Hiroshi Amanuma

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) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R+ Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R+ Env protein but not that induced by the R− Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R+ Env protein. The R+ Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R+ Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R+ Env protein.

Oligomerization of friend spleen focus-forming virus (SFFV) env glycoproteins

The glycoprotein gp55 and its processed form gp65, which are encoded by the env gene of Friend spleen focus-forming virus (SFFV), have been implicated in the initiation of the murine acute erythroleukemia induced by Friend virus (FV). Analyses of these glycoproteins by chemical crosslinking and nonreducing/reducing two-dimensional electrophoresis showed that both gp55 and gp65 exist as monomer and disulfide-bonded dimer and trimer. These oligomers could be detected in various FV-infected erythroleukemia cell lines, as well as in the spleen cells of FV-induced erythroleukemic mice, suggesting that oligomerization is an intrinsic feature of SFFV env glycoproteins.

Decrease in glucose transport activity of Friend erythroleukemia cell caused by dimethylsulfoxide, a differentiation-inducing reagent

A transport system for D-glucose was found in a Friend erythroleukemia cell line, T-3-C1-2-O and was characterized as a facilitated diffusion system. D-Glucose transport activity showed a half-saturation concentration of 2.2 mM and was inhibited by mercuric ions, cytochalasin B, phloretin, and stilbestrol, but was not strongly inhibited by phloridzin. Transport of 3-O-methyl-D-glucose was faster than D-glucose and the intracellular concentration of the sugar was found to reach the concentration in the assay medium. The treatment of cells with a differentiation-inducing reagent, dimethylsulfoxide(Me2SO), for 24 h caused a marked decrease in glucose transport activity due to a decrease in Vmax. In an induction-insensitive Friend cell line, T-3-K-1, D-glucose transport activity was low in untreated cells and Me2SO treatment did not cause a significant decrease in transport activity. The results obtained in this study indicate that the decrease in glucose transport activity is not due to the direct effect of Me2SO on transport activity, but is associated with the induction of differentiation. By immunoblotting cell lysates of T-3-C1-2-O cells using antibody to human erythrocyte glucose transporter, a single major band having a molecular weight of 52,000 was detected, which may be a glucose transporter in Friend cells.

Identification of putative endogenous proviral templates for progenitor mink cell focus-forming (MCF) MuLV-related RNAs.

Murine leukemia virus (MuLV)-related RNAs exhibiting different env deletions are believed to participate in the generation of leukemogenic mink cell focus-forming (MCF) viruses. We have cloned an endogenous MuLV provirus from AKR/J mouse DNA, designated as A-2, which may serve as template for the env-deleted E2 MuLV RNA, expressed in GIX+ mice (D.E. Levy et al., J. Virol. 56, 691-700 (1985]. We have also isolated an endogenous MCF-related DNA, A-1, which shared close sequence homology with the 7.2-kb RNA expressed in AKR mice (F. Laigret et al., J. Virol. 62, 376-386 (1988] and sustained an identical env deletion. The data indicate that putative precursor MCF-related RNAs are transcribed from a heterogenous family of env-deleted endogenous MuLV DNAs.

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus‐forming virus (F‐SFFV) is a replication‐defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)‐like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus‐inducing (MCF) murine leukemia virus (MuLV) Env gp70‐derived sequence in its amino‐terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino‐terminal region. XE gp55 displayed almost the same biological activities as the wild‐type gp55, excluding the above possibility.