The first step in the retroviral life cycle involves binding of viral envelope (Env) glycoproteins to specific host cell surface receptors, and post-binding events leading to virus-cell membrane fusion and viral entry.
The aim of the proposed research is to further our understanding of the early events in viral infection by characterizing the subgroup A-avian leukosis virus (ALSV-A) receptor, which is a protein related to the low density lipoprotein receptor. The first goal of the proposed research is to identify the functional domain(s) on the ALSV-A receptor. To facilitate these experiments we have constructed synthetic receptor genes which will be used for a comprehensive site-directed mutagenesis protocol to identify the amino acid residues that are critical for viral infectivity. A related goal is to identify and characterize the different isoforms of the receptor that are generated from alternatively spliced mRNA transcripts in chicken cells and to test the ability of the different receptor products to mediate viral infection. Another major focus of this research is to use a secreted version of the receptor, comprising the extracellular protein domains fused to an antibody-epitope tag and six histidine residues to study virus-receptor interactions. The secreted receptor has been shown to bind specifically to ALSV-A and block infection. This reagent will be used to determine the binding affinity of the interaction with subgroup A viral Env proteins. It will also be used to select variants of ALSV-A that cannot be inhibited by this protein, and the mutations in Env that are responsible for this altered phenotype will be identified. The secreted receptor will also be tested for its ability to induce structural changes in Env that are predicted to occur prior to membrane fusion. These alterations will be detected by monitoring changes in the sensitivity of Env to proteolytic digestion, and using antibodies raised to amino acid sequences which flank the hydrophobic fusion domain of the ALSV TM Env protein. To test directly whether the receptor protein can by itself drive membrane fusion, this protein will be overexpressed and purified from tissue culture cells and reconstituted into liposomes. The proteoliposomes will be tested for fusion with octadecylrhodamine-labeled ALSV-A and membrane fusion measured by a fluorescence dequenching assay. Finally, the proposed studies aim to understand the mechanism of ALSV-A Env-induced receptor downregulation which occurs after viral infection.Together, these studies will yield fundamental insights into the pH-independent mechanism of retroviral entry into cells, and in addition might yield some information about the post-binding events required for viral entry. This, in turn, could lead to the design of novel therapeutic strategies aimed at preventing these first steps of viral infection.
| Zingler, K; Young, J A (1996) Residue Trp-48 of Tva is critical for viral entry but not for high-affinity binding to the SU glycoprotein of subgroup A avian leukosis and sarcoma viruses. J Virol 70:7510-6 |
| Zingler, K; Belanger, C a; Peters, R et al. (1995) Identification and characterization of the viral interaction determinant of the subgroup A avian leukosis virus receptor. J Virol 69:4261-6 |
| Belanger, C; Zingler, K; Young, J A (1995) Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry. J Virol 69:1019-24 |
