Description of Research Expertise

Research interests
Genetic approaches to the study of host-virus interactions, genetics of susceptibility to virus infection, immune response to virus infection.

Keywords: retrovirus, transferrin receptor, superantigen, toll receptor, mouse genetics, breast cancer.

Research Details
All animals, including humans, show differential susceptibility to infection with viruses. Study of the genetics of susceptibility or resistance to specific pathogens is most easily studied in inbred mice. We are using mouse mammary tumor virus (MMTV) to study virus/host interactions. MMTV is a retrovirus that causes breast cancer in mice by inserting next to cellular oncogenes and activating their expression. MMTV is both inherited through the germ line and passed as an infectious virus from mothers to offspring through milk. Our studies have focused on understanding the mechanisms that determine genetic susceptibility to MMTV-induced mammary tumors.

The ultimate target tissue for MMTV is the mammary gland. However, MMTV also utilizes the cells of the immune system in its journey from milk to the mammary gland of offspring, in part because it encodes a superantigen gene that causes T cell stimulation. An ongoing project is to determine the type of lymphocytes that deliver MMTV to the mammary cell and how they accomplish this transfer. The role of cell-cell interaction in the transfer of MMTV from lymphocytes to mammary epithelial cells and how infected lymphocytes traffic to the mammary gland are being studied in vivo; specifically, we are determining whether homing to mucosal lymphoid tissue plays a role in virus dissemination.

A better understanding of how MMTV infects these different cell types and reaches its target tissue is dependent on identifying the cell surface receptor(s) that the virus uses to infect cells. We recently found mouse transferrin receptor 1 (TrfR1) is the MMTV cell entry receptor. TrfR1 is the major means by which most cells take up iron and it binds transferrin at the cell surface, traffics to the early acidic endosome where it releases iron, and then recycles back to the cell surface and releases transferrin. The identification of a protein that traffics to the acidic endosome upon ligand binding is important because MMTV infection is also dependent on acidified endosomes. We are currently studying the physical interaction between the MMTV envelope protein and TrfR1 and their trafficking in cells. We are also determining why the human TrfR1 does not function as a virus receptor. This is important to determine because several labs have recently implicated MMTV-like elements in human breast cancer.

A final area of interest in the lab is how retroviruses infect their initial targets in vivo. Although most retroviruses require activated cells as their targets for infection, it is not known how this is achieved in vivo. A candidate protein for the activation of B cells by mouse mammary tumor virus (MMTV) is the toll-like receptor 4 (TLR4), a component of the innate immune system. C3H/HeN mice are resistant to ligands that bind TLR4, like lipopolysaccaride from gram negative bacteria, because of a dominant negative mutation in their TLR4 gene. They have also been reported to be less susceptible to MMTV-induced mammary tumors. We recently showed that B and dendritic cells from C3H/HeN mice were activated by MMTV in vivo, in contrast to those from C3H/HeJ mice. We also determined that the MMTV envelope protein bound to TLR4. More recently, we have discovered that MMTV also works through an additional toll-like receptor, TLR2. Our current focus is on understanding the outcome on viral infection of signaling through TLR pathways.

Rotation Projects
1. Introduction of the human transferrin receptor coding region into mouse embryonic stem cells. Mice with targeted deletion of the transferrin receptor are embryonic lethal. We would like to try to rescue these mice with the human transferrin receptor. The project is to insert the human transferrin receptor cDNA into a retroviral vector and then infect mouse embryonic stem cells and select for cells that contain the receptor. Techniques: subcloning, tissue culture, DNA transfection, production of pseudoviruses, FACS.

2. Analysis of novel oncogenes in MMTV-induced mammary tumors. We have recently carried out high through-put analysis of MMTV integration sites in MMTV-induced tumors and identified a number of potential novel oncogenes. The rotation project would be to determine whether expression of these genes is up-regulated or down-regulated in tumors relative to normal mammary tissue. Techniques: DNA and RNA isolation; RT-PCR; real-time PCR.

Lab personnel:
Cecilia Courreges, Ph.D. – Laboratory Manager
Chioma Okeoma, Ph.D – Postdoc
Enxiu Wang, Ph.D- Postdoc
Dionne Robinson, B.S. – Postbaccalaureate Student
Stacy Hultine, M.S. – Research Specialist
Qihua Ying, M.S. – Research Specialist