This project uses genetic, biochemical and structural approaches to understand how autonomously replicating parvoviruses target and enter their host cells and establish infection. It will provide new information on the control of tissue tropis and on structural transitions in the virion that regulate successive steps in the infectious entry pathway of these ubiquitous viruses, particularly relating to an unexpected mode of genome exposure that could allow the capsid to fulfill novel intranuclear functions. The autonomously replicating parvoviruses are rugged and genetically simple single-stranded DNA viruses that are non-transforming because they are unable to activate resting cells to re-enter the cell cycle, and thus depend upon the host cell's regulation of cell cycle progression. In addition, many parvovirus species that infect rodents are inherently oncoselective, and preferentially infect transformed human cells, suggesting that they could be developed as therapeutic agents to target human tumors such as melanoma. The project pursues four approaches to understanding and exploiting such target specificity. 1. The targeting functions of the parvoviral shell will be identified by mapping the fortuitous tropism of parvovirus LuIII for human melanoma cells. This will involve making capsid chimeras with the non- melanotropic murine virus. Since LuIII is unlikely to be optimally melanotropic, we will use DNA shuffling to ask whether this property can be enhanced. 2. Differential qPCR analysis of sub-fractions of infected cells, expression of dominant-negative host genes and a novel in situ hybridization approach will be used to follow virions as they hijack specific intracellular trafficking pathways to gain access to the host nucleus. 3. Collaborative structural studies will be combined with genetic and biochemical approaches to investigate sequential conformational changes in the virion that facilitate ordered progress through its lifecycle. We have shown that genome uncoating is not a simple reversal of the packaging process, and will use these combined approaches to determine how the structurally symmetrical parvoviral capsid packages, retains and then releases its genome in the appropriate cellular compartments. 4. Optimized capsids will be used to package vectors designed to deliver heterologous genes into human and mouse melanoma cells. We will test the efficacy of a parvoviral vector packaged in an appropriately optimized capsid for targeting a transplantable murine melanoma in vivo. The vector expresses the B7-1 co-stimulatory molecule, and will potentially activate cytotoxic killer T-cells specific for melanoma tumor antigens, resulting in tumor eradication.

Public Health Relevance

Advanced melanomas are stubbornly resistant to current therapeutic strategies, exhibit an aggressive clinical course and commonly give rise to late metastases. Despite recent advances in understanding the molecular basis of this cancer, new therapy options are urgently needed. The proposed studies are directed toward the understanding, development and use of parvoviral vectors designed to enhance the immunogenicity of melanoma cells, while providing important insight into fundamental mechanisms governing the establishment of parvoviral infection.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01CA029303-33
Application #
8657786
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Daschner, Phillip J
Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
City
New Haven
State
CT
Country
United States
Zip Code
06510
Cotmore, Susan F; Agbandje-McKenna, Mavis; Chiorini, John A et al. (2014) The family Parvoviridae. Arch Virol 159:1239-47
Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter (2014) Complementation for an essential ancillary non-structural protein function across parvovirus genera. Virology 468-470:226-37
Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie et al. (2014) Profiling of glycan receptors for minute virus of mice in permissive cell lines towards understanding the mechanism of cell recognition. PLoS One 9:e86909
Vollmers, Ellen M; Tattersall, Peter (2013) Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses. Virology 446:37-48
Mattei, Lisa M; Cotmore, Susan F; Li, Lei et al. (2013) Toll-like receptor 9 in plasmacytoid dendritic cells fails to detect parvoviruses. J Virol 87:3605-8
Li, Lei; Cotmore, Susan F; Tattersall, Peter (2013) Parvoviral left-end hairpin ears are essential during infection for establishing a functional intranuclear transcription template and for efficient progeny genome encapsidation. J Virol 87:10501-14
Mattei, Lisa M; Cotmore, Susan F; Tattersall, Peter et al. (2013) Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts. Virology 442:20-7
Paglino, Justin C; Ozduman, Koray; van den Pol, Anthony N (2012) LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells. J Virol 86:7280-91
Cotmore, Susan F; Tattersall, Peter (2012) Mutations at the base of the icosahedral five-fold cylinders of minute virus of mice induce 3'-to-5' genome uncoating and critically impair entry functions. J Virol 86:69-80
Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F et al. (2011) Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM). Virology 410:375-84

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