Dataset Information

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.

ABSTRACT: Previously, mutant Tva receptors were classified as either partially or completely defective in mediating subgroup A avian leukosis and sarcoma virus (ALSV-A) entry (C. Bélanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995; K. Zingler, C. Bélanger, R. Peters, D. Agard, and J. A. T. Young, J. Virol. 69:4261-4266, 1995). To specifically test the abilities of these mutant Tva proteins to bind ALSV-A surface (SU) protein, binding studies were performed with a subgroup A SU-immunoadhesin. This fusion protein is composed of the subgroup A Schmidt-Ruppin SU protein fused in frame to a rabbit immunoglobulin constant region. This reagent was conjugated to fluorescein isothiocyanate and 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 KD of approximately 1.5 nM. Amino acid substitutions 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, drastically reduced the binding affinity for the SU-immunoadhesin. Thus, the effects on viral entry of some mutations could be explained solely by changes in the binding affinity for ALSV-A SU. However, this was not true for other mutations tested, especially those with amino acid substitutions that replaced Trp-48. Compared with the wild-type receptor, these latter mutations led to approximately 43- to 200-fold reductions in viral infectivity but only to approximately 2.5- to 3.4-fold reductions in the binding affinity for the SU-immunoadhesin. These results support a role for Trp-48 of Tva in mediating steps of viral entry subsequent to binding ALSV-A SU.

SUBMITTER: Zingler K

PROVIDER: S-EPMC190818 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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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.

Zingler K K Young J A JA

Journal of virology 19961101 11

Previously, mutant Tva receptors were classified as either partially or completely defective in mediating subgroup A avian leukosis and sarcoma virus (ALSV-A) entry (C. Bélanger, K. Zingler, and J. A. T. Young, J. Virol. 69:1019-1024, 1995; K. Zingler, C. Bélanger, R. Peters, D. Agard, and J. A. T. Young, J. Virol. 69:4261-4266, 1995). To specifically test the abilities of these mutant Tva proteins to bind ALSV-A surface (SU) protein, binding studies were performed with a subgroup A SU-immunoadh ...[more]

PMID: 8892869

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Project description:The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Project description:Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Dataset Information

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.

Publications

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.

Similar Datasets

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