The SV40 Late Protein VP4 Is a Viroporin that Forms Pores to Disrupt Membranes for Viral Release

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsulated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release

Cholesterol Exposure at the Membrane Surface Is Necessary and Sufficient to Trigger Perfringolysin O Binding

Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin

Perfringolysin O as a useful tool to study human sperm physiology

Objective: To evaluate perfringolysin O, a cholesterol-dependent pore-forming cytolysin, as a tool to study several aspects of human sperm physiology. Design: Prospective study. Setting: Basic research laboratory. Patient(s): Human semen samples with normal parameters obtained from healthy donors. Intervention(s): Interaction of recombinant perfringolysin O with human spermatozoa. Main Outcome Measure(s): Assessment of perfringolysin O binding to spermatozoa, tests for acrosome and plasma membrane integrity, and acrosomal exocytosis assays. Result(s): Perfringolysin O associated with human spermatozoa at 4 C. The binding was sensitive to changes in cholesterol concentrations and distribution occurring in the plasma membrane of these cells during capacitation. When perfringolysin O–treated sperm were incubated at 37 C, the plasma membrane became permeable, whereas the acrosome membrane remained intact. Permeabilized spermatozoa were able to respond to exocytic stimuli. The process was inhibited by proteins that interfere with membrane fusion, indicating that large molecules, including antibodies, were able to permeate into the spermatozoa. Conclusion(s): PFO is a useful probe to assess changes in the amount and distribution of the active sterol fraction present in the sperm plasma membrane. The toxin can be used for the efficient and selective permeabilization of this membrane, rendering a flexible experimental model suitable for studying molecular processes occurring in the sperm cytoplasm. (Fertil Steril 2013;99:99–106. 2013 by American Society for Reproductive Medicine).Fil: Pocognoni, Cristián Adrián. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: de Blas, Gerardo Andrés. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Heuck, Alejandro P.. University of Massachusett. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Belmonte, Silvia Alejandra. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; ArgentinaFil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Tecnicas. Centro Cientifico Tecnologico Mendoza. Instituto Histologia y Embriologia de Mendoza "Dr. M. Burgos"; Argentin