"Positive-strand RNA [(+)RNA] viruses, the largest class of viruses infecting humans, animals and plants, share common replication mechanisms. A feature conserved among these viruses is the association of the viral replication complex with specific intracellular membranes, which are rearranged and are induced to proliferate to form partially closed vesicles in which virus replication takes place. The (+)RNA Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae) replication in plants occurs in membranous structures derived from modified mitochondria. The determinants targeting CIRV replication complex to the mitochondrial outer membrane are contained in the non structural protein p36, which is indispensable for virus replication. However, the function and biological role of p36 are still elusive. Many DNA and RNA virus proteins behave as proapoptotic or antiapoptotic viral factors, either by direct or indirect interaction with the mitochondrial outer membrane. The yeast Saccharomyces cerevisiae has been widely used as a model host to study the replication of (+)RNA viruses, as well as cell death and survival processes. In view of the interaction of CIRV p36 with the mitochondrial outer membrane and the known role of mitochondria in cell fate decisions, some biological effects of p36 expression in yeast cells were investigated along with the possible effect of CIRV p36 on cell survival and death. The viability of yeast cells expressing p36 cloned under the inducible GAL1 promoter, in the absence or presence of 160 mM acetic acid (AA), was analyzed as a function of time by counting colony forming units (cfu) on YPD plates after 48 h growth at 30°C. Plasma membrane integrity was measured by propidium iodide (PI) staining. Phosphatidylserine (PS) exposure and membrane integrity were detected by a FITCcoupled Annexin V reaction. Cells were observed in an Axioplan 2 microscope (Zeiss). Protein analyses were performed either on whole cell lysates or after spheroplasting and separation into a membrane-enriched pellet and a supernatant fractions. It was shown that p36 expression was stable over time and affected cell growth rate, but did not promote cell death. In addition, p36 could change 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. Acetic acid treatment did not impair the strict association of p36 with mitochondrial membranes, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria. Yeast proved to be 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, for fundamental studies and for the development of antiviral strategies.
Saccharomyces cerevisiae: a model system for the study of virus replication
Rubino L;Guaragnella N;Antonacci A;Giannattasio S
2017
Abstract
"Positive-strand RNA [(+)RNA] viruses, the largest class of viruses infecting humans, animals and plants, share common replication mechanisms. A feature conserved among these viruses is the association of the viral replication complex with specific intracellular membranes, which are rearranged and are induced to proliferate to form partially closed vesicles in which virus replication takes place. The (+)RNA Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae) replication in plants occurs in membranous structures derived from modified mitochondria. The determinants targeting CIRV replication complex to the mitochondrial outer membrane are contained in the non structural protein p36, which is indispensable for virus replication. However, the function and biological role of p36 are still elusive. Many DNA and RNA virus proteins behave as proapoptotic or antiapoptotic viral factors, either by direct or indirect interaction with the mitochondrial outer membrane. The yeast Saccharomyces cerevisiae has been widely used as a model host to study the replication of (+)RNA viruses, as well as cell death and survival processes. In view of the interaction of CIRV p36 with the mitochondrial outer membrane and the known role of mitochondria in cell fate decisions, some biological effects of p36 expression in yeast cells were investigated along with the possible effect of CIRV p36 on cell survival and death. The viability of yeast cells expressing p36 cloned under the inducible GAL1 promoter, in the absence or presence of 160 mM acetic acid (AA), was analyzed as a function of time by counting colony forming units (cfu) on YPD plates after 48 h growth at 30°C. Plasma membrane integrity was measured by propidium iodide (PI) staining. Phosphatidylserine (PS) exposure and membrane integrity were detected by a FITCcoupled Annexin V reaction. Cells were observed in an Axioplan 2 microscope (Zeiss). Protein analyses were performed either on whole cell lysates or after spheroplasting and separation into a membrane-enriched pellet and a supernatant fractions. It was shown that p36 expression was stable over time and affected cell growth rate, but did not promote cell death. In addition, p36 could change 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. Acetic acid treatment did not impair the strict association of p36 with mitochondrial membranes, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria. Yeast proved to be 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, for fundamental studies and for the development of antiviral strategies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
