Retargeting of Rat Parvovirus H-1PV to Cancer Cells through Genetic Engineering of the Viral Capsid

Abstract

The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds ␣ v ␤ 3 and ␣ v ␤ 5 integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing ␣ v ␤ 5 integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

“…Virus variants with an enhanced ability to replicate lytically and spread in tumor cell populations are being isolated through natural selection or site-directed mutagenesis (55,56). To minimize PV sequestration by normal tissues, PV capsids are first detargeted and then retargeted to cancer cells through the display of specific peptide ligands (57). The ability of PVs to act as adjuvants to boost the host antitumor response can also be increased by arming them with immunostimulatory molecular patterns (43).…”

Characteristics of Oncolytic Vesicular Stomatitis Virus Displaying Tumor-Targeting Ligands

“…The RGD motif has been displayed previously on the surfaces of many viruses (measles virus [45], parvovirus [27], adenovirus [46][47][48], and adeno-associated virus [49]) with the goal of enhancing their oncolytic specificities and/or transduction efficiencies. In selecting potential insertion sites for the VSV G protein, we relied heavily on the crystal structure of the protein and sequence comparisons with the G proteins of various related vesiculoviruses.…”

“…RGD has been extensively studied in targeting and killing tumor cells (26). Early studies demonstrated that displaying the RGD peptide on the surface of oncolytic parvoviruses, adenoviruses, or measles viruses enhanced their interaction with tumor vasculature and/or tumor cells (19,27,28). cRGD also has been used to label gold nanoparticles to target tumor cells for destruction or imaging (29,30).…”

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

“…For other parvoviruses, SIA interactions are reported for bovine parvovirus (40), canine parvovirus (CPV), feline panleukopenia virus (FPV) (41), H-1 parvovirus (H-1PV) (42), and porcine parvovirus (43) in addition to MVM (38). The amino acids utilized for binding SIA are unknown for bovine and porcine parvovirus, but like MVM, CPV, FPV, and H-1PV bind SIA in or close to the 2-fold depression.…”

“…The amino acids utilized for binding SIA are unknown for bovine and porcine parvovirus, but like MVM, CPV, FPV, and H-1PV bind SIA in or close to the 2-fold depression. For MVM and H-1PV, residues in the vicinity of the SIA binding site also play a role in tissue tropism determination (38,42), and for MVM, they also dictated virulence and pathogenicity (38). However, in CPV and FPV, the SIA interaction either alone or in the context of their receptor transferrin does not seem to be important for infection or host range (44).…”