Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology

Abstract

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus

“…3B). These observations were similar to those 314 reported for the smaller replicase proteins of MNSV and CIRV though such 315 polypeptides were in general more resistant to membrane extraction through 316 biochemical treatments (Mochizuki et al, 2009;Rubino et al, 2000). We concluded 317 from these results that p27 was associated to membranes through a mechanism that 318 imparted significant stability to protein-membrane interactions though its nature as 319 integral membrane protein could not be confirmed.…”

“…The plasmid p36K-GFP, allowing expression of 168 protein p36 of Carnation italian ringspot virus (Rubino et al, 2000), was also included 169 for comparison purposes. Transformation of plasmids was done with the lithium 170 acetate-polyethylene glycol method (Ito et al, 1983).…”

Expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA

“…Protein extraction, electrophoresis and Western blot analysis were done as previously described (Rubino et al, 2000), except that the replicase proteins were detected using anti-c-Myc or anti-HA antibodies (Santa Cruz Biotechnology) (Pantaleo et al, 2004).…”

“…Total yeast RNA was extracted and analysed as described previously (Pantaleo et al, 2003). Riboprobes for positive-and negative-strand DI or sat RNAs were obtained by cloning approximately 300 nt of the 39-terminal regions of DI or sat RNAs in the vector pTL7SN (Oh & Carrington, 1989) in the appropriate orientation.Protein extraction, electrophoresis and Western blot analysis were done as previously described (Rubino et al, 2000), except that the replicase proteins were detected using anti-c-Myc or anti-HA antibodies (Santa Cruz Biotechnology) (Pantaleo et al, 2004).Plant growth, agroinfiltration and protoplasts. Transgenic Nicotiana benthamiana plant line 92KA11, expressing the full-length CymRSV replicase gene (Rubino et al, 1993), and non-transgenic plants were grown in growth chambers at 25 uC with a 14 h photoperiod.…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…Entre los miembros del género Tombusvirus, se ha descrito la asociación de la proteína p33 del TBSV, el CymRSV y el CNV a membranas peroxisomales [Navarro et al, 2004;Panavas et al, 2005a;McCartney et al, 2005], mientras que la proteína homóloga del CIRV, p36, se localiza en mitocondrias [Rubino et al, 2000. Tanto la proteína p33 como la proteína p36 inducen la formación de múltiples vesículas esféricas u ovoides (MVB) resultado de la invaginación de la membrana externa del orgánulo con el que se asocian.…”