Our research efforts are directed at understanding the molecular mechanisms by which mammalian parvoviruses target particular cell types, express their genes, take over their host cells and replicate their own DNA. Eukaryotic and prokaryotic expression systems, coupled with directed mutagenesis, are currently being used to separate the various functions of the complex, multi-functional parvoviral gene products, in order to understand how the virus subverts the macromolecular metabolism of its target host cell to its own ends. We are currently applying this knowledge to the construction and validation of vectors designed to target tumor cells, and deliver immunomodulatory proteins directly within the tumor microenvironment, initially as a therapeutic strategy against melanoma.
Specialized Terms: Biochemistry; Genetics; Virology; DNA replication; Oncolytic virus; Vaccines; Vectors; Melanoma
Manipulating the oncoselectivity of parvoviruses in human tumor models
Most of the rodent parvoviruses will bind to and enter human cells with high efficiency, but fail to initiate gene expression, replicate their genomes, generate progeny or spread through the culture, unless the host cell is neoplastically transformed. As a consequence, these viruses are promising candidates as oncolytic agents for cancer therapy, particularly in situations where other treatments have proven ineffective. Our current efforts are directed toward understanding, at the molecular level, why cellular changes that accompany oncogenic transformation promote viral growth, and how we can use this knowledge to further improve the efficacy of the virus in tumor eradication. Since tumorigenesis normally involves loss of genomic integrity, tumor cells carry many mutations that are secondary to those causing the transformed phenotype. To avoid studying or selecting for viral traits that represent adaptations to such “collateral” transformed cell properties, we are using host cells that have been transformed in a stepwise fashion with activated oncogenes and/or tumor suppressor knock-downs. Currently we are exploring the contribution of the viral capsid and initiating promoter to the discrimination between normal and transformed cells, using stepwise transformed human fibroblasts and melanocytes, the latter being a model for malignant melanoma. These studies are directing strategies for selecting more oncotropic versions of these critical oncoselective elements, using gene shuffling and degenerate promoter library approaches.
Induction of immunogenic cell death by oncosuppressive parvoviruses
Many of the autonomously replicating rodent parvoviruses can enter human cells, generate progeny and spread through the culture only if the host cell is neoplastically transformed, making these viruses promising candidates as oncolytic agents. Parvoviral induction of complete tumor regression has been achieved in several syngeneic transplantable tumor models in immunocompetent rodent hosts, and often results in immunization of the animal against subsequent transplantation of cells of the same tumor, even at high input numbers, suggesting that some aspect of parvovirus infection elicits a strong anti-tumor immune response. This project utilizes a mouse melanoma model system to explore whether parvovirus-induced cell death proceeds via an immunogenic, rather than tolerogenic, pathway, by examining the expression of phagocytic engulfment signals on the infected cell surface, coupled with the secretion of soluble damage-associated molecular pattern (DAMP) molecules, such as HMGB1 and Hsp72.
How parvoviruses enter their host cell and traffic to the nucleus
Parvoviruses do not have a lipid envelope, and so cannot deliver their virions into the host cell by fusing with its plasma or endosomal membranes. These viruses have developed an alternative strategy to breach their host cell's outer membrane and gain entry into the cytoplasm. We have shown that the compact, icosahedral virion of the murine parvovirus Minute Virus of Mice, MVM deploys a lipolytic enzyme, phospholipase A2 (PLA2) that is expressed at the N-terminus of the minor coat protein, VP1. This region of VP1 is normally sequestered within the viral shell, but is extruded during the entry process as a capsid-tethered domain, via an 8Å pore that extends through the prominent 5-fold cylinder. [Figure] In addition to the PLA2 domain, the extruded VP1 N-terminus also displays a number of small protein interaction domains predicted to engage both ubiquitin ligases of the NEDD4 family, involved in endocytosis and vesicle trafficking, and nuclear transport proteins of the alpha-importin family. The sequential conformational shifts within the particle that allow these transitions to occur as the virion transits its entry pathway, exposing first its VP2 N-termini, then its VP1 N-termini and ultimately its DNA are being analyzed using X-ray crystallography and asymmetric cryo-electron microscopy, in collaborations with Drs. Susan Hafenstein at Hershey Medical School and Mavis Agbandje-McKenna at the University of Florida, Gainesville. Finally, we are using reverse genetics combined with differential real-time PCR, sub-cellular fractionation and in situ imaging techniques, such as Proximity Ligation, to explore the roles of the VP1 N-terminal domain in the trans-cytosolic trafficking and nuclear import of MVM virions.
Characterizing the unique chromatin assembled during parvoviral DNA replication
During the S-phase following infection, autonomous parvoviruses inveigle host cells to replicate the linear, single-stranded viral DNA chromosome, instead of the cellular genome. Part of the virus’ replication strategy involves the elaboration of a unique form of chromatin, which ChIP analysis suggests incorporates both cellular histones and many copies of NS1, the major viral non-structural protein. This project explores the replication of an otherwise wildtype viral genome rendered artificially devoid of NS1 binding sites throughout its entire NS1 gene, capsid gene and/or 3’ untranslated region. 2D gel electrophoresis and nuclease protection assays will be used to look for stalling or pausing of replication forks through the capsid region, and to characterize packaging intermediates generated by mutant, compared to wildtype, virus.
Dependovirus; Biochemistry; Eukaryotic Cells; Genetics; Melanoma, Experimental; Mice, Mutant Strains; Medical Laboratory Science; Parvovirus; Erythrovirus; Oncolytic Virotherapy; Oncolytic Viruses; Mice; Bocavirus; Viral Structures; Immunomodulation