In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1

Abstract

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein {SU, residues 1 to 236 [SU:(1-236)]} and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (K d , 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain. MATERIALS AND METHODS Construction of expression plasmids encoding MCAT-1 and Friend SU. The vector pSP64T (16) was modified by (i) insertion of the BamHI-to-XbaI portion of the polylinker from pCDNA3 (Invitrogen) at the BglII/SalI site, (ii) reinsertion of the deleted BglII-to-SmaI fragment of pSP64T at the BamHI/PvuII site and addition of a second polylinker containing NsiI, BglII, ApaI, and PstI sites at the PstI site, and (iii) insertion of a 140-bp fragment encoding (in tandem) a factor Xa protease site (amino acids IEGR), three copies of the hemagglutinin (HA) peptide (YPYDVPYA [10]) recognized by monoclonal antibody 12CA5 (Boehringer Mannheim), and a six-histidine motif at the XhoI/PstI site in the polylinker (Flu 3-his 6 tag). The new vector was designated pRD67:Flu 3-his 6. A clone encoding envelope SU of Friend MLV (reference 12; GenBank accession no. J02192) in plasmid pUC13 (a gift from A. Pinter) was modified by addition of a 5Ј EcoRI site at bp Ϫ40 relative to the initiator ATG and a 3Ј XhoI site at bp 810 that permitted insertion into pRD67:Flu 3-his 6 with an in frame fusion at the 3Ј end to the sequences encoding the protease cleavage site and Flu 3-his 6 tag. A cDNA encoding the ecotropic retrovirus receptor (MCAT-1) with the Cterminal protease cleavage site and Flu 3-his 6 tag (MCAT-1t) was constructed by using PCR to place an EcoRI site at bp Ϫ50 relative to the initiator ATG and to replace the stop codon of MCAT-1 (residue 622) with an in-frame XhoI recognition site and inserted into the polylinker of pRD67:Flu 3-his 6. To avert the possibility of PCR-induced mutations, the BamHI-to-SphI fragment of this plasmid was excised and replaced by the equivalent fragment from the original MCAT-1 cDNA clone. The absence of Taq polymerase-induced changes in the remaining portion (Ͻ10% of the coding region) was confirmed by nucleotide sequencing. Expression of recombinant proteins in oocytes and mammalian cells. Capped mRNAs encoding MCAT-1 or MCAT-1t were transcribed and injected into Xenopus laevis oocytes, and after 3 days, transport of L-[ 14 C]arginine and exogenous binding of 125 I-SU proteins were measured as described elsewhere (11). In some experiments, detergent extracts of oocytes were prepared and solubilized proteins separa...

Analysis of Receptor Usage by Ecotropic Murine Retroviruses, Using Green Fluorescent Protein-Tagged Cationic Amino Acid Transporters

“…In previous studies of the structure-function relationship of mCAT1, the level of exogenous mCAT1 expression was estimated by the arginine uptake assay (20,25), which required radioactive materials and relatively complicated procedures. In another study, mCAT1 was tagged with the antigenic epitope of influenza virus HA in order to facilitate immunological detection (8). However, microscopic detection of epitope-tagged mCAT1 would require immunostaining procedures.…”

“…Therefore, dimerization of CAT molecules revealed in this study may be biologically significant. It has previously been shown that the RBD of the MuLV SU protein and mCAT1 interact with a stoichiometry of 1:1 in vitro (8). Since crystallographic studies strongly suggested trimer formation of MuLV envelope proteins (10), it is possible that mCAT1 is expressed on the cell surface as a trimer that is partially disassembled during protein sample preparation.…”

Identification of a Receptor-Binding Pocket on the Envelope Protein of Friend Murine Leukemia Virus

“…Mouse-derived fibroblast cell lines, NIH 3T3, NFM, and mCAT-1 Ϫ/Ϫ (27), were grown in Dulbecco minimal essential medium (DMEM) and fetal calf serum (FCS; 10%). NFM was created by clonal selection after transfection with an expression plasmid (pCDNA3; Invitrogen) encoding mCAT-1 bearing an influenza virus HA epitope tag at the carboxyl terminus (11). The packaging cell line Anjou, which expresses MLV Gag and Pol proteins, was obtained from Warren Pear (26) and also grown in DMEM and fetal calf serum (10%).…”

“…Although the receptors for type C MLVs (2,31,33) and HIV (15) have been identified, as yet these studies have provided little insight into how binding to SU results in fusion. Toward this end, the receptor-binding domain in the SU protein of Friend 57 MLV (Fr57-MLV) has been identified (residues 1 to 236 of SU; Fr57-RBD), the stoichiometry and binding affinity have been determined by using purified proteins (11), and the structure has been resolved to 2.0-Å resolution by X-ray crystallography (13). Residues conserved in the SU proteins of ecotropic MLV (5), which utilize mCAT-1 as a receptor, were found among intertwined loops and helices (variable region A [VRA] domain) which abut a "stalk"-like ␤-barrel (see Fig.…”

Sequential activation of the three protomers in the Moloney murine leukemia virus Env

“…The Mo-MLV fusion protein is called Env (envelope protein). It binds to its receptor (murine cationic amino acid transporter 1) on the cell surface by the N-terminal domain (the receptor binding domain, RBD) of the peripheral or surface subunit (SU) (14)(15)(16). The RBD then transmits an activation signal to the TM via the C-terminal SU domain (17,18).…”