Infection Of Human Cell Lines Research Articles

Human monocytic cell lines transformed in vitro by Epstein-Barr virus display a type II latency and LMP-1-dependent proliferation.

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.

Varicella-zoster virus (VZV) mediates a delayed host shutoff independent of open reading frame (ORF) 17 expression.

Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor. We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.

Association of the Epstein-Barr virus with breast cancer: in vivo and in vitro studies

Epstein-Barr virus (EBV) may be a cofactor in the development of different malignancies, including several types of carcinomas. We demonstrated the presence of EBV in human breast cancers. We detected the EBV genome by PCR in 51% of the tumor biopsies. In 90% of the cases studied, the virus was not detected in healthy tissue. The presence of the EBV genome in breast tumors was confirmed by Southern-blot analysis. The EBV latent protein EBNA-1 was observed in a fraction (5-30%) of tumor epithelial cells. Expression of the EBV genes BNLF1 and BARF0 will be reported. A statistical relationship was established between the presence of EBV and several poor prognostic factors. EBV may be a cofactor in the development of a subset of breast cancers. Latently EBV infected breast undifferentiated human epithelial cell line, MDA-MB-231, was obtained and injected into nude mice. Tumors were obtained in which EBV persists. The persistence of EBV in nude mice tumors, in the absence of any selection, suggests that mammary epithelial cells could be a natural host for EBV. These models will be used for the elaboration of specific therapeutic targets. Normal breast mammary epithelial cells are now being infected by EBV in order to investigate the oncogenic potential of EBV in those epithelial cells.

Adenoviral vector expressing ICP47 inhibits adenovirus-specific cytotoxic T lymphocytes in nonhuman primates.

Studies from several laboratories have shown that administration of E1-deleted Ad vectors results only in transient transgene expression in the lungs of immunocompetent animals. This is due, at least in part, to destruction of vector-transduced cells by host cellular immune responses (predominantly CD8(+) CTLs) directed against viral proteins and/or immunogenic transgene products. We have previously demonstrated that E1-deleted Ad vectors can lead to persistent expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) in the lungs of several strains of immunocompetent mice, despite the presence of Ad-specific CTLs. However, we found that these same vectors gave rise only to transient hCFTR expression in the lungs of rhesus monkeys. We have constructed new Ad vectors that coexpress both hCFTR and the ICP47 gene from herpes simplex virus. ICP47 has been shown to inhibit the transporter associated with antigen presentation, thus blocking major histocompatibility antigen I (MHC class I)-mediated antigen presentation to CD8(+) T cells. The Ad/hCFTR/ICP47 vector decreased levels of cell-surface MHC class I molecules on infected monkey and human cell lines. Similar results were obtained with primary human cells and primary monkey airway epithelial cells. In vitro studies showed that the Ad/hCFTR/ICP47 vector decreased cytolysis by both monkey and human CTLs. When Ad/hCFTR/ICP47 was administered to the lungs of rhesus monkeys, it inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced in monkeys treated with the Ad/hCFTR/ICP47 vector.

6-Aminoquinolones as new potential anti-HIV agents.

A series of 6-aminoquinolone compounds were evaluated for their in vitro activity against human immunodeficiency virus type 1 (HIV-1). Compound 12a, bearing a methyl substituent at the N-1 position and a 4-(2-pyridyl)-1-piperazine moiety at the C-7 position, was the most active in inhibiting HIV-1 replication on de novo infected C8166 human lymphoblastoid cell lines. The 12a EC(50) value was 0.1 microM, a 7-20-fold lower concentration relative to that for compounds 8a and 7a containing a cyclopropyl and tert-butyl substituent at the N-1 position, respectively. When the C-6 amino group was replaced with a fluorine atom, a decreased antiviral effect was observed. The observed effects are selective, since potency is substantially reduced when testing the compounds against the herpes simplex virus type 1 (HSV-1). Active quinolone derivatives very efficiently interact with TAR RNA, which suggests a nucleic acid-targeted mechanism of action.

