Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Development of a novel screen for protease inhibitors.

We have developed a novel plasmid-based, quantitative, in vitro screen to test the protease-inhibiting activities of existing and newly discovered agents.

Mechanism of interference mediated by human parainfluenza virus type 3 infection.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.

Inhibition of HIV-1 replication by combined expression of gag dominant negative mutant and a human ribonuclease in a tightly controlled HIV-1 inducible vector.

An HIV-1-based expression vector has been constructed that produces protective genes tightly regulated by HIV-1 Tat and Rev proteins. The vector contains either a single protective gene (HIV-1 gag dominant negative mutant (delta-gag)) or a combination of two different protective genes (delta-gag and eosinophil-derived neurotoxin (EDN), a human ribonuclease) which are expressed from a dicistronic mRNA. After stable transfection of CEM T cells and following challenge with HIV-1, viral production was completely inhibited in cells transduced with the vector producing both delta-gag and EDN and delayed in cells producing delta-gag alone. In addition, cotransfection of HeLa-Tat cells with an infectious HIV-1 molecular clone and either protective vector demonstrated that the HIV-1 packaging signals present in the constructs were functional and allowed the efficient assembly of the protective RNAs into HIV-1 virions, thus potentially transmitting protection to the HIV-1 target cells.

IL-6 signals inhibition of cell adhesion in melanoma A375-C6.

IL-6 has been found to be a potent inhibitor of melanoma A375-C6 cell adhesion, in addition to its known action in arresting cells at G1/G0 phase of the cell cycle IL-6 treated melanoma cells were found to round up and to lose the ability to adhere to fibronectin, laminin, collagen, and tenascin over 72 to 96 hours of IL-6 treatment, a time course similar to that seen for cell cycle inhibition. Cell cycle inhibition and loss of adhesion were found, however, to be independent effects of IL-6. Analysis of cell surface integrins indicated significant changes in the expression of several integrins including downregulation of a3 and av beta 5 and upregulation of a3. However, the changes in integrin expression did not correlate with loss of adhesion to relevant ligands. Three A375 melanoma clones varying in metastatic potential also demonstrated inhibition of both cell proliferation and matrix adhesion by IL-6.

Leukemia inhibitory factor induces interleukin-8 and monocyte chemotactic and activating factor in human monocytes: differential regulation by interferon-gamma.

Leukemia inhibitory factor (LIF) is a cytokine released at the site of injuries where there is a recruitment of monocytes and polymorphonuclear cells. We analyzed the effect of LIF on human monocytes, which are a major source of chemotactic factors. We showed that supernatants of monocytes treated with LIF (50 ng/mL) for 18 hours had chemotactic activity for neutrophils and monocytes that was neutralized by anti-interleukin-8 (anti-IL-8) and anti-monocyte chemotactic and activating factor (anti-MCAF) neutralizing antibodies. Northern blot analysis showed induction of IL-8 and MCAF RNA in monocytes treated with LIF. Both IL-8 and MCAF mRNA were induced within 3 hours of stimulation. IL-8 and MCAF mRNAs expression peaked at 6 hours and 18 hours, respectively. Interferon-gamma (IFN-gamma), a potent monocyte activator, inhibited IL-8 induction by LIF. On the contrary, IFN-gamma by itself induced MCAF and did not affect the LIF-induced MCAF. These results indicate that LIF released at the site of injury by inducing IL-8 and MCAF can play an important role in recruiting leukocytes and that IFN-gamma can differentially regulate this recruitment.

Interleukin-2 and human monocyte activation.

The recognition of the monocyte/macrophage-activating properties of IL-2 has broadened our image of the biological effects of this lymphokine from those of a T cell growth factor to those of a molecule with pleiotropic effects. The detailed analysis of the mechanisms of action of IL-2 including its biological effects on different cell types and the regulation of its receptors has increased dramatically the spectrum of the biological responses that can be modified by IL-2. The regulation of the expression of the IL-2 receptor subunits differs in terms of response to extracellular stimuli and intracellular control, suggesting that the response to IL-2 will vary depending on the nature and extent of environmental stimulation. Furthermore, the fact that the IL-2R gamma chain can be part of the receptor for IL-4, IL-7, and perhaps other cytokines indicates that IL-2 may modulate the response of monocytes simply by binding or releasing the IL-2R gamma chain and thus modulating the responsiveness to IL-4 or IL-7. Conversely, the extent of utilization of IL-2R gamma chain by various cytokines may dictate the monocytic response to IL-2. In fact, the availability of IL-2R gamma chain seems to be the limiting factor in the response of monocytes to IL-2. Modulation of cytokine receptors is an integral part of the control of the IL-2 response. The induction of CSF-1 receptor by IL-2 and the positive effect of CSF-1 on the duration of the cytotoxic response in IL-2-stimulated monocytes are an interesting example of a synergistic interaction of potential physiological relevance. The response of monocytes to IL-2 can also be modulated by inhibitory circuits, such as those involving TGF-beta 1, IFN-gamma, and IL-4. However, IFN-gamma and IL-4 can also activate monocytes and the timing and relative concentrations of the various cytokines may be critical variables in determining the ultimate monocyte phenotype. These studies have given us a glimpse of a very complex picture composed of multiple backgrounds and several players. However, the present information is not sufficient to make meaningful predictions of the resulting monocyte phenotype in an inflammatory reaction in which multiple cytokines are involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Potential requirement of a functional double-stranded RNA-dependent protein kinase (PKR) for the tumoricidal activation of macrophages by lipopolysaccharide or IFN-alpha beta, but not IFN-gamma.

