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 ANDSARCOMA-VIRUSES

Previously, mutant Tva receptors were classified as either partially o r completely defective in mediating subgroup A avian leukosis and sarc oma virus (ALSV-A) entry (C. Belanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995; K. Zingler, C. Belanger, R. Peters, D. Agard, and J. A. T. Young, J. Virol. 69:4261-4266, 1995), To specifica lly test the abilities of these mutant Tva proteins to bind ALSV-A sur face (SU) protein, binding studies were performed with a subgroup A SU -immunoadhesin, This fusion protein is composed of the subgroup A Schm idt-Ruppin SU protein fused in frame to a rabbit immunoglobulin consta nt region, This reagent was conjugated to fluorescein isothiocyanate a nd used for flow cytometric analysis with transfected human 293 cells expressing different forms of Tva. The SU immunoadhesin bound the wild -type Tva protein with a K-D of approximately 1.5 nM, Amino acid subst itutions that reduced viral entry at Asp-46 and at Cys-35 and Cys-50, which are predicted to form an intrachain disulfide bond in Tva, drast ically reduced the binding affinity for the SU-immunoadhesin. Thus, th e effects on viral entry of some mutations could be explained solely b y changes in the binding affinity for ALSV-A SU, However, this was not true for other mutations tested, especially those with amino acid sub stitutions that replaced Trp-48. Compared with the wild-type receptor, these latter mutations led to approximately 43- to 200-fold reduction s in viral infectivity but only to approximately 2.5- to 3.4-fold redu ctions in the binding affinity for the SU-immunoadhesin, These results support a role for Trp-48 of Tva in mediating steps of viral entry su bsequent to binding ALSV-A SU.