Oncolytic H-1 parvovirus binds to sialic acid on laminins for cell attachment and entry

Abstract

H-1 parvovirus (H-1PV) is a promising anticancer therapy. However, in-depth understanding of its life cycle, including the host cell factors needed for infectivity and oncolysis, is lacking. This understanding may guide the rational design of combination strategies, aid development of more effective viruses, and help identify biomarkers of susceptibility to H-1PV treatment. To identify the host cell factors involved, we carry out siRNA library screening using a druggable genome library. We identify one crucial modulator of H-1PV infection: laminin γ1 (LAMC1). Using loss- and gain-of-function studies, competition experiments, and ELISA, we validate LAMC1 and laminin family members as being essential to H-1PV cell attachment and entry. H-1PV binding to laminins is dependent on their sialic acid moieties and is inhibited by heparin. We show that laminins are differentially expressed in various tumour entities, including glioblastoma. We confirm the expression pattern of laminin γ1 in glioblastoma biopsies by immunohistochemistry. We also provide evidence of a direct correlation between LAMC1 expression levels and H-1PV oncolytic activity in 59 cancer cell lines and in 3D organotypic spheroid cultures with different sensitivities to H-1PV infection. These results support the idea that tumours with elevated levels of γ1 containing laminins are more susceptible to H-1PV-based therapies.

Introduction

Oncolytic viruses (OVs) selectively replicate in and destroy tumour cells without harming normal healthy tissues. They act in a multimodal fashion by inducing lysis of the cells and anticancer immunity^1,2,3,4. More than 40 OVs from at least nine virus families are currently being tested against various malignancies in early- or late-phase clinical trials. In addition, the engineered herpes simplex virus encoding granulocyte–macrophage colony-stimulating factor (talimogene laherparepvec, Imlygic^TM) was granted approval in 2015 both in the USA and in Europe for use against malignant metastatic melanoma⁵. There is optimism that other OVs may be approved in the near future for the treatment of other cancers⁶. However, OVs as a standalone therapy have rarely been reported to induce the complete regression of tumours. Major efforts to improve the clinical outcome of OV treatments are directed towards the search for anticancer modalities that synergise with OVs to kill cancer cells without toxic side-effects. One promising avenue is the combination of OVs with other forms of immunotherapy (e.g., checkpoint blockade)^{3,7,8,9,10,11}. Another is to identify patients with tumours whose genetic characteristics are favourable to the virus life cycle and who are thus most likely to benefit from OV treatment. This could lead to the design of ‘smart’ clinical trials that reduce clinical costs and approval times. A better understanding of the OV life cycle and the identification of host cellular determinants that contribute to virus infection are crucial to guide both the rational design of combination treatments and the identification of biomarkers that could be used for patient stratification.

Rat protoparvovirus H-1PV is a clinically relevant OV whose anticancer potential has been demonstrated at the preclinical level in a number of in vitro cell systems and animal models^12,13. The first phase I/IIa trial in patients with recurrent glioblastoma (GBM) showed that H-1PV treatment as a standalone therapy is safe, well tolerated and associated with first evidence of efficacy, including: (i) ability to cross the blood–brain (tumour) barrier after intravenous delivery; (ii) widespread intratumoural distribution and expression; (iii) immunoconversion of tumour microenvironment; and (iv) extended median progression-free/overall survival in comparison with historical controls^14,15. A second clinical trial in patients with pancreatic carcinomas is now in its evaluation phase¹⁶.

H-1PV is a small, non-enveloped, single-stranded DNA virus. Its 5.1 kb genome is organised into the NS and VP gene units, whose expression is regulated by the P4 and P38 promoters, respectively. The NS gene unit encodes the NS1, NS2 and NS3 proteins, whereas the VP gene unit encodes the VP1 and VP2 capsid proteins and the SAT non-structural protein. NS1 is a multifunctional protein that regulates virus DNA replication and gene transcription, and it is the major effector of H-1PV oncolysis^13,17,18.

H-1PV’s DNA replication and gene expression depend on host cell factors. For instance, replication relies on the E2 family of transcription factors, cAMP response element binding protein, activating transcription factors and cyclin A^12,17, which are normally overexpressed in fast-proliferating cancer cells and are therefore important determinants of virus oncotropism. In addition, NS1 activity is modulated by post-translational modifications such as phosphorylation and acetylation^17,19,20.

Many of the host cell factors that have a role in the H-1PV life cycle are yet to be identified. It is largely unknown why some cancer cell lines are highly susceptible to H-1PV infection, whereas others derived from the same tumour entity are less sensitive or even completely refractory. Differences in permissiveness are thought to be regulated by cell–host interactions. Non-permissive tumour cells may lack important factors needed for different stages of the virus life cycle, e.g., virus cell attachment and entry.

The extracellular matrix (ECM) is a three-dimensional interlocking mesh of extracellular macromolecules, encompassing laminins, fibronectin, collagen, elastin, heparan sulphate, chondroitin sulphate, keratan sulphate and hyaluronic acid, which provide structural and biochemical support to the surrounding cells. The ECM often represents a barrier to a virus on its way into the host cell. Some viruses (and other microorganisms) have developed strategies to bind to specific ECM components^21,22. This leads to the accumulation of a large quantity of virus particles at the host cell surface, thereby facilitating efficient engagement with host cell transmembrane receptor(s) or receptor complex(es). The attachment to ECM proteins is therefore an essential event that primes the virus to recognise cell surface molecules, initiates virus infection, and hence represents a key determinant of virus tropism and infectivity. Although attachment factors and functional receptors have been identified for some of the members of the Parvoviridae family (e.g., transferrin receptor for the canine and feline parvoviruses; several receptors and attachment factors for a number of adeno-associated virus serotypes^23,24,25,26), the attachment receptor (complex) involved in H-1PV ECM and cell membrane recognition is still unknown. An essential component needed for H-1PV cell attachment is sialic acid (SA); two residues on the viral capsid are involved in SA interaction^27,28. This property is shared with other protoparvoviruses, such as the minute virus of mice²⁹ and porcine parvovirus³⁰. However, it remains to be characterised whether SA alone is sufficient to mediate H-1PV cell membrane recognition and entry or if the virus requires additional interaction(s) with other proteins present at the cell surface (sialylated or not). Recently, we showed that after binding to the cell membrane, H-1PV enters cells via clathrin-mediated endocytosis³¹, a property shared with other protoparvoviruses³².

In this study, we searched for novel host cell factors involved in the H-1PV life cycle with a primary focus on the sialylated proteins required for H-1PV attachment at the cellular membrane. We identified laminins as key mediators of virus cell attachment and entry.