Capsid structural tethers and cellular glycan network constrains drive parvovirus tropism

Parvoviruses are small non-enveloped DNA viruses that infect humans and animals, causing a variety of diseases. Parvoviruses may show a marked tropism for specific cell types, such as the Erythrovirus B19 for human erythroid progenitor cells, which is primarily determined by the interaction of their icosahedral capsid with cell surface receptors. The capsid of many parvoviruses, such as that of the Protoparvovirus Minute Virus of Mice (MVM), is composed of two structural proteins, VP1 and VP2, which share a common C-terminal domain but differ in their N-terminal domains. The VP1 protein contains a unique region (VP1u) that harbors important functions for virus entry and infection, such as phospholipase A2 activity and nuclear localization signals. The VP1u is hidden inside the capsid and becomes exposed upon receptor binding and subsequent endosomal acidification. Parvoviruses are promising candidates for anticancer and gene therapy applications, owing to their ability to infect dividing cells and to elicit immune responses. Parvoviruses such as adeno-associated viruses (AAVs), are commonly used as vectors for gene therapy. They can be modified to carry therapeutic genes into target cells, delivering the desired genetic material to treat genetic disorders. AAV-based gene therapies have shown promise in treating conditions like inherited retinal diseases, spinal muscular atrophy, and hemophilia. Another promising medical application is Oncolytic Virotherapy, where parvoviruses can be engineered to selectively infect and kill cancer cells while sparing normal physiological cells. These oncolytic parvoviruses are being tested as targeted treatment for various types of cancers. They replicate within cancer cells, leading to their destruction, and can also stimulate the immune system to mount an anti-tumor response. Clinical trials are underway to explore the potential of oncolytic parvoviruses in cancer treatment. However, the natural tropism and host range of parvoviruses limit their therapeutic potential and pose challenges for their delivery. Therefore, engineering the capsid to modify its receptor binding, intracellular trafficking, immune evasion and transgene expression, are desirable strategies to improve parvovirus vectors. Several approaches have been used to engineer the parvovirus capsid, such as peptide insertion, site-directed mutagenesis, DNA shuffling and directed evolution. These methods have resulted in novel capsids with altered tropism, enhanced transduction efficiency, reduced immunogenicity and increased gene expression. However, the molecular mechanisms underlying these changes are not fully understood and require further investigation.

In a new study published in the journal, Frontiers in Microbiology, Spanish scientists: Dr. Tania Calvo-López, Dr. Esther Grueso, Dr. Cristina Sánchez-Martínez and Dr. José M. Almendral from the Centro de Biología Molecular Severo Ochoa (CSIC-UAM) and the Departamento de Biología Molecular, Universidad Autónoma de Madrid, investigated on the mechanism how the cell type specific sialic acid receptors influence the intracellular traffic of the parvovirus virion to the endosome, and how this process affects viral tropism, thereby their ability to preferably infect certain cell types. Parvoviruses can display disparate tropism despite high genetic similarity, as exemplified by the the two MVMp and MVMi strains, although the molecular mechanisms underlying this feature is not fully understood. Using MVM as a model, the study hypothesizes that different sialic acid receptors on the cell surface mediate the entry, endocytosis and traffic of the parvovirus virion, and that this determines viral tropism and pathogenicity.

The research team attempted to retarget the oncolytic parvovirus minute virus of mice (MVMp strain) to the tumor vasculature by engineering its capsid with peptides competing with the vascular endothelial growth factor (VEGF). They generated chimeric virions by substituting residues at three functional domains of the capsid and tested their attachment and infection properties in different cell types. They found that substitutions in a “dimple” at the twofold axis of the capsid that allocates sialic acid (SIA) receptors altered viral tropism, as one chimeric virion showed reduced infection of murine fibroblasts and enhanced the ratio of infection of human glioblastoma cells as compared to MVMp. They also found that the chimeric virion was efficiently attached to α2-linked SIA moieties on the cell surface, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner.

The authors tracked the virus by fluorescence to follow its intracellular traffic and found that the chimeric virion was internalized by clathrin-mediated endocytosis but was restricted to reach early endosomes of human fibroblasts fully permissive to the MVMp parental virus. In contrast, in human glioblastoma cells, the chimeric virion was able to efficiently reach the early endosomes, where it underwent a drastic capsid structural transition that enabled its escape and infection. The capsid structural transition involved the cleavage of some VP2-Nt sequences and the exposure of the VP1u sequence that contains a phospholipase A2 domain essential for endosomal escape. Previous work from the same authors probed that VP2-Nt cleavage and VP1u exposure were necessary for infection. The authors also showed that neuraminidase treatment of restrictive fibroblasts removed inhibitory SIA moieties and enhanced the infection by the chimeric virion, correlating with its restored traffic to the endosome and the extent of VP2-Nt cleavage/VP1u exposure. Therefore, the authors concluded that the attachment to cell type-specific SIAs plays a major role in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range (See attached Figure reproduced with permission from Calvo-López et al., Front. Microb. 2023).

It is important to note that while parvoviruses hold promise for medical applications, further research and clinical trials are necessary to ensure their safety and efficacy. In conclusion, Dr. José M. Almendral and colleagues revealed the molecular mechanisms underlying the tropism and host range of parvoviruses, and the challenges and opportunities of capsid engineering for therapeutic applications. The authors showed that the attachment to cell type-specific sialic acid receptors determines the intracellular virus traffic to the endosome, where a capsid structural transition enables the infection. They also showed that modifying a dimple at the twofold axis of the capsid that allocates sialic acid receptors alters viral tropism and infection efficiency. This study illustrates the potential of parvovirus capsid engineering for anticancer and gene therapy, as well as the structural constraints and glycan network interactions that need to be considered.