The first 6 weeks of the treatment of a psychotic episode: Know more by better measuring?

The expression of type I interferon (IFN) is one of the most potent innate defences against viral infection in higher vertebrates. Borna disease virus (BDV) establishes persistent, noncytolytic infections in animals and in cultured cells. Early studies have shown that the BDV phosphoprotein can inhibit the activation of type I IFN through the TBK1–IRF3 pathway. The function of the BDV nucleoprotein in the inhibition of IFN activity is not yet clear. In this study, we demonstrated IRF7 activation and increased IFN-α/β expression in a BDV-persistently infected human oligodendroglia cell line following RNA interference-mediated BDV nucleoprotein silencing. Furthermore, we showed that BDV nucleoprotein prevented the nuclear localisation of IRF7 and inhibited endogenous IFN induction by poly(I:C), coxsackie virus B3 and IFN-β. Our findings provide evidence for a previously undescribed mechanism by which the BDV nucleoprotein inhibits type I IFN expression by interfering with the IRF7 pathway.

Transgenic mice expressing human HIV receptors become persistently recipient of HIV DNA after injection with infected human cell lines.

Transgenic mice expressing human HIV receptors become persistently recipient of HIV DNA after injection with infected human cell lines.

The efficacy of nucleoside analogs against JC virus multiplication in a persistently infected human fetal brain cell line.

The effectiveness of nucleoside analogs in blocking viral multiplication was evaluated using an immortalized human neuroglial cell line capable of sustaining a persistent JCV infection, SVG-JC. Results from in situ DNA hybridization and hemagglutination assays performed on drug treated cultures were used as a measure of viral DNA replication and multiplication, respectively. Of the three drugs tested, Ara-C (cytosine arabinoside), AZT (3'-azido-3'-deoxythymidine), and cidofovir (S)-1-[3-hydroxy-2-(phosphonylmethoxypropyl] cytosine), only Ara-C showed a significant effect in decreasing active JCV replication and multiplication. In vitro data, using different cell types and virus strains have shown that specific drugs can indeed modulate viral infection. However, such modulation has not previously been demonstrated in those cells of the CNS which are specifically targeted by JCV. The SVG-JC cells represent a unique system with which further studies can be conducted on the effects of drugs on brain derived cells that are susceptible to viral infection.

Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression.

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.

Inhibition of Respiratory Syncytial Virus by Double Termini-Protected 2–5A Antisense Chimeras

Respiratory syncytial virus (RSV) replication was reduced by greater than 90% after treatment of infected human tracheal epithelial cell line, 9HTE, with double termini-protected 2–5 A antisense chimeras.

Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies. Induction of HIV-1 expression, measured by p24 core antigen capture enzyme-linked immunosorbent assay (ELISA), was found to be exclusively triggered by TNFR-p55 stimulation. However, our results also showed that the addition of TNFR-p75-specific ligands negatively modulated the HIV-1 expression induced via TNFR-p55.

Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.

Superantigens Activate HIV-1 Gene Expression in Monocytic Cells

Binding of superantigens to MHC class II molecules results in transduction of biochemical signals leading to cellular activation and gene expression. We demonstrate that the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA) activate HIV-1-LTR-driven transcription of chloramphenicol acetyl transferase in the human monocytic cell line THP-1. Induction of HIV-1-LTR-driven transcription in THP-1 cells hy superantigens was associated with the induction of nuclear factor-κB DNA-binding activity. Superantigens also increased viral protein secretion from the granulocyte-macrophage colony-stimulating factor-pretreated chronically infected human monocytic cell line U1. Induction of HIV-1 gene expression in monocytic cells by superantigens occurred via tumor necrosis factor-α-dependent and -independent mechanisms. Our results suggest that superantigens and other MHC class II ligands may activate HIV-1 gene expression in monocytes/macrophages.

Interleukin 1 induces expression of the human immunodeficiency virus alone and in synergy with interleukin 6 in chronically infected U1 cells: inhibition of inductive effects by the interleukin 1 receptor antagonist.

