Triatoma Virus Recombinant VP4 Protein Induces Membrane Permeability through Dynamic Pores

Abstract

ABSTRACTIn naked viruses, membrane breaching is a key step that must be performed for genome transfer into the target cells. Despite its importance, the mechanisms behind this process remain poorly understood. The small protein VP4, encoded by the genomes of most viruses of the orderPicornavirales, has been shown to be involved in membrane alterations. Here we analyzed the permeabilization activity of the natively nonmyristoylated VP4 protein from triatoma virus (TrV), a virus belonging to theDicistroviridaefamily within thePicornaviralesorder. The VP4 protein was produced as a C-terminal maltose binding protein (MBP) fusion to achieve its successful expression. This recombinant VP4 protein is able to produce membrane permeabilization in model membranes in a membrane composition-dependent manner. The induced permeability was also influenced by the pH, being greater at higher pH values. We demonstrate that the permeabilization activity elicited by the protein occurs through discrete pores that are inserted on the membrane. Sizing experiments using fluorescent dextrans, cryo-electron microscopy imaging, and other, additional techniques showed that recombinant VP4 forms heterogeneous proteolipidic pores rather than common proteinaceous channels. These results suggest that the VP4 protein may be involved in the membrane alterations required for genome transfer or cell entry steps during dicistrovirus infection.IMPORTANCEDuring viral infection, viruses need to overcome the membrane barrier in order to enter the cell and replicate their genome. In nonenveloped viruses membrane fusion is not possible, and hence, other mechanisms are implemented. Among other proteins, like the capsid-forming proteins and the proteins required for viral replication, several viruses of the orderPicornaviridaecontain a small protein called VP4 that has been shown to be involved in membrane alterations. Here we show that the triatoma virus VP4 protein is able to produce membrane permeabilization in model membranes by the formation of heterogeneous dynamic pores. These pores formed by VP4 may be involved in the genome transfer or cell entry steps during viral infection.