Sodium-dependent neutral amino acid transporter type 1 is an auxiliary receptor for baboon endogenous retrovirus

The baboon endogenous retrovirus (BaEV) belongs to a large, widely dispersed interference group that includes the RD114 feline endogenous virus and primate type D retroviruses. Recently, we and another laboratory independently cloned a human receptor for these viruses and identified it as the human sodium-dependent neutral amino acid transporter type 2 (hASCT2). Interestingly, mouse and rat cells are efficiently infected by BaEV but only become susceptible to RD114 and type D retroviruses if the cells are pretreated with tunicamycin, an inhibitor of protein N-linked glycosylation. To investigate this host range difference, we cloned and analyzed NIH Swiss mouse ASCT2 (mASCT2). Surprisingly, mASCT2 did not mediate BaEV infection, which implied that mouse cells might have an alternative receptor for this virus. In addition, elimination of the two N-linked oligosaccharides from mASCT2 by mutagenesis, as substantiated by protein N-glycosidase F digestions and Western immunoblotting, did not enable it to function as a receptor for RD114 or type D retroviruses. Based on these results, we found that the related ASCT1 transporters of humans and mice are efficient receptors for BaEV but are relatively inactive for RD114 and type D retroviruses. Furthermore, elimination of the two N-linked oligosaccharides from extracellular loop 2 of mASCT1 by mutagenesis enabled it to function as an efficient receptor for RD114 and type D retroviruses. Thus, we infer that the tunicamycin-dependent infection of mouse cells by RD114 and type D retroviruses is caused by deglycosylation of mASCT1, which unmasks previously buried sites for viral interactions. In contrast, BaEV efficiently employs the glycosylated forms of mASCT1 that occur normally in untreated mouse cells.     

Localization of the receptor gene for type D simian retroviruses on human chromosome 19.

Simian retrovirus (SRV) serotypes 1 to 5 are exogenous type D viruses causing immune suppression in macaque monkeys. These viruses exhibit receptor interference with each other, with two endogenous type D viruses of the langur (PO-1-Lu) and squirrel monkey, and with two type C retroviruses, feline endogenous virus (RD114/CCC) and baboon endogenous virus (BaEV), indicating that each utilizes the same cell surface receptor (M. A. Sommerfelt and R. A. Weiss, Virology 176:58-69, 1990). Vesicular stomatitis virus pseudotype particles bearing envelope glycoproteins of RD114, BaEV, and the seven SRV strains were employed to detect receptors expressed in human-rodent somatic cell hybrids segregating human chromosomes. The only human chromosome common to all the susceptible hybrids was chromosome 19. By using hybrids retaining different fragments of chromosome 19, a provisional subchromosomal localization of the receptor gene was made to 19q13.1-13.2. Antibodies previously reported to be specific to a BaEV receptor (L. Thiry, J. Cogniaux-Leclerc, R. Olislager, S. Sprecher-Goldberger, and P. Burkens, J. Virol. 48:697-708, 1983) did not block BaEV, RD114, or SRV pseudotypes or syncytia. Antibodies to known surface markers determined by genes mapped to chromosome 19 did not block virus-receptor interaction. The identity of the receptor remains to be determined.     

