. Avian reovirus: structure and biology. Virus Res. 123, 105–119. Benavente, J., Martinez-Costas, J., 2006b. Early steps in avian reovirus morphogenesis. Curr. Topics Microbiol. Immunol. 309, 67–85. ˜ Brandariz-Nunez, A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010a. Avian reovirus microNS protein forms homo-oligomeric inclusions in a microtubule-independent fashion, which involves specific regions of its Cterminal domain. J. Virol. 84, 4289–4301. ˜ A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010b. Brandariz-Nunez, IC-tagging and protein relocation to ARV muNS inclusions: a method to study protein-protein interactions in the cytoplasm or nucleus of living cells. PLoS One 5 (11), e13785. ˜ Brandariz-Nunez, A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010c. A versatile molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein immobilization and purification. PLoS One 5 (11), e13961. Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., Tsien, R.Y., 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U. S. A. 99, 7877–7882. Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., Lukyanov, S.A., 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973. Sacchetti, A., Subramaniam, V., Jovin, T.M., Alberti, S., 2002. Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore. FEBS Lett. 525, 13–19. Touris-Otero, F., Cortez-San Martin, M., Martinez-Costas, J., Benavente, J., 2004a. Avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of sigmaNS and lambdaA to microNS inclusions. J. Mol. Biol. 341, 361–374. Touris-Otero, F., Martinez-Costas, J., Vakharia, V.N., Benavente, J., 2004b. Avian reovirus non-structural protein microNS forms viroplasm-like inclusions and recruits protein sigmaNS to these structures. Virology 319, 94–106. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V., Remington, S.J., 2001. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A˚ resolution. Proc. Natl. Acad. Sci. U. S. A. 98, 462–467.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions ˜ 1,2 , Iria Otero-Romero 1 , Javier Benavente, Jose M. Martinez-Costas ∗ Alberto Brandariz-Nunez Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia y Centro Singular de Investigación en Química Biológica y Materiales Moleculares (CIQUS), na), Spain Universidad de Santiago de Compostela, Campus Vida S/N, 15782-Santiago de Compostela (A Coru˜
a r t i c l e
i n f o
Article history: Received 18 May 2011 Received in revised form 5 July 2011 Accepted 11 July 2011 Available online 22 July 2011 Keywords: Protein–protein interactions IC-tagging Particulate protein inclusions Reovirus
a b s t r a c t We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore. © 2011 Elsevier B.V. All rights reserved.
1. Results and discussion Avian reovirus (ARV) viral factories are dense globular cytoplasmic structures where the different viral components required for viral replication and morphogenesis are sequentially and specifically attracted to increase the overall efficiency of the replication process (Benavente, 2006a,b; Touris-Otero et al., 2004a). The viral protein muNS is believed to form the matrix of such factories because of its ability to form globular inclusions in the cytoplasm of eukaryotic cells in absence of any other viral protein (TourisOtero et al., 2004b). We have recently developed a versatile tagging system (IC-tagging) that uses the IC domain of the viral protein muNS. As the IC domain is an interaction domain involved in muNS homo-oligomer formation, it causes relocation of the IC-tagged proteins to ARV muNS-derived intracellular globular inclusions ˜ (Brandariz-Nunez et al., 2010a,b,c). We have also demonstrated the utility of our method for purifying foreign proteins (Brandariz˜ et al., 2010c). Proteins purified with the IC-tagging system Nunez can be obtained in soluble form or integrated into particulate pellets formed by protein muNS or its derivatives. Pellet-integrated enzymes have the additional advantage that they can be re-used or eliminated from solution by simple centrifugation or filtration.
