Characterisation of Oncolytic H-1 Parvovirus Cell Entry Pathways

H-1 protoparvovirus (H-1PV) is a self-propagating virus, non-pathogenic in humans, endowed with oncolytic and oncosuppressive activities. H-1PV is the only member of the Parvoviridae family to be tested as an anticancer agent in a clinical setting. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma showed that virus treatment is safe and well-tolerated. Virus treatment was associated with first signs of efficacy, including immune conversion of tumour microenvironment, good virus distribution in the tumour bed, as well as improved patient overall survival compared with historical controls. However, monotherapeutic use of the virus was not sufficient to eradicate the tumours. In this manner, my approach consists of further understanding the virus life cycle in order to improve H-1PV-based anticancer therapies. This knowledge can provide hints on which drugs or treatment modalities could be combined with the virus in order to enhance its oncotoxicity. In addition, a deeper understanding of H-1PV life cycle can help to identify biomarkers capable of predicting which patients would most likely benefit from virus treatment. To achieve this goal, previous members of the laboratory performed a druggable genome-wide siRNA library screening to identify putative modulators of H-1PV infection. Focusing on cellular factors potentially involved at the early steps of H-1PV infection, three top activators were identified: LAMC1, LGALS1 and AP2M1. (i) Laminin containing the γ1 chain, encoded by LAMC1, had previously been demonstrated to play a pivotal role at the level of binding and entry into cancer cells. Building on these results, I provide direct evidence that H-1PV binds to laminins through the sialic acid moieties present in these molecules. (ii) Galectin-1, encoded by LGALS1, is here shown to interact directly with H-1PV at the cell surface and promote the efficient virus internalisation into cancer cells. Knock-down/out of LGALS1 strongly decreases the ability of H-1PV to infect and kill cancer cells. These properties are rescued by the re-introduction of LGALS1 into cancer cells. The in silico analysis reveals that LGALS1 is overexpressed in glioblastoma and pancreatic carcinoma. In collaboration with Dr. Miletic (University of Bergen, Norway), we also show by immunohistochemistry analysis on 122 glioblastoma biopsies that galectin-1 protein levels vary across the different tumours with higher levels detected in glioblastoma than normal tissues, and higher in recurrent in comparison with primary glioblastoma tumours. We also found a direct correlation between LGALS1 transcript levels and H-1PV oncolytic activity in 59 cancer cell lines from different tumour origins. Together these results suggest that tumours with higher galectin-1 content may be a more suitable target for H-1PV. Strikingly, the addition of purified galectin-1 sensitises poorly susceptible glioma cell lines to H-1PV killing activity by rescuing virus cell entry. (iii) AP2μ1, encoded by AP2M1, is a subunit from the adaptor 2, a key regulator of clathrin-mediated endocytosis. Indeed, siRNA-mediated knockdown of AP2M1 or chemical inhibition of clathrin-mediated endocytosis strongly decreased H-1PV entry. Using electron and confocal microscopy, H-1PV particles were detected within clathrin-coated pits and vesicles, further corroborating that H-1PV uses clathrin-mediated endocytosis for cell x entry. In contrast, I observed no evidence of viral entry through caveolae-mediated endocytosis. I also show that H-1PV internalisation depends on dynamin activity, and that viral trafficking occurs from early to late endosomes, with low endosomal pH required for a successful infection. Based on the body of evidence gathered during this dissertation, I propose a model where H-1PV binds to the sialic acid present in laminins containing γ1 chains, and then galectin-1 promotes the efficient internalisation of virus particles through clathrin-mediated endocytosis. For the first time, this dissertation describes the cell entry pathways of oncolytic H-1PV.