Conserved Region of Mammalian Retrovirus RNA

The viral RNAs of various mammalian retroviruses contain highly conserved sequences close to their 3' ends. This was demonstrated by interviral molecular hybridization between fractionated viral complementary DNA (cDNA) and RNA. cDNA near the 3' end (cDNA(3')) from a rat virus (RPL strain) was fractionated by size and mixed with mouse virus RNA (Rauscher leukemia virus). No hybridization occurred with total cDNA (cDNA(total)), in agreement with previous results, but a cross-reacting sequence was found with the fractionated cDNA(3'). The sequences between 50 to 400 nucleotides from the 3' terminus of heteropolymeric RNA were most hybridizable. The rat viral cDNA(3') hybridized with mouse virus RNA more extensively than with RNA of remotely related retroviruses. The related viral sequence of the rodent viruses (mouse and rat) showed as much divergence in heteroduplex thermal denaturation profiles as did the unique sequence DNA of these two rodents. This suggests that over a period of time, rodent viruses have preserved a sequence with changes correlated to phylogenetic distance of hosts. The cross-reacting sequence of replication-competent retroviruses was conserved even in the genome of the replication-defective sarcoma virus and was also located in these genomes near the 3' end of 30S RNA. A fraction of RD114 cDNA(3'), corresponding to the conserved region, cross-hybridized extensively with RNA of a baboon endogenous virus (M7). Fractions of similar size prepared from cDNA(3') of MPMV, a primate type D virus, hybridized with M7 RNA to a lesser extent. Hybridization was not observed between Mason-Pfizer monkey virus and M7 if total cDNA's were incubated with viral RNAs. The degree of cross-reaction of the shared sequence appeared to be influenced by viral ancestral relatedness and host cell phylogenetic relationships. Thus, the strikingly high extent of cross-reaction at the conserved region between rodent viruses and simian sarcoma virus and between baboon virus and RD114 virus may reflect ancestral relatedness of the viruses. Slight cross-reaction at the site between type B and C viruses of rodents (mouse mammary tumor virus and RPL virus, 58-2T) or type C and D viruses of primates (M7, RD114, and Mason-Pfizer monkey virus) may have arisen at the conserved region through a mechanism that depends more on the phylogenetic relatedness of the host cells than on the viral type or origin. Determining the sequence of the conserved region may help elucidate this mechanism. The conserved sequences in retroviruses described here may be an important functional unit for the life cycle of many retroviruses.     

N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions

A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2. Removal of this N-linked oligosaccharide by mutagenesis enabled hamster ASCT1 to function as a receptor for all viruses of this interference group. These results strongly suggest that combinations of amino acid sequence changes and N-linked oligosaccharides in a critical carboxyl-terminal region of ECL2 control retroviral utilization of both the ASCT1 and ASCT2 receptors.     

Primate retroviruses: envelope glycoproteins of endogenous type C and type D viruses possess common interspecies antigenic determinants.

The major 70,000- to 80,000-molecular-weight envelope glycoproteins of the squirrel monkey retrovirus, Mason-Pfizer monkey virus, and M7 baboon virus and the related endogenous feline virus, RD114, were isolated and immunologically characterized. Immunoprecipitation and competition immunoassay analysis revealed these viral envelope glycoproteins to possess several distinct classes of immunological determinants. These include species-specific determinants, group-specific antigenic determinants unique to endogenous primate type C viruses, and group-specific determinants for type D viruses such as Mason-Pfizer monkey virus and squirrel monkey retrovirus. In addition, a class of broadly reactive antigenic determinants shared by envelope glycoproteins of both type C viruses of the baboon/RD114 group and type D viruses of the Mason-Pfizer monkey virus/squirrel monkey virus group are described. Other mammalian oncornaviruses tested, including isolates of nonprimate origin and representative type B viruses, lacked these determinants. The demonstration of antigenic determinants specific to envelope glycoproteins of type C and type D primate viruses indicates either that these viruses are evolutionarily related or that genetic recombination occurred between their progenitors. Alternatively, endogenous type D oncornaviruses may be replication defective, and acquisition of endogenous type C viral genetic sequences coding for envelope glycoprotein determinants may be necessary for their isolation as infectious virus.     

Evolutionary relationships between gag gene-coded proteins of murine and primate endogenous type C RNA viruses.

Several low molecular weight proteins of endogenous type C viruses of the RD114/baboon group are compared with the gag gene translational products of endogenous type C viruses of murine origin. The p10 proteins of each virus group are shown to be immunologically and biochemically related, while the p12 proteins of RD114/baboon viruses are demonstrated to share antigenic determinants with murine viral p15. Moreover, highly type-specific phosphoproteins, p15 of RD114/baboon viruses and p12 of murine viruses, are shown to possess very similar biochemical properties. These findings, along with previous studies indicating immunologic cross-reactivity between their major internal antigens, p30, demonstrate that each of the gag gene-coded proteins of murine type C viruses has a analogue in viruses of the RD114/baboon group. The immunologic and biochemical relatedness of their gag gene translational products supports the concept of a common progenitor in the evolution of these endogenous viruses.     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Spleen necrosis virus, an avian immunosuppressive retrovirus, shares a receptor with the type D simian retroviruses.

