Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Identification of a hepatic factor capable of supporting hepatitis C virus replication in a nonpermissive cell line

Although hepatitis C virus E2 protein can bind to human cells by interacting with a putative viral receptor, CD81, the interaction alone is not sufficient to establish permissiveness for hepatitis C virus infection. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a cDNA capable of supporting hepatitis C virus replication in an otherwise nonpermissive cell line. This cDNA encodes a protein exhibiting homology to a group of proteins derived from various evolutionarily distant species, including Oryza sativa submergence-induced protein 2A. The mRNAs encoding this factor are heterogeneous at the 5' ends and are ubiquitously expressed in multiple tissues, albeit in a very small amount. The longest mRNA contains an in-frame and upstream initiation codon and codes for a larger protein. This 5'-extended form of mRNA was detected in hepatocellular carcinoma, but not in normal liver tissue. Immunofluorescence analysis demonstrated that the hepatic factor was distributed evenly in cells, but occasionally formed aggregations in the peri- or intranuclear areas. In summary, we have identified a hepatic factor capable of supporting hepatitis C virus replication in an otherwise nonpermissive cell line. This factor belongs to a previously uncharacterized protein family. The physiological function of this protein awaits further study.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Herpes simplex virus 1 recombinants with noninverting genomes frozen in different isomeric arrangements are capable of independent replication.

Herpes simplex virus 1 genome consists of two covalently linked components, L and S, that invert relative to each other to yield four equimolar isomeric populations designated P (prototype), Is (inversion of S component), Il (inversion of L component), and Ils (inversion of L and S components) differing in the orientation of the two components. Previous studies have yielded recombinant genomes frozen in the P isomeric arrangement, reinforcing suggestions that the four isomers may not be functionally equivalent. We report the isolation of recombinants produced by insertional mutagenesis with alpha TK mini-Mu that are frozen in Is and Ils arrangements. Thus, all isomeric forms of herpes simplex virus DNA appear to be capable of independent replication and must be considered as functionally equivalent.     

Polyomavirus-plasmid recombinants capable of replicating have an enhanced transforming potential.

The frequency of transformation of rodent fibroblasts by polyomavirus is enhanced by a viral gene product, large T-antigen. However, this effect of large T-antigen cannot be demonstrated with pBR322-cloned viral DNA. Recently, it was discovered that pBR322 contains cis-acting sequences inhibitory to DNA replication in mammalian cells. Because polyomavirus large T-antigen is required for viral DNA replication, we examined the possibility that our inability to demonstrate a requirement for large T-antigen in transformation with pBR322-cloned viral DNA was due to the failure of the chimeric DNA to replicate in the transfected cells. To this end we constructed polyomavirus recombinant molecules with a plasmid (pML-2) that lacks these "poison" sequences and measured their capacity to transform cells. Here we report that recombinant plasmids capable of replicating in the transfected cells transform these cells at frequencies approximately sixfold greater than their replication-defective counterparts.     

Isolation of influenza C virus recombinants.

Recombinants between two different influenza C viruses were isolated. In MDCK (canine kidney) cells, one strain, C/JJ/50, caused lytic plaques, whereas C/JHG/66 virus did not produce clear plaques. From a mixed infection of MDCK cells with C/JHG/66 virus and UV-inactivated C/JJ/50 virus, clones were isolated which possessed the clear-plaque phenotype. Fingerprint analyses indicated that the RNAs of parent viruses had different oligonucleotide patterns and that one of the clones derived from the mixed infection was formed by reassortment of parental genes. This recombinant clone most likely inherited RNAs 1, 2, 3, 6, and 7 from C/JGH/66 virus and RNAs 4 and 5 from C/JJ/50 virus.     

Isolation and characterization of revertant cell lines. IV. Direct selection of serum-revertant sublines of SV40-transformed 3T3 mouse cells

SV101, the SV40-transformed subline of the mouse fibroblast line 3T3, is both serum- and density transformed, since it grows in both 1% and 10% calf serum, and grows beyond confluence in 10% calf serum. Negative selection at low cell density in 1% calf serum or in 10% agamma-depleted serum permits direct recovery of serum-revertant sublines of SV101. These sublines are unable to grow in 1% calf serum. Although negative selection at high cell density in 10% calf serum is known to permit recovery of density-revertant sublines of SV101, most density-revertants are not serum-revertant. However, all serum-revertants isolated so far are density-revertant as well.     

