Effects of the ligand sequence modifications on the retargeted transduction by the retroviral vector having a ligand-chimeric Env protein.

There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1alpha) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 10(4) c.f.u. ml(-1). Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 10(6) c.f.u. ml(-1). The SDF-1alpha ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3-51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.

Mechanisms underlying glycosylation-mediated loss of ecotropic receptor function in murine MDTF cells and implications for receptor evolution.

A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus-host interactions.

Determinant for the inhibition of ecotropic murine leukemia virus infection by N-linked glycosylation of the rat receptor.

Ecotropic murine leukemia viruses (MLVs) recognize the third extracellular loop of the receptor, cationic amino acid transporter type 1 (CAT1). The CAT1 protein contains two conserved N-linked glycosylation sites in the third extracellular loops of the mouse, rat, and hamster receptors (mCAT1, rCAT1, and hCAT1, respectively). Glycosylation of the rCAT1 and hCAT1 receptors inhibits ecotropic MLV infection of CAT1-expressing cells, but that of the mCAT1 does not afford the cells this protection. As compared to the mCAT1 protein, the rCAT1 and hCAT1 proteins possess three and six amino acid insertions, respectively, in the third extracellular loop. To determine whether these inserted amino acids are associated with ecotropic MLV infection inhibition by glycosylation, several mutants of mCAT1 and rCAT1 receptors were constructed. Of all the mutants generated in the present study, only rCAT1 mutant 1 exhibited detectable protein expression levels. The rCAT1 mutant 1-expressing human NP2 cells were more susceptible to transduction by ecotropic MLV vectors than the wild-type rCAT1-expressing cells. Tunicamycin, an N-glycosylation inhibitor, increased transduction titer in the wild-type rCAT1-expressing cells, but did not do so in the cells expressing either the mCAT1 or rCAT1 mutation 1. An amino acid substitution in the glycosylation site of the wild-type rCAT1 conferred higher infection susceptibility, but that of the rCAT1 mutant 1 did not. As with the wild-type mCAT1 and rCAT1 proteins, the rCAT1 mutants were detected on the cell surface by immunofluorescence microscopy. Tunicamycin treatment did not affect cellular distribution of the rCAT1 mutant 1, wild-type mCAT1 or rCAT1 proteins. These results indicate that the extra amino acids in the rCAT1 (as compared to the mCAT1) are associated with inhibition of ecotropic MLV infection by the rCAT1 glycosylation.

Retinoic acid controls blood vessel formation by modulating endothelial and mural cell interaction via suppression of Tie2 signaling in vascular progenitor cells.

Inhibition by all-trans retinoic acid (atRA) of the microvasculature formation in chicken chorioallantoic membrane (CAM) accompanied remarkably reduced numbers of endothelial cells (ECs) and increased numbers of mural cells (MCs) under the chorionic epithelial layer. Ro41-5253 (retinoid antagonist) exerted the opposite effect. Although atRA did not affect the differentiation of murine embryonic stem cell-derived vascular progenitor cells (VPCs) into ECs or MCs, atRA suppressed EC-MC interaction, leading to impaired branching. In both atRA-treated VPC cultures and CAM tissues underneath the chorionic epithelial layer, the expression of angiopoietin-2 (Ang-2; competitor for Ang-1) was enhanced, whereas that of Tie2 (a receptor for Angs) was reduced. Simultaneous treatment with Ang-1 partially blocked RA induction of EC-MC malinteraction and reduction in blood vessel formation. These results suggest that retinoid(s) may reduce EC-MC interaction by down-regulating Tie2 signaling as well as decreased EC numbers, which lead to impaired vascular remodeling.

An essential role for the His-8 residue of the SDF-1alpha-chimeric, tropism-redirected Env protein of the Moloney murine leukemia virus in regulating postbinding fusion events.

BACKGROUND: To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1. METHODS: We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated. RESULTS: All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion. CONCLUSIONS: Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.

Mutational analysis of the R peptide cleavage site of Moloney murine leukaemia virus envelope protein.

Moloney murine leukaemia virus (MoMLV) enters host cells by membrane fusion between the viral envelope and the host cell membrane. The cytoplasmic tail (R peptide) of the MoMLV envelope protein (Env) is cleaved by the viral protease during virion maturation. R peptide-truncated Env induces syncytia in susceptible cells but R peptide-containing Env does not, indicating that the R peptide inhibits membrane fusion. To examine the function of amino acid residues at the R peptide cleavage site in virus entry, mutant Env expression plasmids containing amino acid substitutions at these cleavage site residues were constructed. Some of these mutants induced syncytia in NIH 3T3 cells, even though they expressed the R peptide, indicating the importance of these residues for membrane fusion inhibition by the R peptide. Some mutants in which R peptide cleavage was detected had comparable transduction efficiency to wild-type Env, but mutants in which R peptide cleavage was not detected had lower transduction efficiency than wild-type Env. This result strongly supports that R peptide cleavage is required for virus entry.

N-Linked glycosylation is required for XC cell-specific syncytium formation by the R peptide-containing envelope protein of ecotropic murine leukemia viruses.

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.

A glycosylation-defective variant of the ecotropic murine retrovirus receptor is expressed in rat XC cells.

The XC rat sarcoma cell line undergoes extensive cell-to-cell fusion (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 (Env) protein 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 assess whether this property of the XC cell line arises from differences in its ecotropic MLV receptor, CAT1, we isolated CAT1 cDNA clones from XC cells. A variant CAT1 (xcCAT1) was found together with the wild-type rat CAT1 (rCAT1). xcCAT1 cDNA encodes a protein with a single amino acid change that destroys a conserved N-linked glycosylation site proximal to the Env-binding motif. We found that xcCAT1 expressed in Chinese hamster ovary (CHO) cells undergoes less glycosylation than rCAT1 and that the expression of xcCAT1 rendered the CHO cells more susceptible to infection with Moloney MLV. Thus, N-glycosylation negatively regulates the receptor activity of rCAT1. This is supported by the observation that treatment of rat F10 cells with the N-glycosylation inhibitor tunicamycin enhanced their susceptibility to Mo-MLV. However, xcCAT1-expressing CHO cells did not fuse with 293T cells expressing R+ Env, indicating that xcCAT1 expression is not sufficient to induce the XC cell-specific characteristic of forming syncytia in response to R+ Env.

Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein.

To develop a reliable strategy for cell-specific delivery of retroviral vectors, we genetically modified the envelope (Env) protein of the ecotropic Moloney murine leukemia virus. We found a site in the variable region A, where the insertion of ligands, epidermal growth factor (EGF) and stromal-derived factor-1 alpha (SDF-1 alpha), was possible without abolishing virion incorporation of the Env protein and its ecotropic entry function. The vector containing the EGF-Env did not show the EGF receptor-dependent transduction. The vector containing the SDF-1 alpha-Env, however, specifically transduced human cells expressing CXCR4, the receptor for SDF-1 alpha, at titers of 10(3)-10(4) c.f.u./ml. Further experiments showed that the CXCR4-dependent transduction was based on the specific interaction between the SDF-1 alpha moiety of the SDF-1 alpha-Env and CXCR4 and was independent of the ecotropic entry function. The direct targeting of the retroviral vector may be possible if the proper chimeric Env structure and the appropriate ligand-receptor system are employed.

Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray.

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones--Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin--which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.