N-Linked Glycosylation Is Required for XC Cell-Specific Syncytium Formation by the R Peptide-Containing Envelope Protein of Ecotropic Murine Leukemia Viruses

Abstract

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) Ecotropic murine leukemia viruses (MLVs) enter host cells mediated by fusion between the viral envelope and the cellular membrane following the recognition of the viral envelope glycoprotein (Env) by the cellular receptor, CAT1 (1). The Env protein of the MLVs is synthesized as a precursor polyprotein and is cleaved to surface (SU) and transmembrane (TM) subunits in infected cells by a cellular protease (25). The C-terminal 16 amino acids of the TM protein (R peptide) are further cleaved off during virion maturation by the viral protease (5, 7). The R peptide-truncated Env protein (R Ϫ Env) has membrane fusion activity to induce syncytium formation when it is expressed in susceptible cells, but the R peptide-containing Env protein (R ϩ Env) does not. This result indicates that the R peptide inhibits membrane fusion (14,23,24). Because cells expressing the R Ϫ Env protein will form syncytia and die, the selective truncation of the R peptide may be an adaptive mechanism that allows ecotropic MLVs to delay membrane fusion until the viruses leave the cells.Rat XC cells, which are widely used to measure ecotropic MLV titers (12), are more susceptible to ecotropic MLV infection than other rat cells, such as F10 cells (13,28). In addition to the R Ϫ Env protein, R ϩ Env can induce syncytia in XC cells but not in other susceptible cells (9). Recently, it was found that some CAT1 cDNA clones isolated from XC cells have an amino acid substitution of aspartic acid for asparagine at one of two N-linked glycosylation sites, and the XC cell receptor was designated xcCAT1 (13). CHO cells expressing xcCAT1 were more susceptible to Moloney MLV (Mo-MLV) vector transduction than those expressing the wild-type rat CAT1 (rCAT1). In addition, F10 cells treated with tunicamycin, an inhibitor of protein N glycosylation, were more susceptible to Mo-MLV vector transduction than untreated F10 cells. These results indicate that the N glycosylation of rCAT1 inhibits Mo-MLV vector infection. In CHO cells expressing xc-CAT1, however, the R ϩ Env protein does not induce syncytia, but R Ϫ Env does. This result indicates that the amino acid substitution detected in xcCAT1 is not sufficient for XC cellspecific syncytium formation by the R ϩ Env protein and that another mechanism is necessary.To analyze the ecotropic MLV receptor in XC cells, a retroviral vector encoding a mouse CAT1 (mCAT1) protein Cterminally tagged with the influenza virus hemagglutinin (HA) epitope was inoculated into XC and NIH 3T3 cells. The molecular size of the HA-tagged mCAT1 (mCAT1-HA) expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Tunicamycin treatment of XC cells significantly inhibited XC cell-specific syncytium formation by the R ϩ Env protein but not that by ...

Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

“…It is thought that the mechanism of Eco-MLV entry into rat XC cells is distinct from other susceptible cells. pH-independent cell-cell fusion is induced in XC cells by the Eco-MLV infection [16], or by the R peptide-containing Eco-MLV Env protein [17], [18], but not in other susceptible cells. In addition, ammonium chloride, which increases the pH of endosomes, inhibits the Eco-MLV infection in many susceptible cells, but not in XC cells, indicating that the Eco-MLV infection in XC cells is independent of low pH in endosomes [11].…”

Cathepsin L is required for ecotropic murine leukemia virus infection in NIH3T3 cells

“…Cathepsin B is involved in syncytium formation during myogenesis (Gogos et al, 1996;Jane et al, 2002), and is expressed in XC cells in which the R peptide-containing Env protein can induce syncytium formation (Jones and Risser, 1993;Kubo, Ishimoto, and Amanuma, 2003). As mentioned above, cathepsin B expression is significantly activated in transformed NIH3T3 cells (Chambers et al, 1992; He et al, 2004).…”

“…Human 293T (Pear et al, 1993), TE671, TELCeB6 (Cosset et al, 1995), NP2 (Soda et al, 1999), mouse NIH3T3, and rat XC (Kubo, Ishimoto, and Amanuma, 2003) cells were cultured in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) supplemented with 8% fetal bovine serum (Biosource, Rockville, MD) at 37°C in 5% CO 2 . Friend Eco-MLV vector-producing cells were constructed by stable transfection of TELCeB6 cells with the Friend MLV Env expression plasmid (Kubo et al, 2004).…”

“…However, the R peptide-containing Env protein successfully induces syncytia in rat XC and fu-1 cells (Jones and Risser, 1993;Wong, Yuen, and Kaufman, 1977). We have previously reported that treatment of XC cells with tunicamycin, an N-linked glycosylation inhibitor, significantly suppresses the syncytium formation of XC cells by the R peptidecontaining Env protein, but does not that by the R peptide-truncated Env protein, suggesting that an unknown glycosylated cellular protein is involved in the XC cell-specific syncytium formation by the R peptide-containing Env protein (Kubo, Ishimoto, and Amanuma, 2003). Because XC and fu-1 cells were both derived from muscle tissues, it was speculated that the cellular factor is also associated with syncytium formation in myogenesis.…”

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor

“…An expression plasmid of the ecotropic Friend MLV Env has been already described [11]. A VSV-G expression plasmid was obtained from Dr. L. Chang through the AIDS Research and Reference Reagent Program, NIAID, NIH, USA.…”