Identification of a Critical Basic Residue on the Ecotropic Murine Leukemia Virus Receptor

Abstract

Susceptibility to ecotropic murine leukemia viruses (MLV) is restricted to mice and rats at the level of virus binding to the host cell receptor. Asparagine 232, valine 233, tyrosine 235, and glutamic acid 237 in the third extracellular domain (EL3) of the receptor are critical determinants of the host range difference between mice and humans. However, placing these residues in the human homolog confers only partial binding, indicating that other divergent sequences are involved. We sought to determine if the other sequences lie within or outside EL3. Here we report the identification of lysine 234 as another critical residue that influences virus binding and infection, as well as evidence that the unidentified sequences lie outside EL3. Each of the four basic residues in the third extracellular domain were changed to an acidic residue and initially examined in combination with a change at position 235 or position 237. Substitution of lysine 211, 215, or 222 combined with substitution of the critical tyrosine 235 or glutamic acid 237 did not affect virus infection. However, combined substitution of lysine 234, a conserved residue between mice and humans, and tyrosine 235 resulted in a marked decrease in virus infection and binding. A lysine 234 change alone reduced virus binding, contrary to previous observations that at least two of the other four residues must be changed before binding is reduced. Interestingly, there was no decrease in infection when lysine 234 was replaced in combination with glutamic acid 237. This result suggests that residue 234 may act by influencing the local structure of residues 233 to 235, whereas the presence of a glycine at position 236 may prevent this influence from extending to residue 237. With this report, the involvement of all the residues divergent between mice and humans in the third extracellular domain has been ruled out, suggesting that as yet unidentified determinants lie in other extracellular domains.The host range of the ecotropic murine leukemia virus (MLV) is restricted to mice and rats by the availability of their cell surface receptor ATRC1, an integral membrane protein with 14 membrane-spanning domains (3). In the host cell, ATRC1 functions as the principal transporter of cationic amino acids (9, 18). Other mammals, including humans, have homologous proteins that function as cationic amino acid transporters but lack ecotropic MLV receptor capability (1, 2).Homology scanning mutagenesis of the receptor and its human homolog led to the identification of residues 211 to 239 as the putative virus binding domain (2, 21) (Fig. 1A). This domain resides on the extracellular side of the plasma membrane since the two N-linked glycosylation sites (asparagines 223 and 229) within it are glycosylated in vivo (10). Replacing two or more of asparagine 232, valine 233, tyrosine 235, and glutamate 237 results in loss of virus surface protein (SU) binding and virus infection, although single substitution of any of these residues did not reduce binding or infection (2), indicatin...

Complementation of a Binding-Defective Retrovirus by a Host Cell Receptor Mutant

“…Virus binding assays were performed exactly as previously described (16). Briefly, virus stock was concentrated and shedded SU was removed using a Centricon Plus-80 device (100-kDa molecular mass cutoff).…”

“…Although infection via mutant receptors carrying a lysine 234-to-alanine (rK234A), a lysine-toglutamate (rK234E), or a lysine-to-aspartate (rK234D) change was comparable to that via WT receptor, virus binding was reduced (16). In addition, 293 cells expressing double mutant rY235A plus rK234D receptors showed greatly reduced infection and loss of binding, whereas infection was not affected by double mutant rE237A plus rK234D (16).…”

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

“…The nucleotide sequences of the original cDNA and the optimized cDNA encoding the receptor binding domains (RBD) were deposited in the GenBank database; sequences outside the RBD are identical to those of the previously published wild-type Moloney murine leukemia virus Env (J02255). Control chimeras Sst-NH 2 and Sst-PRR were made by ligating an Sst-encoding fragment into the NotI restriction site in two previously described plasmids, pcDNA-MolMLV-Nflex and pcDNA-MoMLV-PRR, respectively (44). The Sst-230 chimeric sequence was constructed by ligating an Sst-encoding fragment into the EcoO109I site at codon 230 of the MoMLV Env cDNA.…”

“…The relative levels of surface expression of SSTR2a on transiently transfected and stable cell lines were determined as previously described (44) with the following modifications. SSTR2a protein was detected using rabbit anti-SSTR2a antiserum (1:500 dilution) incubated overnight at 4°C.…”

Comprehensive Mapping of Receptor-Functioning Domains in Feline Leukemia Virus Subgroup C Receptor FLVCR1

“…The only exception is the interaction of ecotropic murine leukemia virus with the cationic amino acid transporter, CAT1 (3,13,36). To date, only presumptive extracellular loop 3 (ECL3) has been identified as critical for virus infection (2,9,23,38).…”