In the present study we have observed that interleukin (IL) 1 alpha or IL-1 beta directly induced expression of human immunodeficiency virus (HIV) in the latently infected human promonocytic cell line U1. In addition, IL-1 synergized with IL-6, but not with tumor necrosis factor, in the upregulation of virus expression in U1 cells as measured by accumulation of steady-state mRNAs and production of reverse transcriptase activity. The HIV inductive effect of IL-1 was blocked by transforming growth factor beta, anti-IL-1 antibodies, or monoclonal antibodies directed to the type 1, but not to the type 2, cell surface receptor for IL-1; the latter actually caused enhancement of the IL-1-mediated effect. Unlike tumor necrosis factor alpha, IL-1 either alone or in combination with IL-6 did not induce activation of the transcription activating factor NF-kappa B above the constitutive levels of unstimulated U1 cells. Finally, the IL-1 receptor antagonist effectively blocked IL-1-mediated direct and synergistic inductive effects on virus production. Thus, IL-1 may be an important mediator of HIV expression, and blocking of IL-1 expression and/or its effects may have a potential therapeutic role in the inhibition of HIV expression in infected individuals.

Soluble tumor necrosis factor receptor: inhibition of human immunodeficiency virus activation.

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to stimulate human immunodeficiency virus type 1 (HIV-1) replication in both chronically and acutely infected T lymphocytes and monocytes. Transcriptional activation of the HIV long terminal repeat and subsequent increase in virus production are linked to TNF activation of the cellular transcription factor NF-kappa B. Here we report the use of two forms of soluble recombinant type 1 (p80) TNF receptor to inhibit TNF-induced HIV activation in vitro. One receptor form is a monomer containing the entire 236 residues of the extracellular (ligand-binding) portion of p80. A second receptor form is a chimeric homodimer containing these residues fused to a truncated human IgG1 immunoglobulin heavy chain and, thus, resembles a bivalent antibody without light chains. These recombinant receptor proteins were tested for their ability to inhibit TNF-alpha-induced expression of HIV-1 in chronically infected human cell lines. We also examined the ability of the soluble receptors to limit the activation of the HIV-long terminal repeat transcription. The soluble TNF receptor dimer was most effective at blocking TNF-alpha-induced HIV-1 expression in both monocytoid and lymphoid cells. The molar ratio of TNF-receptor dimer to TNF-alpha found to be most effective was, at least, 5:1. We conclude that at specific TNF/soluble TNF-receptor dimer ratios, TNF-alpha-induced HIV-1 transcription and expression can be limited in vitro.

Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-α

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-α had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-α production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-α production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.

4-oxatetradecanoic acid is fungicidal for Cryptococcus neoformans and inhibits replication of human immunodeficiency virus I.

Candida albicans and Cryptococcus neoformans are major causes of systemic fungal infections, particularly in patients with acquired immunodeficiency syndrome. Metabolic labeling studies revealed that these organisms synthesize a small number of N-myristoylproteins, the most prominent being 20-kDa ADP-ribosylation factors (Arfs). C. albicans Arf has approximately 80% identity with the essential Arf1 and Arf2 proteins of Saccharomyces cerevisiae. [3H]Myristic acid analogs with oxygen for -CH2- substitutions at C4, C6, C11, and C13 are incorporated into cellular N-myristoylproteins, phospholipids, and neutral lipids produced by these three yeasts during exponential growth at 30 degrees C in complex media. Analog- and organism-specific differences in the efficiency of labeling of proteins and lipid classes were observed. The effects of oxatetradecanoic acids with oxygen for -CH2- substitutions at C3-C13 on C. neoformans, C. albicans, and S. cerevisiae were assessed during mid-log phase growth at 30 degrees C. A single dose of 3-oxa-, 4-oxa-, 5-oxa- or 6-oxatetradecanoic acid (O3-O6, final concentration = 300 microM) was able to inhibit growth of C. neoformans in the order O4 greater than O5 greater than O3 approximately O6. The other compounds were inactive. 4-Oxatetradecanoic acid was fungicidal, producing a 10,000-fold reduction in viable cell number 1 h after administration and continued suppression of cell growth for 7 h. A clear dose response was observed over a concentration range of 100-300 microM. 4-Oxatridecanoic acid was 100-fold less potent in reducing cell viability than 4-oxatetradecanoic acid but more potent than 5-oxatridecanoic acid. O4 produced approximately 10-100-fold reductions in the viability of C. albicans and S. cerevisiae at 300-500 microM, respectively, whereas O5 and O6 were less active. Since N-myristoylation of the Pr55gag polyprotein precursor produced by human immunodeficiency virus I (HIV-I) is essential for its assembly, we also assessed the antiviral effects of 4-oxatetradecanoic acid. O4 is able to produce a 50% reduction in the replication of HIV-I in acutely infected human T-lymphocyte cell lines at a concentration of 18 microM. Together, these data suggest that (i) the position of the oxygen for methylene substitution is a critical determinant of the fungicidal activity of O4 and (ii) NMT may be an attractive therapeutic target for treating opportunistic fungal infections in patients infected with HIV-I.