∗ Corresponding author. Tel.: +34 981599157; fax: +34 981599157. E-mail address: [email protected] (J.M. Martinez-Costas). 1 These authors contributed equally to this work. 2 Present address: Microbiology and Immunology Department, Albert Einstein College of Medicine, New York, USA. 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.07.010
Additionally, muNS purified inclusions can also be used as T-cell response-inducer vaccination vectors, when decorated with appropriate foreign IC-tagged epitopes. The IC-tagging method can also be used for detecting or validating protein-protein interactions in the cytoplasm or nucleus of living cells because IC-tagged proteins are able to attract their untagged binding partners to the muNS˜ inclusions (Brandariz-Nunez et al., 2010b). However, we do not know if the IC-tagged molecules are able to interact between them while integrated in the inclusions as they are directed to specific places inside the inclusions guided by the IC-tag. We have proposed that the IC-tagging system could be used for recruiting and concentrating the components of supra-molecular complexes in the inclusions to increase the efficiency of complex formation, mimicking the natural activity of muNS in the viral factories but, as said before, we showed no proof yet of the interaction between IC-tagged proteins. To test this hypothesis we explored the ability of DsRed protein to fluoresce when integrated into muNSderived inclusions. We used DsRed, because it displays strong red fluorescence emission (Matz et al., 1999), and also because it is an obligate tetramer whose oligomerization is essential for the formation of a functional red fluorescent chromophore (Baird et al., 2000; Sacchetti et al., 2002). The crystal structure for DsRed revealed a tetrameric quaternary structure where the N-terminus of each protomer points towards the surface of the tetramer, while the Cterminus is directly involved in dimer to dimer contacts (Fig. 1A). Such contacts seemed essential for tetramerization, which lead the authors to predict the non-functionality of any construct that would involve the fusion of the tagged protein to the carboxyl-terminus of DsRed (Yarbrough et al., 2001). In fact, successful DsRed tagging
A. Brandariz-Nu˜ nez et al. / Journal of Biotechnology 155 (2011) 284–286
Fig. 1. DsRed tagging and intracellular distribution. (A) Ribbon representation of the DsRed tetramer produced with Chimera (http://www.cgl.ucsf.edu/chimera) using the PDB file 1G7K (Yarbrough et al., 2001). Monomers are labelled in capital letters and their C-termini with lower case letters. (B) Intracellular distribution and fluorescence ability of untagged and IC-tagged DsRed proteins. Vero cells were transfected with plasmids expressing the proteins indicated at the right of the figure. DsRed was visualized without antibodies (red), and the IC-tag with antibodies against muNS (muNS). Nuclei were counterstained blue with DAPI.
was only reported for proteins fused to the N-terminus of the fluorescent protein, and point mutations that abolished the formation of the tetramers, were also non-fluorescent (Campbell et al., 2002; Sacchetti et al., 2002). These reasons prompted us to use DsRed as a suitable model to test whether IC-tagged proteins contact with each other when integrated into muNS-derived inclusions. To this end, we first fused the IC-Tag to either end of the DsRed protein, and checked the ability of the tagged proteins to emit red fluorescence when transiently expressed in mammalian Vero cells. For expressing IC-
285
DsRed, the IC-encoding sequence within plasmid pCDNA3.1/Zeo-IC ˜ et al., 2010c) was amplified and cloned between (Brandariz-Nunez the BglII and EcoRI sites of plasmid pDsRed1-N1 (Clontech, Saint-Germain en Laye, France). For expressing DsRed-IC, the DsRed-encoding sequence within plasmid pDsRed1-N1 was ampli˜ fied and cloned into pCDNA3.1/Zeo-IC (Brandariz-Nunez et al., 2010c). Plasmids expressing untagged and IC-tagged DsRed were transfected into Vero cell monolayers and the cells were analyzed by fluorescence microscopy. As expected, DsRed and IC-DsRed proteins emitted red fluorescence, whereas DsRed-IC did not (Fig. 1B, red panels), confirming that the presence of a C-terminal tag hinders DsRed tetramerization and/or chromophore evolution (Fig. 1B, panel 3). The inability of DsRed-IC to emit red fluorescence was not due to reduced protein expression since the use of antibodies directed against muNS that recognize the IC tag, revealed similar intracellular levels of both tagged proteins (Fig. 1B, ␣-muNS panels). Next, we checked the fluorescence ability of the IC-tagged constructs when integrated into muNS cytoplasmic inclusions. For this, Vero cells were co-transfected with the DsRed-expressing constructs shown in Fig. 1B, and plasmid pCINeo-muNS, which ˜ expresses full-length ARV muNS (Brandariz-Nunez et al., 2010b). The results shown in Fig. 2 revealed