The use of both genetic and molecular approaches to the study of virus-host interactions in vivo provides us with insight into the processes that determine the susceptibility and resistance of individuals to viral infection and virus-induced cancer (approximately 20% of human cancers). Our interests lie in determining why viruses infect specific hosts and how in turn, host genes confer resistance to this infection. Our lab studies 2 different types of viruses, retroviruses like mouse mammary tumor virus (MMTV) and murine leukemia virus (MLV) which cause cancer in mice, and the new world arenaviruses, which cause hemorrhagic fever in humans.
The genetics of susceptibility is easily studied with naturally-occurring pathogens in inbred and genetically-manipulated mice. MMTV is an endemic oncogenic retrovirus that has been an infectious agent in mice for > 20 million years, while MLV has been in mice ~ 3 million years. Infectious MMTV is passed from mothers to offspring through milk and first spreads in lymphoid cells before infecting mammary epithelial cells; MLV is probably also milk-transmitted. These viruses thus serve as models for the human milk-borne retroviruses HIV-1 and HTLV1. MMTV causes breast cancer and MLV causes lymphomas when the viral genome inserts next to cellular oncogenes by activating their expression. Our studies focus on understanding the mechanisms that determine susceptibility to MMTV infection and virus-induced mammary tumors and we have identified a number of genes and mechanisms that confer resistance to infection by MMTV and MLV.
Genes of the immune system play a major role in susceptibility to infection, and one gene which we recently discovered is involved in the control of MMTV and MLV infection is Apobec3. All mammals encode Apobec3 genes which play a role in intrinsic cellular immunity to a number of viruses, including human immunodeficiency virus type 1. APOBEC3 proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytosine on the negative strand DNA intermediates. We found that mouse APOBEC3 protein is packaged into MMTV and MLV particles in vitro and dramatically reduces viral titers. Most importantly, APOBEC3 knockout mice are more susceptible to MMTV and MLV infection compared to their wild type littermates. These findings indicate that the APOBEC3 provides protection to mice against retroviral infection and represent the first demonstration that it functions during retroviral infection in vivo. We are currently studying how genetic variation in the mouse APOBEC3 genes affects their ability to inhibit infection and whether APOBEC3 can be used as an anti-retroviral therapeutic target.
We have recently extended our studies to new world arenaviruses like Junín virus. These viruses are endemic in new world rodents in South America and are spread to humans via aerosolization. Interestingly, both Junín virus and MMTV use transferrin receptor 1 (TfR1) for entry. We are currently studying how MMTV and Junín virus use TfR1 to enter cells and how the iron metabolic pathway intersects with infection by these viruses. In addition, we are studying different host genes that confer resistance or susceptibility Junín virus, and whether polymorphisms in these genes in humans alter infection. These studies will help us identify host molecules involved in cell- and disease-tropism and help us to develop new anti-viral therapies.
"ES cell line XN450 with the gene trap vector in intron 4 of the mouse APOBEC3 (mA3) gene was obtained from BayGenomics Gene Trap Project (http://baygenomiccs.ucsf.edu) and injected into the blastocysts by Transgenic Knockout Shared Resource at UC Davis Mouse Biology Program, Davis, CA, to create chimeric mice. Chimeric mice were backcrossed to C57BL/6J mice (Jackson Laboratory, Jackson, ME) for nine generations and heterozygotes were crossed with each other to generate A3 -/- mice. Heterozygous mice were generated by crossing A3 -/- to C57BL/6J mice."
The transgene in this strain contains an entire copy of a genetically engineered MMTV provirus in which the 3' half (including 3' LTR, env, and sag genes) was derived from C3H exogenous virus and the 5' half (including 5' LTR, gag, and pol genes) came from the Mtv-1 endogenous locus in C3H/HeN mice.
The plasmid hybrid MMTV (a gift from G.M. Shackleford, University of California, Los Angeles) was linearized, then microinjected into the pronucleus of fertilized one-cell eggs recovered from the oviducts of females that had mated with males of the same strain the previous night. Microinjected C3H/HeN MTV- fertilized zygotes were implanted into SW pseudopregnant foster mothers. The strain was carried by breeding heterozygous transgenic males to C3H/HeN females.
This mouse line has wild type mA3.