The SAT Protein of the Minute Virus of Mice Induces the Lysis of the Cell through the Formation of Viroporin-like Structures

Abstract

The prototype minute virus of mice (MVMp) and the H-1 virus belong to the family of Parvoviridae and can be used as oncolytic agents that infect, replicate in and kill human cancer cells. In preclinical trials the viruses were shown to be very successful to eradicate tumors in mice and rats. A phase I/IIa clinical trial with patients suffering from recurrent glioblastoma multiforme showed that parvovirus is safe to use in humans and that the therapy induces an infiltration of immune cells into the tumor tissue. \n \nMany aspects of the parvoviral life cycle have been studied over the last decades. The entry, replication, transcription and packaging mechanisms of the virus are well understood. However, the process of the virally-induced lysis of the cell is still unknown. The viral non structural protein 1 (NS1) was suggested to have cytotoxic features but a direct lytic mechanism could not be shown. Recently, the porcine parvovirus (PPV) was found to express the short transmembrane protein SAT. Meszaros and coworkers showed that a knockout of SAT led to a reduced lytic capacity of the virus (Meszaros et al., 2017). In the current study we show that the SAT protein of MVMp is also important for the lysis of the cell. A knockout of SAT in the genome of MVMp reduced the lytic capacity of the virus and SAT-ko viruses were also found to be less infectious than the wild type virus. The sole expression of SAT induced lysis and it exceeded the lytic capacity of NS1 by multiple times. Meszaros and coworkers suspected SAT to induce an irreversible stress in the endoplasmic reticulum which eventually led to the death of the cell. However, a direct mechanism of this process could not be shown. We suspect SAT to function as a viroporin - a class of virally-encoded transmembrane proteins that oligomerise to form pores through membranes. We could show that SAT is transported to the plasma membrane where it oligomerises in multimers and makes the plasma membrane permeable to the small molecule Hygromycin B. A computer simulation of the protein confirmed that SAT oligomerises in symmetrical complexes. However, pore-like structures were not observed. Furthermore, an increase in the permeability of ions such as calcium and sodium, which is often seen for other viroporins, was not found. \n \nIn an attempt to increase the lytic capacity of MVMp we created the SUPER virus. The position of SAT was altered in the genome of SUPER in order to increase its translation. We could show that the translation of SAT was indeed increased for the SUPER virus which led to an accelerated lysis of the cells. However, the production of the SUPER virus was not very efficient. In order to increase the production of SUPER, we constructed shRNA constructs to knockdown SAT. Although the production of SAT was decreased, this approach did not increase the production of the virus. Nevertheless, the SUPER virus could be a promising candidate for the therapy with oncolytic parvoviruses. We suspect its increased lytic potential to release tumor antigens more efficiently compared to the lytic potential of the wild type virus. The released tumor antigens are suspected to stimulate cells of the immune system such as dendritic cells and cytotoxic T-cells in order to recognise and attack non-infected tumor cells.