that, while untagged DsRed protein does not co-localize with muNS inclusions (lane 1), ICDsRed readily associates with these structures (Fig. 2, lane 2). The strong red fluorescence detected in muNS inclusions not only demonstrates that the IC tag is able to drive fused DsRed to these structures, but also suggests that the IC-DsRed monomers are able to interact with each other to form a functional oligomer when inclusion-associated. However, the possibility still exist that some soluble IC-DsRed monomers are recruited to the inclusions by inclusion-integrated ones. Thus, the observed fluorescence might not really be due to the interaction between monomers that are all associated with the inclusion through their IC-tag. Strikingly, DsRed-IC, which does no emit fluorescence when expressed in the absence of muNS, emitted strong red fluorescence when associated to muNS inclusions (Fig. 2, lane 3). The ability of DsRed-IC proteins to fluoresce only when they are inclusion-integrated, indicates that their recruitment to inclusions by the IC-tag renders the DsRed monomers free to productively interact to each other and evolve a functional chomophore. Thus, our results demonstrate (i) that the IC tag successfully directs re-location to muNS-derived inclusions regardless of its position at the N or C-terminus of the tagged protein, which is very useful for problematic proteins as the DsRed presented here, and (ii) that proteins contained within muNS inclu-
Fig. 2. Association of tagged DsRed with muNS-derived cytoplasmic inclusions. Vero cells were co-transfected with plasmids expressing the proteins indicated on the left of the figure and immunostained with anti-muNS antibodies (muNS). DsRed was visualized without antibodies (red). Nuclei were counterstained blue with DAPI.
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sions are able to interact with each other and catalyze complex reactions as the tetrameric chromophore evolution of DsRed. Competing interests ˜ Javier Benavente Martínez and Jose Alberto Brandariz Nunez, M. Martinez-Costas have a patent application on the IC-tagging system. Acknowledgments This work was supported by grants from the European Commission under contracts ERAS-CT-2003-980409 (as part of the European Science Foundation EUROCORES Programme EuroSCOPE, web – http://www.esf.org/euroscope); the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330, BFU 205-24982-E, web: http://www.mityc.es/) and Xunta de Galicia (08CSA009203PR, web – http://www.conselleriaiei.org/ga/dxidi/index.php). ABN and IOR were recipients of a predoctoral FPI fellowship from the Spanish Ministerio de Ciencia y Tecnología. We thank Rebeca Menaya Vargas and Leticia Barcia Castro for excellent technical assistance. References Baird, G.S., Zacharias, D.A., Tsien, R.Y., 2000. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. U. S. A. 97, 11984–11989.
Benavente, J., Martinez-Costas, J., 2006a. Avian reovirus: structure and biology. Virus Res. 123, 105–119. Benavente, J., Martinez-Costas, J., 2006b. Early steps in avian reovirus morphogenesis. Curr. Topics Microbiol. Immunol. 309, 67–85. ˜ Brandariz-Nunez, A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010a. Avian reovirus microNS protein forms homo-oligomeric inclusions in a microtubule-independent fashion, which involves specific regions of its Cterminal domain. J. Virol. 84, 4289–4301. ˜ A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010b. Brandariz-Nunez, IC-tagging and protein relocation to ARV muNS inclusions: a method to study protein-protein interactions in the cytoplasm or nucleus of living cells. PLoS One 5 (11), e13785. ˜ Brandariz-Nunez, A., Menaya-Vargas, R., Benavente, J., Martinez-Costas, J., 2010c. A versatile molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein immobilization and purification. PLoS One 5 (11), e13961. Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., Tsien, R.Y., 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U. S. A. 99, 7877–7882. Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L., Lukyanov, S.A., 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973. Sacchetti, A., Subramaniam, V., Jovin, T.M., Alberti, S., 2002. Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore. FEBS Lett. 525, 13–19. Touris-Otero, F., Cortez-San Martin, M., Martinez-Costas, J., Benavente, J., 2004a. Avian reovirus morphogenesis occurs within viral factories and begins with the selective recruitment of sigmaNS and lambdaA to microNS inclusions. J. Mol. Biol. 341, 361–374. Touris-Otero, F., Martinez-Costas, J., Vakharia, V.N., Benavente, J., 2004b. Avian reovirus non-structural protein microNS forms viroplasm-like inclusions and recruits protein sigmaNS to these structures. Virology 319, 94–106. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V., Remington, S.J., 2001. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A˚ resolution. Proc. Natl. Acad. Sci. U. S. A. 98, 462–467.