Lorraine M. Albritton, Ph.D.
858 Madison Ave.
601F Molecular Science Building
Memphis, TN 38163
Two major interests drive my research programs – identifying host barriers to retroviral entry and infection, and developing viral vectors for gene therapy of cancer and human genetic diseases. As a model system we study infection of the ecotropic murine leukemia viruses (MLV) via the natural virus receptor, focusing on identifying important properties of receptors and cellular factors that influence entry. The membrane fusion function of ecotropic Env is activated by receptor binding. An additional role for a cellular protease has been suggested by others but no protease had yet been identified. We recently determined that a cellular protease, cathepsin B, is involved in the membrane fusion step that occurs after activation by the receptor. Inhibition of cathepsin B reduces infection in a dose-dependent manner and murine embryonic fibroblasts from cathepsin B knock-out mice show greatly reduced infection. We are currently investigating the mechanism by which this cathepsin influences membrane fusion. Future studies will test the hypothesis that specific cleavage of the viral Env is required for the completion of conformation changes initiated by receptor binding. We have identified two putative cleavage sites in the surface subunit of ecotropic Env and are investigating their relevance to virus entry.
Figure 1. Left panels, Cathepsin B cleaves the surface subunit (SU) of Env into discrete fragments but does not digest the transmembrane subunit (TM) or the viral capsid protein (CA). Cleavage is specific to cathepsin B since addition of CA 074 Me inhibits digestion. Right panel, Hypothetical model for a tole of cathepsin B cleavage of SU in potentiating fusion of the viral membrane (bottom) with the host cell membrane (upper).
In other studies we are investigating the influence of membrane microdomains in which the receptor resides and the role of cellular proteases. The retroviral receptor clusters primarily in detergent resistant membranes or lipid rafts on murine NIH 3T3 fibroblasts and when expressed on human and canine polarized kidney epithelial cells. This cellular location of the receptor, the sensitivity of infection to cholesterol depletion and the lack of sensitivity to dominant negative clathrin suggested that ecotropic MLV enter host cells via caveolae-mediated endocytosis. Entry does not occur exclusively through this pathway, since susceptible rat XC cells lack caveolae. However, the initial kinetics of virus-cell membrane fusion are substantially slower in XC cells compared to NIH 3T3 cells. We are currently investigating the hypothesis that localization of the receptor to caveolae influences infection by providing a high local density of receptors and rapid endocytosis.
Our efforts at developing gene therapy vectors are also focused on retroviral and lentiviral vectors. My identification and molecular cloning of the cellular receptor for ecotropic MLV as a post-doctoral fellow led to discovery of its normal cellular function as the principal transporter of cationic amino acids, as well as the identity of important residues in the putative virus binding site. Later efforts discovered functional domains in the retroviral envelope protein (Env), including evidence that an interaction between aspartic acid 84 (D84) on Env and lysine 234 on the receptor makes a key contribution to virus binding. Identification of D84 as a central residue in the receptor binding sequence (RBS) of Env enabled us to reduce to practice a novel concept for attaining targeted entry: replacement of the RBS with the sequences of a target cell-specific peptide ligand. The prototype construct contains the sequence of the peptide hormone somatostatin replacing the RBS. Retroviral vectors coated with this modified Env infect cells expressing somatostatin receptor-5 at titers of over 105 transducing units/ml, a level that approaches 50% the level of infection of the wildtype virus using the natural retroviral receptor. Currently, we are determining the effectiveness of this targeted virus in animals and constructing new Env modified by other peptide ligand sequences to expand the number of different cell types that can be targeted.
Figure 2. A new concept in targeting the entry step of infection by retroviral vectors. Left panel, The natural receptor binding sequence (RBS, residues shown in light and dark gray) are replaced by the peptide hormone somatostatin to construct the modified Env referred to as Sst-RBS. Right panels, Sst-RBS virus infects approximately half of the target somatotstatin receptor positive cells to deliver the lacZ marker transgene, whereas, the parent ecotropic virus (MoMLV) is unable to infect these human cells.
- Ph.D., University of Tennessee-Oak Ridge National Laboratories, Bicmedical Sciences, 1986
- B.S., Northeast Louisiana University, Physics/Mathematics, 1972