Cell receptors for baboon endogenous virus recognized by monoclonal antibodies.

Hybridomas from mice immunized with baboon endogenous virus (BaEV) from A204(M7) cells produced several antiviral monoclonal antibodies and, in addition, antibodies D-12 and E-4, which appeared to be virus specific because they reacted with BaEV but not with Mason-Pfizer virus or RD-114 virus. However, they also bound to human virus-free cells, and they did not recognize BaEV from bat or canine host cells. Cell membrane targets for these antibodies comigrated with an 18,000-dalton protein, which may contain specific determinants of BaEV receptors since antibody masking of these cell sites prevented BaEV but not Mason-Pfizer virus or RD-114 virus adsorption. However, RD-114 virus interfered with BaEV adsorption. Thus, the two viral receptors must be adjacent, but the antibody D-12 and E-4 targets are not within the active site of RD-114 virus receptor. Conversely, cell coating with BaEV from bat or canine hosts inhibited antibody D-12 binding. Noncultivated human lymphocytes and cells from fetal organs bound much less antibody D-12 than did cells from established cell lines, with a correlation between amounts of antibody D-12 acceptor sites and BaEV receptors. Thus, in vivo, BaEV infection of human cells may be inefficient. In vitro, antibody D-12 treatment of chronically infected A204(M7) cells caused intracellular accumulation of viral proteins and decreased virus release, with no such effect on RD-114 virus-producing cells. Canine cells bound antibody D-12 only if coated with BaEV from A204(M7) cells, indicating that the human determinant coadsorbed with the virions to animal cells. Possibly, determinants of cell receptors participate in BaEV maturation and become associated with the virions.     

Baboon Virus Isolate M-7 with Properties Similar to Feline Virus RD-114

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.     

Expression of interferon-inducible genes in RD-114 cells.

RD-114 is a cell line which is partially responsive to interferon (IFN). Although both IFN-alpha and IFN gamma inhibit production of the resident retrovirus, they do not inhibit replication of other viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, in these cells. In the studies reported here, we studied the characteristics of induction of seven IFN-inducible mRNAs in RD-114 cells. We observed that mRNAs 561, 6-16, 1-8, 2A, and 6-26 have similar induction characteristics in RD-114 cells and in HeLa cells, a fully responsive line. mRNA 2'-5'-oligo-adenylate synthetase (2-5(A) synthetase), however, was induced more efficiently by IFN-alpha in HeLa cells than in RD-114 cells. The same was true for the induction of metallothionein II mRNA by IFN-gamma. However, the latter mRNA was induced equally strongly in both lines when ZnCl2 was used as the inducer, suggesting that the gene is not defective in RD-114 cells. Although IFN-alpha induced 2-5(A) synthetase mRNA poorly and IFN-gamma did not induce it at all in these cells, a mixture of IFN-alpha and IFN-gamma induced this mRNA quite effectively, to a level of induction comparable to that in HeLa cells. Only 1 U of IFN-gamma per ml was sufficient to elicit this synergism, and the data suggested that an IFN-gamma-inducible protein was needed for this process. Induction of mRNA 561 by IFN-alpha in RD-114 cells, unlike that in HeLa cells, did not need ongoing protein synthesis. Once induced, this mRNA turned over rapidly in both cell lines, and this turnover could be slowed down by inhibiting protein synthesis in either cell line. IFN-induced mRNAs, such as 561 and 1-8, were polysome associated in IFN-treated RD-114 cells, suggesting that they were actively translated. Therefore, it is unlikely that the products of these IFN-inducible genes, by themselves, mediate the inhibition of replication of those viruses which are insensitive to IFN action in RD-114 cells.     

35S]S-[5-(4-benzoylphenyl)pentyl]glutathione: a versatile radioligand targeting GS-X pumps with the ability of photoaffinity labeling.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione (GIF-0017) as a biochemical probe targeting the ATP-dependent organic anion transporters GS-X pumps was synthesized by the reaction of [35S]glutathione and excess 4-(5-bromo)pentylbenzophenone under alkaline conditions, with the radiochemical yield of 24-33% after HPLC purification. Photolysis of the mixture of [35S]GIF-0017 and plasma membrane vesicles prepared from the MRP1 cDNA-transfected LLC-PK1 cells resulted in radio-labeling of a 180-kDa membrane protein. Immunoprecipitation and western blotting using an anti-MRP1 monoclonal antibody confirmed that the [35S]GIF-0017-labeled protein was the MRPI/GS-X pump.     