The reticuloendotheliosis viruses (REV) are a family of highly related retroviruses isolated from gallinaceous birds. On the basis of sequence comparison and overall genome organization, these viruses are more similar to the mammalian type C retroviruses than to the avian sarcoma/leukemia viruses. The envelope of a member of the REV family, spleen necrosis virus (SNV), is about 50% identical in amino acid sequence to the envelope of the type D simian retroviruses. Although SNV does not productively infect primate or murine cells, the receptor for SNV is present on a variety of human and murine cells. Moreover, interference assays show that the receptor for SNV is the same as the receptor for the type D simian retroviruses. We propose that adaptation of a mammalian type C virus to an avian host provided the REV progenitor.     

Novel Endogenous Type C Retrovirus in Baboons: Complete Sequence, Providing Evidence for Baboon Endogenous Virus gag-pol Ancestry

A complete endogenous type C viral genome has been isolated from a baboon genomic library. The provirus, Papio cynocephalus endogenous retrovirus (PcEV), is 8,572 nucleotides long, and 38 to 59 proviral copies per baboon genome are found. The PcEV provirus possesses the typical simple retroviral gene organization, including two long terminal repeats and genes encoding gag, pol, and env proteins. The open reading frames for gag-pol and env are complete but have premature stop codons or frameshift mutations. The primer binding site of PcEV is complementary to tRNAGly. The gag and pol genes of PcEV are closely related to those of the baboon endogenous virus (BaEV). The env coding region of PcEV is related to the env genes of type C retroviruses. This suggests that PcEV is one of the ancestors of BaEV contributing the type C gag-pol genome fragment to the type C/D recombinant virus BaEV. Earlier it was shown that another endogenous type D virus (simian endogenous retrovirus) provided the env gene for BaEV (A. C. van der Kuyl et al., J. Virol. 71:3666-3676, 1997).     

Ancient and modern retroviruses

Retroviruses are transmitted in two distinct ways: as infectious particles and as 'endogenous' proviral DNA integrated in the germ line of the host. Modern infectious viruses such as HIV-1 and HIV-2 recently infected mankind from chimpanzee and simian hosts, whereas human endogenous retroviral genomes have been present throughout old world primate evolution. Human T-cell leukemia viruses (HTLV-1 and II) have a much older human provenance than HIV, although new zoonoses from simians may also occur. We have recently characterized new retroviruses in pigs and humans. Porcine endogenous retroviral (PERV) genomes are carried in chromosomal DNA but can be activated to produce virions that are infectious for human cells, which has raised concern over human xenotransplantation using pig tissues. Human retrovirus 5 (HRV-5) is detected as an exogenous genome in association with arthritis and systemic lupus erythematosus.     

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.     

Replication-competent chimeric lenti-oncovirus with expanded host cell tropism.

Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.     

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.     

Reticuloendotheliosis type C and primate type D oncoretroviruses are members of the same receptor interference group.

The reticuloendotheliosis viruses (REVs), originally isolated from avian species, constitute a group of retroviruses which are more closely related to mammalian retroviruses than to other avian retroviruses. The envelope glycoproteins of members of the REV group display a striking amino acid sequence identity with a group of primate oncoretroviruses which belong to a single receptor interference group and include all of the type D and some type C primate oncoretroviruses. Members of the REV group also have a broad host range which covers most avian cells and some mammalian cells, including those of simian and human origin. In view of this broad host range and the envelope sequence similarities, we investigated the cross-interference pattern between REV and primate virus groups to determine whether they utilized the same receptor. Superinfection experiments using a vector virus containing an Escherichia coli lacZ gene showed that reticuloendotheliosis and simian oncoretroviruses constitute a single receptor interference group on both human and canine cells and indicate that the viruses bind to the same receptor to initiate infection. These results suggest that this receptor binding specificity has been maintained over a wide range of retroviruses and may be responsible for the broad spread of these retroviruses between different orders of vertebrates.     

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.     

Suppression of human lymphocyte mitogen response by disrupted primate retroviruses of type C (baboon endogenous virus) and type D (PMFV)

Disrupted primate retroviruses of type C (baboon endogenous virus, BaEV) and type D (human cell line-derived isolate PMFV) considerably suppressed Concanavalin A - induced blastogenic response of human lymphocytes. Rauscher mouse leukemia virus (RLV) displayed a suppressive activity on murine splenic lymphocytes when tested under analogous conditions. The immunosuppressive activities were shown not to result from cytotoxicity or from virus-mitogen binding.