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. 2012;7(4):e34307.
doi: 10.1371/journal.pone.0034307. Epub 2012 Apr 10.

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome

Tuomas Rönnberg  1 , Kirsi Jääskeläinen, Guillaume Blot, Ville Parviainen, Antti Vaheri, Risto Renkonen, Michele Bouloy, Alexander Plyusnin
Affiliations

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome

Tuomas Rönnberg et al. PLoS One. 2012.

Abstract

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Interactome (human) of TULV and PUUV NSs proteins.
Proteins of the human interactome are arranged into clusters: 1. Cellular structure, 2. Transport, 3. Proteasome, 4. Signaling, 5. Transcription & translation, 6. Histone remodeling. The size of a node is relative to the number of connections it has, the color is related to the number of connections its neighbors have (from green, few to red, many). Primary nodes are shown as triangles, secondary as circles.
Figure 2
Figure 2. Intersection proteins.
Intersection of closest shared TULV and PUUV NSs -linked nodes. Primary nodes are in red, secondary nodes in pink. Secondary nodes that do not directly connect to two different primary nodes are in white. Metanodes are represented by pink diamonds.
Figure 3
Figure 3. Results of FRET assay.
Primary cells were infected with TULV strain Lodz for 9 days and fixed on coverslips. Cells were stained for the viral NSs protein, which was seen as bright spots around perinuclear area (A), and ACBD3 protein (B). FRET assay: Dpre, donor intensity before bleaching, Dpost, donor intensity after bleaching, Apre, acceptor intensity before bleaching, Apost, acceptor intensity after bleaching, FRETeff, calculated efficiency of FRET.
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References

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