Enhancement of the growth of human osteosarcoma cells by human interferon-gamma.

It is well known that interferons inhibit cell growth. However, we found that human interferon-gamma (HuIFN-gamma) enhanced the growth of human osteosarcoma cells, HOS-Y1 cells, in a dose-dependent manner. This enhancing effect was found only under the following conditions: when the cells were precultured for 2 or 3 days and then treated with HuIFN-gamma for 2, 3, or 4 days, and when the cells were seeded at a density of 1,000 or 2,000 cells/well. The degree of enhancement of cell growth was maximum when the cells were precultured at a density of 1,000 cells/well for 3 days and then treated with HuIFN-gamma for 2 days. The enhancing effect of HuIFN-gamma disappeared in the presence of anti-HuIFN-gamma antibody. In addition, it was found that the conditioned medium from HOS-Y1 cells enhanced the growth of HOS-Y1 cells, and that the conditioned medium from HOS-Y1 cells cultured with HuIFN-gamma enhanced the cell growth more than that from cells cultured without HuIFN-gamma. Epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta 1(TGF-beta 1) did not enhance the growth of HOS-Y1 cells. These results suggest that HuIFN-gamma enhanced the cell growth by augmenting the production of unknown growth factor(s) in HOS-Y1 cells via an autocrine mechanism.     

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.     

RD 114 Virus-Specific Sequences in Feline Cellular RNA: Detection and Characterization

RNA extracted from cat cells contains sequences homologous to RD-114 viral RNA. The sequences are measured by molecular hybridization with a single-stranded DNA probe synthesized by the virion polymerase using the endogenous viral RNA as template. Viral-specific RNA has been detected in all cells of cat origin tested thus far, but not in cells of other animals, except for the virus-producing human rhabdomyosarcoma cell, RD-114. The extent of hybridization of the DNA probe to cellular RNA was equivalent to that obtained with viral 70S RNA indicating that an equal extent of viral specific sequences is present in all cat cells as well as in RD-114 cells. The amounts of this viral RNA reach approximately 100 copies per cell in cat cells, while virus-producing RD-114 cells contain about 1,000 copies per cell. The viral RNA is present in cat cells in two distinct sizes of about 35S and 18S, whereas in RD-114 cells virus-specific RNA is quite heterogeneous in size.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

Discovery of a New Endogenous Type C Retrovirus (FcEV) in Cats: Evidence for RD-114 Being an FcEVGag-Pol/Baboon Endogenous Virus BaEVEnv Recombinant

Analysis of a cat genomic DNA library showed that cats harbor a previously unrecognized endogenous type C retrovirus, whose env gene has homology to the murine Fv-4 resistance gene. This unique retrovirus, designated FcEV (Felis catus endogenous retrovirus), has a type C pol gene, closely related to the primate Papio cynocephalus endogenous virus (PcEV) pol, not overlapping the env gene, unlike in other type C retroviruses, and is presumably present in a higher copy number than RD-114. Phylogenetic analysis of FcEV and RD-114 fragments amplified from cat species and comparison with baboon endogenous virus (BaEV) fragments from monkeys suggested that RD-114 does not represent the cat strain of BaEV but is actually a new recombinant between FcEV type C genes and the env gene of BaEV. Although BaEV did appear to have infected an ancestor of the domestic cat lineage, it was a de novo recombinant that made its way into the cat germ line.     

Heteroduplex study of the sequence relations between RD-114 and baboon viral RNAs.

The regions of sequence homology and nonhomology between the RNA genomes of RD-114 and baboon endogenous type C viruses have been mapped by an electron microscope heteroduplex study. Short complementary DNA (cDNA) copies (approximately 150 to 200 nucleotides in length) of RD-114 RNA were prepared by an endogenous synthesis; labels of polydeoxythymidylic acid [poly(dT)] were attached to the 3' ends of the cDNA molecules by a reaction catalyzed by deoxynucleotidyl terminal transferase. The cDNA-poly(dT) was hybridized to RD-114 RNA and to baboon viral RNA dimer (50 to 70S) units, and the position- of the poly(dT) labels were observed by electron microscopy. With RD-114, labels were distributed uniformly along the genome. With baboon virus RNA (monomer length, 9.5 kilobases [kb]), the regions of high homology with RD-114 cDNA were observed to lie in the intervals from 1.5 to 2.5 kb and from 3.7 to 5.5 kb from the 5' end. The relations of these heteroduplex maps to the known antigenic similarities and differences among the several viral proteins and to the genetic maps of the viruses are discussed.     

