GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus.

The human gene GLVR1 has been shown to render mouse cells sensitive to infection by gibbon ape leukemia virus. This indication that the GLVR1 protein acts as a virus receptor does not reveal the protein's normal physiological role. We now report that GLVR1 is homologous to pho-4+, a phosphate permease of Neurospora crassa, at a level sufficiently high to predict that GLVR1 is also a transport protein, although the substrate transported remains unknown. To characterize the gene further, we have cloned cDNA for the mouse homolog of the gene, Glvr-1. The sequence of the murine protein differs from that of the human protein in 10% of residues, and it may be presumed that some of these differences are responsible for the inability of gibbon ape leukemia virus to infect mouse fibroblasts. Glvr-1 RNA is most abundant in mouse brain and thymus, although it is present in all tissues examined. The pattern of RNA expression found in mouse tissues was also found in rat tissues, in which the RNA was expressed at high levels in all compartments of the brain except the caudate nucleus and was expressed most abundantly early in embryogenesis. Thus, high-level expression of Glvr-1 appears to be restricted to specific tissues and may have developmental consequences.     

Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.

10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.     

Definition of a domain of GLVR1 which is necessary for infection by gibbon ape leukemia virus and which is highly polymorphic between species.

Expression of human GLVR1 in mouse cells confers susceptibility to infection by gibbon ape leukemia virus (GALV), while the normally expressed mouse Glvr-1 does not. Since human and murine GLVR1 proteins differ at 64 positions in their sequences, some of the residues differing between the two proteins are critical for infection. To identify these, a series of hybrids and in vitro-constructed mutants were tested for the ability to confer susceptibility to infection. The results indicated that human GLVR1 residues 550 to 551, located in a cluster of seven of the sites that differ between the human and mouse proteins, are the only residues differing between the two which must be in the human protein form to allow infection. Sequencing of a portion of GLVR1 from the rat (which is infectible) confirmed the importance of this cluster in that it contained the only notable differences between the rat and mouse proteins. This region, which also differs substantially between the rat and the human proteins, therefore exhibits a pronounced tendency for polymorphism.     

Localization of the human gene allowing infection by gibbon ape leukemia virus to human chromosome region 2q11-q14 and to the homologous region on mouse chromosome 2.

Retrovirus receptors remain a largely unexplored group of proteins. Of the receptors which allow infection of human and murine cells by various retroviruses, only three have been identified at the molecular level. These receptors include CD4 for human immunodeficiency virus, Rec-1 for murine ecotropic virus, and GLVR1 for gibbon ape leukemia virus. These three proteins show no homology to one another at the DNA or protein level. Therefore, work to date has not shown any general relationship or structural theme shared by retroviral receptors. Genes for two of these receptors (CD4 and Rec-1) and several others which have not yet been cloned have been localized to specific chromosomes. In order to assess the relationship between GLVR1 and other retroviral receptors, we mapped the chromosome location of GLVR1 in human and mouse. GLVR1 was found to map to human chromosome 2q11-q14 by in situ hybridization and somatic-cell hybrid analysis. This location is distinct from those known for receptors for retroviruses infecting human cells. Glvr-1 was then mapped in the mouse by interspecies backcrosses and found to map to chromosome 2 in a region of linkage conservation with human chromosome 2. This mouse chromosome carries Rec-2, the likely receptor for M813, a retrovirus derived from a feral Asian mouse. These data raise the interesting possibility that Rec-2 and Glvr-1 are structurally related.     

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.     

Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system

We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.     

Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein.

A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.