Mechanisms of Ageing and Development 161 (2017) 255–261 Contents lists available at ScienceDirect Mechanisms of Ageing and Development jou rn al hom ep age: www.elsevier.com/locate/mechagedev Original article Heterologous expression of carnation Italian ringspot virus p36 protein enhances necrotic cell death in response to acetic acid in Saccharomyces cerevisiae Luisa Rubino a , Nicoletta Guaragnella b , Sergio Giannattasio b,∗ a Istituto di Protezione Sostenibile delle Piante, CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy b Istituto di Biomembrane e Bioenergetica, CNR, Via Amendola 165/A, 70126 Bari, Italy a r t i c l e i n f o Article history: Received 15 March 2016 Received in revised form 5 September 2016 Accepted 12 September 2016 Available online 13 September 2016 Keywords: (+)RNA virus CIRV Yeast Programmed cell death Acetic acid a b s t r a c t A universal feature of the replication of positive-strand RNA viruses is the association with intracellular membranes. Carnation Italian ringspot virus (CIRV) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. The product encoded by CIRV ORF1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. Here the yeast Saccharomyces cerevisiae was used as a model host to study the effect of CIRV p36 on cell survival and death. It was shown that p36 does not promote cell death, but decreases cell growth rate. In addition, p36 changed the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. The tight association of p36 to membranes was not affected by acetic acid treatment, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria in yeast. This work proved yeast as an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses. © 2016 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Replication of positive-strand RNA [(+)RNA] viruses occurs invariably in association with cell membranes, which are rear- ranged and are induced to proliferate so as to form partially closed vesicles where virus replication takes place. To this effect and depending specifically on the virus, all intracellular membranes, i.e. plasma and vacuolar membrane, endoplasmic reticulum, bounding membrane of mitochondria, peroxisomes, lysosomes and chloro- plasts, may be recruited (den Boon and Ahlquist, 2010; Laliberté and Sanfac ¸ on, 2010). This represents a universal feature shared among positive-strand RNA viruses, which makes them attractive models for fundamental studies and for the development of antivi- ral strategies. The (+)RNA Carnation Italian ringspot virus is a member of the genus Tombusvirus in the family Tombusviridae (Rochon et al., 2012), originally isolated from carnation plants (Dianthus ∗ Corresponding author. E-mail addresses: luisa.rubino@cnr.it (L. Rubino), n.guaragnella@ibbe.cnr.it (N. Guaragnella), s.giannattasio@ibbe.cnr.it (S. Giannattasio). caryophyllus) (Hollings et al., 1970). Icosahedral virus parti- cles package a genome consisting of one single-stranded, 4760 nucleotides messenger-sense RNA molecule, lacking both the 5 ′ cap structure and a 3 ′ poly(A) tail and containing five open read- ing frames (ORFs) (Rubino et al., 1995). The 5 ′ proximal ORF1 is translated at the very early stage of infection and codes for a 36- kDa protein (p36); by read-through of the p36 amber termination codon, the p95 RNA-dependent RNA polymerase (RdRp) product of ORF2 is synthesized. Both p36 and p95 are part of the virus replica- tion complex and are indispensable for virus replication (Dalmay et al., 1993; Russo et al., 1994; White and Nagy, 2004). ORF3 encodes the 41-kDa capsid protein, whereas the nested ORFs 4 and 5 code, respectively, for the 22-kDa cell-to-cell movement protein and for the 19-kDa protein which is involved in symptom elicitation in infected plants (Rubino et al., 1995). CIRV-infected plants show local chlorotic lesions on inoculated leaves in three-four days and systemic mosaic and leaf distortion in one week, followed by apical necrosis and death of the plant in about two weeks. At the ultra- structural level, the major characteristic of CIRV-infected cells is the presence of membranous structures derived from modified mito- chondria. A progressive invagination of the outer membrane leads to the formation of round-to-ovoid vesicles 80–150 nm in diameter, http://dx.doi.org/10.1016/j.mad.2016.09.004 0047-6374/© 2016 Elsevier Ireland Ltd. All rights reserved.