Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry

Abstract

The extracellular domain of the subgroup A avian sarcoma and leukemia virus (ALSV-A) receptor contains a region that is related in sequence to the ligand-binding motifs of the low-density lipoprotein receptor (LDLR).This domain contains six cysteines that are highly conserved between different members of the LDLR protein superfamily, and these residues are presumed to participate in intrachain disulfide bonds. To assess the importance of each cysteine in the ALSV-A receptor, individual or multiple cysteines were mutated to alanines and the altered receptors were tested for the ability to confer susceptibility to viral infection. Receptors bearing single mutations allowed subgroup A viral entry, albeit at less than wild-type levels. Receptors containing two or three substitutions were completely inactive if one of the changed residues was Cys-35 or Cys-50. Of the altered receptors tested, the only exception to this rule was a functional receptor which lacked both Cys-35 and Cys-50, an activity that was dependent on the presence of other cysteines in this protein. Most interestingly, a receptor containing both Cys-35 and Cys-50 but lacking the other four cysteines was completely functional. These results demonstrate the importance of Cys-35 and Cys-50 for viral entry mediated by the ALSV-A receptor and show that in the presence of these two residues, all of the other cysteines in this protein can be removed without loss of this function.

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

“…Construction of the HA-Tva gene. A DNA fragment encoding three copies of the nine-amino-acid HA epitope tag (YPYDVPDYA) was introduced at a ClaI site engineered between the codons for Ser-7 and Leu-8, in the extracellular region of the synthetic quail Tva gene (6,47,55). The ClaI site was introduced by a PCR mutagenesis protocol, and the sequence of the final product was confirmed as described above.…”

“…The epitope-tagged protein was designated HA-Tva. The mutations at residues Asp-46, Glu-47, Trp-48, Cys-28/Cys-35, Cys-41/ Cys-50, and Cys-28/Cys-41, were transferred into HA-Tva from previously described Tva constructs (6,55). The wild-type and mutant HA-Tva genes were placed under the control of the cytomegalovirus early-region promoter in the pCB6 expression plasmid.…”

“…Determinants that are important for viral entry have also been defined for the ATRC1 receptor (1,30,53); for the Tva receptor (6,40,55); for the PiT2, RaPiT2, and HaPiT2 receptors for amphotropic murine leukemia virus (17,34,37); for the PiT1 receptor for gibbon ape leukemia viruses, subgroup B feline leukemia viruses, and simian sarcoma-associated viruses (34,37,46); and for the HaPiT2 receptor for gibbon ape leukemia viruses (17). The precise functions of these determinants during viral entry, however, remain to be established.…”

“…The Tva receptor has a small 83-amino-acid extracellular domain which contains a motif (residues 11 to 50) that is highly related to the seven ligand binding repeat regions of the lowdensity lipoprotein receptor (LDLR) (5). The major viral interaction determinants of Tva are located at the C-terminal end of the LDLR-related motif (6,40) and consist of a threeamino-acid stretch (Asp-46, Glu-47, and Trp-48) and a putative disulfide bond between Cys-35 and Cys-50 (6,55). Although a disulfide bond between Cys-35 and Cys-50 in Tva remains to be formally demonstrated, nuclear magnetic resonance studies have confirmed that the equivalent cysteines in the first and second ligand binding repeat regions of the LDLR form a disulfide bond pair (15,16).…”

Identification of Key Residues in Subgroup A Avian Leukosis Virus Envelope Determining Receptor Binding Affinity and Infectivity of Cells Expressing Chicken or Quail Tva Receptor

“…The overall structure of the Tva cysteine-rich region (amino acids 11 to 50 in quail Tva) is formed by three disulfide bonds (C1-C3, C2-C5, and C4-C6) between the six cysteine residues (Cys-11, Cys-18, Cys-28, Cys-35, Cys-41, and Cys-50) ( Fig. 1C) (9). Three amino acid residues (Asp-46, Glu-47, and Trp-48) (54, 72) contained in the protein loop formed by the C4-C6 disulfide bond, as well as several other residues in the carboxy-terminal half of this region (Leu-34, His-38, and Gly-49) (53), have been identified as critical for efficient interaction between the quail Tva receptor and ALV(A) in a variety of assays.…”