Retrovirus mediated transfer of antisense human c-myc gene into human esophageal cancer cells suppressed cell proliferation and malignancy.

A retroviral vector, called pDAM3, containing the neomycin resistant gene and the antisense human c-myc gene fragment (the third exon and 3' flanking sequence) was constructed. pDAM3 was introduced into amphotropic packaging cells PA317 by the calcium phosphate precipitation method. Several G418-resistant PA317 clones were isolated. The virus titer of these cell lines was determined by infectivity of their culture fluid to NIH/3T3 cells. The highest titer obtained was 8 x 10(5) G418-resistant colony forming units/ml. Clonal and pooled G418-resistant PA317 colonies with high titers were expanded and analyzed by Southern blot for the presence of intact viral sequences. All cell lines were found to harbor the internal sequences of the pDAM3 vector without any rearrangement. Recombinant virus DAM3 infected human esophageal cancer cell line EC8712 efficiently. The DAM3-infected EC8712 (called EC-DAM3) was found to contain the full DAM3 sequence (4.8 kb) by Southern blot analysis. Antisense myc RNA expressed in the EC-DAM3 cell was detected by RNA hybridization. Further studies indicated that [3H]-thymidine incorporation in EC-DAM3 cells was reduced by 45% in average compared to that in untreated EC8712 cells. Growth rate of EC-DAM3 cells also decreased about 50%. DAM3-infected EC8712 cells lost their ability of forming tumor in nude mice. It thus appeared that the antisense myc gene introduced into EC8712 cells via retrovirus vector was capable of inhibiting cell proliferation and malignancy.

Detection of viral surface antigens on HIV-2ben infected human tumor cell lines by flow cytometry.

The human monocytic cell line U-937 clone 2 and two T-cell lines CEM and MOLT-4 clone 8 were infected with HIV-2ben, a recent isolate of HIV-2. Infection and subsequent antigen expression on the cell surface was monitored by flow cytometry using a rabbit-anti-serum against tween-ether-treated HIV-2ben and a fluorescein-isothiocyanate-conjugated IgG against rabbit-IgG. The sensitivity of the three cell lines to infection with HIV-2ben correlated with the percentages of CD4-expressing cells but not with the levels of CD4-expression on the cell. The appearance of viral surface antigens preceded the formation of syncytia and correlated closely with the infecting virus dose. After 1-2 weeks in culture, 20-85% of the cells of each line expressed viral surface antigens. The variation depended on the cell type and cell culture conditions. The MOLT-4 clone 8 and the U-937 clone 2 cells died around 10 or 20 days, respectively, after HIV-2ben infection. Only HIV-2ben infected CEM cells grew permanently. Flow cytometry was an appropriate method to monitor the expression of viral proteins on the cell surface of HIV-infected cell lines. Flow cytometry proved to be more sensitive than determination of RT activity in supernatants of HIV-infected cells and more precise than light microscopy examinations.