Preferential retroviral-mediated transduction of EBV- and CMV-specific T cells after polyclonal T-cell activation.

Graft-versus-host disease, resulting from the T cells present in allogeneic hematopoietic stem cell (HSC) inoculums, can potentially be treated if a suicide gene has been introduced into the donor T cells. However, the diversity and functionality of the transfused T-cell population, including EBV- (EBV-T) and CMV-specific (CMV-T) CD8+ T cells, which are particularly important for immunosuppressed individuals undergoing HSC transplants, are often modified by the gene transfer protocol. Here, we show that following polyclonal T-cell activation, EBV-T and CMV-T cells are preferentially transduced by oncoretroviral vectors, as compared to the bulk CD8+ T-cell population. This preferential transduction is associated with higher surface levels of PiT-2, the receptor for the amphotropic envelope with which the virions are pseudotyped. Moreover, EBV-T and CMV-T cells proliferate more extensively as compared to bulk CD8+ T cells. Thus, retroviral-mediated transduction can be biased toward a given antigenic specificity, even under conditions of polyclonal stimulation.

Precise gene localization by phenotypic assay of radiation hybrid cells.

A high resolution map of the human genome previously has been constructed by using the G3 panel of human/hamster radiation hybrid cell lines and >15,000 unique human genetic markers. By determining whether human DNA sequences are present or absent in each of the hybrids, localization of single genes may routinely be achieved at approximately 250-kb resolution. In this paper we have tested whether similarly precise localization might be achieved by phenotypic screening of the hybrids to facilitate positional cloning of unknown genes. We assayed the susceptibility of each of the hybrid cell lines to transduction by retroviral vectors bearing different retroviral envelope proteins that recognize receptors present on human but not on hamster cells. The results for each of the retroviral vectors were informative and allowed precise localization of the receptor genes for the RD114 cat endogenous retrovirus, xenotropic murine leukemia virus, and type C feline leukemia virus. After cloning of the receptors for these retroviruses, we found that standard genotypic mapping by PCR gave results that were nearly identical to those from phenotypic mapping. These experiments show that precise gene localization by phenotypic assay of radiation hybrids is practical and was not appreciably impacted by the known instability of such hybrid cells. This technique should be applicable to many other human genes having discernible phenotypes in hamster cells and, with completion of the human genome project, will allow rapid identification of unknown genes on the basis of phenotype.

The RD114/simian type D retrovirus receptor is a neutral amino acid transporter.

The RD114/simian type D retroviruses, which include the feline endogenous retrovirus RD114, all strains of simian immunosuppressive type D retroviruses, the avian reticuloendotheliosis group including spleen necrosis virus, and baboon endogenous virus, use a common cell-surface receptor for cell entry. We have used a retroviral cDNA library approach, involving transfer and expression of cDNAs from highly infectable HeLa cells to nonpermissive NIH 3T3 mouse cells, to clone and identify this receptor. The cloned cDNA, denoted RDR, is an allele of the previously cloned neutral amino acid transporter ATB0 (SLC1A5). Both RDR and ATB0 serve as retrovirus receptors and both show specific transport of neutral amino acids. We have localized the receptor by radiation hybrid mapping to a region of about 500-kb pairs on the long arm of human chromosome 19 at q13.3. Infection of cells with RD114/type D retroviruses results in impaired amino acid transport, suggesting a mechanism for virus toxicity and immunosuppression. The identification and functional characterization of this retrovirus receptor provide insight into the retrovirus life cycle and pathogenesis and will be an important tool for optimization of gene therapy using vectors derived from RD114/type D retroviruses.

A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction.

Although present in many copies in the mouse genome, xenotropic murine leukemia viruses cannot infect cells from laboratory mice because of the lack of a functional cell surface receptor required for virus entry. In contrast, cells from many nonmurine species, including human cells, are fully permissive. Using an expression library approach, we isolated a cDNA from HeLa cell RNA that conferred susceptibility to xenotropic envelope protein binding and virus infection when expressed in nonpermissive cells. The deduced product is a 696-aa multiple-membrane spanning molecule, is widely expressed in human tissues, and shares homology with nematode, fly, and plant proteins of unknown function as well as with the yeast SYG1 protein, which has been shown to interact with a G protein. This molecule also acts as a receptor for polytropic murine leukemia viruses, consistent with observed interference between xenotropic and polytropic viruses in some cell types. This xenotropic and polytropic retrovirus receptor (XPR1) is the fourth identified molecule having multiple membrane spanning domains among mammalian type C oncoretrovirus receptors and may play a role in G protein-coupled signal transduction, as do the chemokine receptors required for HIV entry.

Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor.

Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.

Definition of a 14-amino-acid peptide essential for the interaction between the murine leukemia virus amphotropic envelope glycoprotein and its receptor.

Hydrophilic loops in the receptor binding domain of the amphotropic murine leukemia virus (MLV) envelope glycoprotein (SU) are predicted and may participate in SU-receptor interactions. We have replaced five segments of 6 to 15 amino acids located in each of these regions with an 11-amino-acid tag from the vesicular stomatitis virus glycoprotein (VSV-G). Substitution was compatible with envelope processing, transport, and incorporation into virions. However, three substitution mutants showed a temperature-dependent phenotype, suggesting structural unstability. Accessibility of the tagging epitope for a monoclonal anti-VSV-G antibody was greater in oligomeric than in monomeric SUs when insertion was done in VRA, a domain essential for receptor recognition. In contrast, accessibility was independent of structural constraints when insertion was done in VRB, a domain playing an accessory role in receptor binding. Interaction with the amphotropic receptor was investigated by interference assay and study of binding and infection of target cells with MLV particles coated with the substituted envelopes. Envelope-receptor interaction was abolished when substitution was performed in a potential loop-forming segment located at the N-terminal half of VRA. Although interaction was affected to variable extents, depending on the substituted segment, other mutants conserved the ability to interact with the amphotropic receptor. These experiments indicate the 14-amino-acid segment between positions 50 and 64 of SU as an essential determinant of amphotropic-receptor recognition. They also show that a foreign linear epitope can be tolerated in several locations of the amphotropic SU receptor binding site, and this result has implications for the design of targeted retroviral vectors.

Receptor-binding properties of a purified fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein.

A 208-amino-acid amino-terminal fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein contains all of the determinants required to recognize cell surface amphotropic receptors. This fragment was fused with a streptavidin-binding tag, expressed in Sf9 insect cells by using a baculovirus vector, and purified to homogeneity. The (125)I-labeled purified fragment (AS208) specifically bound various cell lines susceptible to amphotropic murine leukemia virus infection. The number of AS208-binding sites was in the range of 7 X 10(4) to 17 X 10(4) per cell. Quantitative analysis of binding revealed that AS208-binding sites are heterogeneous with regard to ligand binding affinity or that cooperativity exists between receptors. Competition experiments showed that the concentration of AS208 required to inhibit virus entry was lower than that required to inhibit the binding of virus particles at the cell surface. Taken together, these data suggested that amphotropic envelope-binding sites present at the cell surface do not act independently and do not participate equally in virus infection.

High-titer packaging cells producing recombinant retroviruses resistant to human serum.

Novel retroviral protein expression constructs were designed to retain minimal retroviral sequences and to express dominant selectable markers by reinitiation of translation after expression of the viral genes. HT1080 cells were selected as producer cells for their ability to release high-titer viruses that are resistant to inactivation by human serum. Two HT1080-based packaging cell lines which produce Moloney murine leukemia virus cores with envelope glycoproteins of either amphotropic murine leukemia virus (FLYA13 line) or cat endogenous virus RD114 (FLYRD18 line) are described. Direct comparison with previous retroviral packaging systems indicated that 100-fold-higher titers of helper-free recombinant viruses were released by the FLYA13 and FLYRD18 lines.

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.

Role of N-linked glycosylation in the activity of the Friend murine leukemia virus SU protein receptor-binding domain.

The 243 N-terminal residues of Friend Murine Leukemia Virus envelope glycoprotein (SU) fold into a structurally and functionally autonomous domain which contains the determinants for binding to the ecotropic virus receptor. The two N-linked glycosylation sites present in this N-terminal portion of the viral SU were removed by site-directed mutagenesis without disturbing its biosynthesis and incorporation into infectious virions. A truncated version of the mutant protein which included only the N-terminal domain was poorly transported but still able to interact with the receptor. Interference assays indicated that the interaction between the mutated protein and the virus receptor was weaker. We conclude that the elimination of N-linked oligosaccharide chains in the envelope N-terminal domain do not prevent receptor interaction but results in subtle conformational changes that may alter recognition and binding.

Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.