A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.     

Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.

Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes

Summary Two long-term cultured cell lines were established from BALB/c mouse axillary and cervical lymph nodes that exhibited a combination of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic unique to endothelial cells. Morphologically the cells were 20–30 μm in diameter and exhibited contact inhibition. The cells were not myeloid in origin because they lacked sodium fluoride-inhibitable nonspecific esterase activity, myeloperoxidase activity, and F4/80 antigen. The cell line phenotypes were compared to high endothelial venule (HEV) cells in tissue sections. HEV cells in lymph node tissue sections expressed endoglin, PECAM-1, ICAM-1, VCAM-1, laminin, fibronectin, collagen IV, H2K^d, MECA 79, MECA 325, and vWF. The cell lines expressed endoglin, VCAM-1, fibronectin, and H2K^d. The cell line derived from cervical lymph nodes also expressed laminin and H2D^d. Neither cell line expressed collagen IV, IA^d, ICAM-1, ICAM-2, dendritic cell antigen, or PECAM-1. They also did not express MECA antigens or intracellular vWF, consistent with reports of many cultured endothelial cells. To further substantiate cell line identification, antiserum generated against the cell lines bound specifically to HEV cells in frozen lymph node tissue sections and to both of the lymph node-derived cell lines but not control cell lines. Thus, the lymph node derived-cell lines expressed molecules found on HEV cellsin vivo and most importantly retained the functions of tubule formation, lymphocyte adhesion, and promotion of lymphocyte migration.     

Cytogenetic replication studies with short thymidine pulses in bromodeoxyuridine-substituted chromosomes of different mouse cell lines

Summary In normal diploid fibroblasts of the mouse, 3T3-, SV-3T3-, and Meth A-cells, the chromosome replication patterns were studied by a bromodeoxyuridine (BrdU)-labelling technique. SV-3T3 is a subline of 3T3 transformed by SV 40 and Meth A is a permanent cell line from Balb c transformed by methylcholanthrene. The use of 1 h thymidine pulses permits high resolution of the S-phase after partial synchronization of the cells at G₁/S in an otherwise BrdU-substituted S-phase. It could be shown that the autosomal heterochromatin of the mouse (Mus musculus) starts replication during the early S-phase (R-band replication), continues while R-band chromatin finishes, and still replicates when G-band chromatin starts. The heterochromatin finishes before the majority of G-bands have been replicated. There is no fundamental difference in the course of chromosome replication between the different cell lines studied here. It is concluded that there are no obligate changes in the course of the S-phase linked to the process of transformation.     

Isolation and characterization of revertant cell lines. 3. Isolation of density-revertants of SV40-transformed 3T3 cells using colchicine

Treatment of the SV40 transformed 3T3 cell line SV101 with colchicine permits the isolation of polyploid revertant sublines Which have lower saturation densities than SV101. These low saturation density lines have also reverted to a high serum requirement for growth, and are unable to form colonies in methocel. Normal SV40 has been recovered from these revertants. 3T3 cells are more resistant to colchicine than SV3T3 cells at all cell densities. Colchicine revertants do not display a 3T3-like resistance to colchicine at low density, but do survive colchicine at confluent cell densities, presumably due to their increased contact inhibition.     

Isolation of a metabolite capable of differentially supporting the growth of nicotinamide adenine dinucleotide auxotrophs of Escherichia coli.

A compound, isolated from the culture fluid of a nadC auxotroph of Escherichia coli grown in a minimal medium, supports the growth of both a nadA and a nadB mutant. This metabolite exhibits an ultraviolet light absorption spectrum and a mass spectrum, different from quinolinic acid. This compound may be the precursor of quinolinic acid, an intermediate in the biosynthesis of nicotinamide adenosine dinucleotide.