Dept. of Microbiology & Immunology

University of Tennessee, Memphis

Lorraine Albritton, Ph.D.

Office: (901) 448-5521

Lab: (901) 448-6687

lalbritton@utmem.edu 

Office: 601F M.S.B.

Lab: 617 M.S.B.

Laboratory web page

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    Our goal is to understand the molecular mechanisms of virus entry and their role in viral pathogenesis. Viruses must begin their life cycles by binding to and penetrating the plasma membrane of a naive host cell. The host cell plasma membrane presents a barrier to this invasion that must be overcome to gain entry into the cell cytoplasm. Viral proteins enlist the aid of normal host cellular activities in accomplishing this formidable task. In retroviruses, the principal proteins involved in entry are the envelope surface and transmembrane proteins. Initially, the surface protein binds to the virus receptor, securely attaching the virion to the host cell. This interaction triggers changes in the structure of both the surface and transmembrane proteins, changes that activate their ability to induce membrane fusion. One of these changes is believed to be the flipping of a stretch of hydrophobic residues on the transmembrane protein outward into the host cell membrane. Intrusion of this "fusion peptide" in the plasma membrane is critical to the formation of a "fusion pore" through which the virus core passes to enter the host cell cytoplasm.

    We would like to identify the host cell proteins and functions subverted by the retrovirus during entry. We would also like to identify the portions of the virus surface and transmembrane proteins involved in receptor binding, especially the residues involved in triggering the extension of the fusion peptide. Using the knowledge gained from these studies, we are designing novel surface proteins for use on retroviral vectors for human gene therapy and addressing the mechanisms of viral pathogenesis with a goal of developing novel therapeutic strategies. Most of our work has focused on studying the murine ecotropic leukemia viruses as a model retrovirus system. More recently, we began working with the amphotropic leukemia virus and Gibbon ape leukemia virus. Although the surface proteins of these retroviruses are very similar, each interacts with a unique cellular receptor protein. We would then like to apply what we learn about the entry of these simple retroviruses toward understanding the entry of the more complex human immunodeficiency virus type 1, whose surface protein interacts with two receptors during entry

The role of the receptor and other host cell factors.   Our attempts to identify the host cell proteins and functions used by the virus during entry have led to novel insights into virus entry. The receptor for the ecotropic viruses normally functions as the principal transporter of cationic amino acids in the cell. It traverses the plasma membrane fourteen times, with residues in its third extracellular loop providing the attachment site for the viral surface protein. The sequence of this loop varies widely between species. Ecotropic retrovirus attaches to the versions found in mouse and rat cells but not to other mammalian cells, explaining why only rodent cells

are susceptible to these viruses. In polarized kidney cells, receptor/transporters show the remarkably regular pattern of distribution in the basolateral plasma membrane seen in the three-dimensional reconstruction of serial confocal images shown in the figure. Its presence in the basolateral membrane results from an association with the cellular cytoskeleton. As seen in the three-paneled figure, the receptor (green image, left panel) colocalizes with a cytoskeletal protein (blue image, middle panel); regions of colocalization appear aqua in the overlay of these kidney cells (right panel). Using co-immunoprecipitation, we recently showed that the association between two cytoskeletal proteins and the receptor/transporter is very strong. Surprisingly, disruption of the cellular cytoskeleton or dissociation of the receptor from the cytoskeletal proteins inhibits ecotropic virus entry but does not affect the ability of the receptor to transport amino acids. We would like to determine exactly what step in virus entry is inhibited by loss of receptor binding to the cytoskeletal protein. Since attachment of ecotropic retrovirus was not reduced, a post-binding step is most likely affected. We are also attempting to identify the binding site on the receptor for the cytoskeletal proteins. These studies led to an explanation for the unusual neuropathogenesis caused by an ecotropic MLV TR1.3 in which hemorrhaging initiates in brain capillary endothelial cells (BCEC) but is not found in the capillary endothelium of other tissues. Because of their central role in forming the blood-brain barrier, BCEC are unique among the endothelia in forming tight junctions that divide their plasma membrane into discreet apical and basolateral membrane. Its localization to the basolateral membrane concentrates receptor, promoting the fusion between adjacent infected BCEC that results in brain hemorrhaging.