Structural Characteristics and Nucleotide Sequence Analysis of Genomic RNA from RD-114 Virus and Feline RNA Tumor Viruses

The results of molecular hybridization experiments have demonstrated that the RNA genome of RD-114 virus has extensive nucleotide sequence homology with the RNA genome of Crandell virus, an endogenous type C virus of cats, but only limited homology with the RNA genomes of feline sarcoma virus and feline leukemia virus. The genomic RNAs of RD-114 virus and Crandell virus also had identical sedimentation coefficients of 50S. A structural rearrangement of genomic RNA did not exist within released RD-114 virions, whereas a structural rearrangement of genomic RNA did occur within feline sarcoma virions and feline leukemia virions after release from virus-producing cells.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Genetic mapping of endogenous RD-114 retroviral sequences of domestic cats.

The RD-114 family of endogenous retroviral sequences in domestic cats has been shown to consist of approximately 20 copies of genetically divergent virogenes per haploid genome. The chromosomal localization for four endogenous sequences (RDV1-4) was accomplished by correlating the occurrence of specific feline chromosomes with diagnostic viral DNA fragments in a panel of cat X rodent somatic cell hybrids. Analysis of the hybrid panel revealed that endogenous RD-114 sequences are dispersed on multiple cat chromosomes, that certain proviral segments are polymorphic with respect to the presence or absence of virus, and that a restriction fragment characteristic of inducible RD-114 resides on a single feline chromosome (B3), probably at a single locus.     

Viral-specific antigen synthesis following de novo RD-114 virus infection of stationary cells

The cell cycle dependence of retrovirus replication was studied. Canine sarcoma (D-17) cells were infected de novo with the xenotropic feline retrovirus RD-114 under conditions previously reported to simultaneously inhibit virus replication and cell DNA synthesis and/or cell division. RD-114 viral antigen synthesis was observed under conditions previously reported to be inhibitory to avian and murine oncornavirus replication, including confluency and serum deprivation, X-irradiation, mitomycin C pretreatment, colchicine, and ethidium bromide treatments of cells. Several mechanisms that could account for viral antigen synthesis under the restrictive conditions used are discussed.     

Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter.

An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes. The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.     

Protection against murine neonatal herpes simplex virus infection by lymphokine-treated human leukocytes

One-week-old mice were protected against a uniformly lethal herpes simplex virus (HSV) infection by IL-2 alone, but especially by the addition of human mononuclear cells (MC) plus IL-2. The dose response of IL-2 was biphasic. The addition of MC from cord blood did not enhance IL-2-mediated survival. Because the effect of IL-2 alone, or IL-2 plus MC, was ablated by anti-IFN-gamma and human neonates have an IFN-gamma production defect, the protective effect of MC plus human IFN-gamma (HuIFN-gamma) was tested. MC from adults cultured for 5 days in HuIFN-gamma afforded protection. At least 1 x 10(6) HuIFN-gamma-treated MC were required with increasing survival to 1 x 10(7) MC. The effector cell activity was ablated by adherence, silica, L-leucine methyl ester treatment or treatment with Leu-M3 plus C (all macrophage markers), and OKT4 plus C treatment (CD4 marker). Use of Leu-11, Leu-7, OKT3, or OKT8 plus C did not inhibit protection and excluded NK or T cell participation. In addition to survival, the ability to produce anti-HSV antibody was reconstituted. For the first time protection was afforded by human cord blood MC after treatment with HuIFN in vitro. We have identified an IFN-gamma-driven protection system against murine neonatal HSV infection mediated by human adult- or cord blood-derived CD4-positive macrophages. Protection is associated with enhanced effector cell function and reconstitution of the neonatal antibody production defect.     

Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.     

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.