The role of virus envelope proteins.  Our attempts at identifying residues in the virus envelope protein involved in receptor binding and in triggering extension of the fusion peptide have also led to new insights into virus entry. We identified a new residue, tryptophan 142, as essential for virus binding and infection. Using a genetic approach, we determined that it interacts directly with a critical tyrosine at position 235 of the receptor. Along with three other hydrophobic amino acids, this tryptophan residue

  forms one side of a shallow pocket or patch on the surface protein that is a prime candidate for the complete binding site for tyrosine 235 on the receptor. Surprisingly, three charged residues previously identified as critical to receptor binding are over 30 Å from the hydrophobic patch (left image). Since the surface and transmembrane proteins form trimers on virus particles, we asked what was the relative positions of the critical residues in an envelope trimer. In the current model for the trimer, the residues are also over 30 Å apart. We generated an alternative trimer model in which the hydrophobic patch on one molecule of surface protein lies on the outer perimeter of the trimer, about 11 Å from the charged residues on the adjacent molecule (right image). Both models place the hydrophobic patch at the interface between adjacent molecules of surface protein, but only the alternative model places the charged residues there as well. This brings up the intriguing possibility that the interaction between a single receptor molecule and the hydrophobic patch and charged residues spanning an intermonomer junction might trigger conformational changes in two envelope protein molecules that induce changes in the third molecule of the trimer.

    Using a genetic selection approach, we showed that there is at least one domain in the surface protein that is involved in fusion; the fusion functions do not reside exclusively in the transmembrane protein as was previously thought. We isolated an infectious retrovirus variant containing three changes in the surface protein -- histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. The histidine 8 change alone resulted in almost complete loss of infection. Virions were proficient at receptor binding but failed to induce membrane fusion, indicating that the histidine at position 8 plays an essential role in penetration of the host cell membrane. In contrast, when all three changes were present virus was highly infectious and induced membrane fusion, indicating that the other two changes suppress the fusion defect caused by loss of histidine 8 function.

Innovative strategies for gene therapy.  The ability to delivery genes or drugs specifically to one type of cell would provide a very potent method of treatment for patients with inherited human diseases or highly aggressive, metastatic cancers. Viruses and liposomes are the most promising vehicles for delivery of genes and drugs but there is no effective method to target their delivery to the desired cell type in the patient. One method for targeting is to modify the surface protein by inserting other proteins into them. For example, an antibody that recognizes human lung cancer cells but not normal cells is inserted into a retrovirus surface protein to target attachment of the virus and delivery of the gene or drug specifically to the lung cancer cells. However, standard modified surface proteins only attach virus to the specific target cell; they fail to deliver the gene or drug inside the cell. This failure results from two problems. First, they are unable to catalyze the fusion of the viral and host cell membranes. Second, they are very unstable. They fall off or shed from virions when subjected to mechanical stress such as storage at low temperatures or vascular flow when injected in a patient. We have designed a novel method of constructing modified surface proteins to solve these problems. Specific residues in the surface protein are changed before inserting the targeting protein. The changes preserve the ability to catalyze membrane fusion and stabilize the modified protein so that it does not shed.

Selected Publications

Zavorotinskaya, T., and L. M. Albritton. Identification of a hydrophobic pocket in ecotropic murine leukemia virus envelope protein as the putative binding site for a critical tryosine residue on the cellular receptor. J. Virol. (In Press, Volume 73, December, 1999).

Chung, M., K. Kizhatil, L. M. Albritton, and G. N. Gaulton. Induction of syncytia by neuropathogenic murine leukemia viruses depends on receptor density, host cell determinants, and the intrinsic fusion potential of envelope protein. J. Virol. 73: 9377-9385 (1999).

Zavorotinskaya, T., and L. M. Albritton. Failure to cleave murine leukemia virus envelope protein does not preclude its incorporation in virions and productive virus-receptor interaction. J. Virol. 73:5621-5629 (1999).

Zavorotinskaya, T., and L. M. Albritton. Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein. J. Virol. 73:5034-542 (1999).

Kizhatil, K., and L. M. Albritton. Requirements for different components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion. J. Virol. 71:7145-7156 (1997).

Zavorotinskaya, T., and L. M. Albritton. Novel retroviral vectors containing chimeric envelope proteins that give high efficiency cell type specific gene transfer. (In Preparation for submission to Gene Therapy, 1999)

Kizhatil, K. and L. M. Albritton. System y+, the cationic amino acid transporter that serves as the ecotropic retrovirus receptor, associates with the cytoskeleton and localizes to the basolateral membrane in epithelial cells. (In Preparation for submission to J. Cell Sc., 1999)

Kizhatil, K. and L. M. Albritton. Ankrin-mediated association of cationic amino acid transporter to the cortical cytoskeleton is critical to its function as the receptor for ecotropic murine leukemia viruses. (In Preparation for submission to J. Biol. Chem., 1999).