We analyzed the expression of the dsRNA-dependent protein kinase (PKR) during the activation of murine macrophages to the tumoricidal state by LPS and/or IFNs. LPS induced PKR expression in a dose-dependent manner at levels that were comparable with those observed in response to IFNs. By using the PKR inhibitor 2-aminopurine (2-AP), we have shown that the pathways of macrophage tumoricidal activation elicited by LPS and IFN-alpha beta, but not by IFN-gamma, included a 2-AP-sensitive step. In fact, LPS- and IFN-alpha beta-induced activation was inhibited by 2-AP, whereas the activation by IFN-gamma was not affected by the presence of the inhibitor. 2-AP did not affect the activation of protein kinase C or protein kinase A in intact cells. In the presence of 2-AP the up-regulation of IFN-beta mRNA by LPS was specifically inhibited, whereas the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA or the induction of PKR remained unchanged, thereby demonstrating that 2-AP inhibited selective macrophage genes. The differential sensitivity to 2-AP suggested that the expression of a functional PKR may be required for the macrophage tumoricidal response triggered by LPS and IFN-alpha beta but not IFN-gamma.

LPS-inducible nuclear factor in human monocytes that binds the negative regulatory element of the HIV LTR.

We studied the constitutive and lipopolysaccharide (LPS)-induced expression of nuclear protein binding to the negative regulatory element (NRE) of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in fresh human monocytes. We demonstrated the existence of a constitutive factor binding to the NRE 73-bp HpaII/HpaII fragment (-216 to -143) whose expression is up-regulated by LPS treatment. Competition experiments with overlapping oligonucleotides covering the HpaII/HpaII fragment and with mutated oligonucleotides mapped the binding within the TTTCATCAC region (-171 to -163). This binding pattern is unique to human monocytes.

Regulation by interleukin-2 (IL-2) and interferon gamma of IL-2 receptor gamma chain gene expression in human monocytes.

The interleukin-2 receptor gamma chain (IL-2R gamma) gene codes for a subunit of the IL-2R and is expressed in human lymphoid cells. The present study was undertaken to determine whether human monocytes expressed the IL-2R gamma gene constitutively or after activation by IL-2 or interferon gamma (IFN gamma). Fresh human monocytes constitutively expressed low but significant levels of IL-2R gamma mRNA, and nuclear run-on experiments showed that IL-2R gamma gene was transcriptionally active. Stimulation with IL-2 or IFN gamma induced a major increase of IL-2R gamma mRNA in a time- and a dose-dependent manner. However, neither cytokine increased the transcriptional activity of the gene. The enhancement of IL-2R gamma mRNA expression by either IL-2 or IFN gamma was concomitant with the stabilization of the mRNA, suggesting a postranscriptional level of control. Finally, the augmented expression of IL-2R gamma in IL-2- and IFN gamma-treated monocytes was associated with an increased IL-2-binding activity, compared with that of unstimulated cells. These results provide the first evidence of the expression of the IL-2R gamma gene in nonlymphoid cells and of its modulation by IL-2 and IFN gamma through posttranscriptional mechanisms.

Interleukin-4 inhibits indoleamine 2,3-dioxygenase expression in human monocytes.

Indoleamine 2,3-dioxygenase (IDO), a flavin-dependent enzyme that catalyzes the conversion of tryptophan to kynurenine, is induced in peripheral blood mononuclear cells by interferon-gamma (IFN gamma). Interleukin-4 (IL-4) is a cytokine that modulates the functional properties of monocytes/macrophages, and we investigated the effects of IL-4 on IDO. We showed that IL-4 inhibited the induction of IDO mRNA and IDO activity by IFN gamma in human monocytes. The inhibitory effect was evident with as little as 2 U/mL of IL-4. These results provide the first evidence that a cytokine can provide a negative signal for IDO expression and that IL-4 can influence the catabolism of tryptophan.

Interferon-gamma upregulates interleukin-8 gene expression in human monocytic cells by a posttranscriptional mechanism.

Interleukin-8 (IL-8) is a neutrophil chemotactic and activating cytokine that is produced in response to several stimuli. Because monocytic cells are important producers of IL-8, we investigated whether interferon-gamma (IFN-gamma), a potent inducer of activation and differentiation of mononuclear phagocytes, affected IL-8 expression in this cell lineage. We found a low constitutive level of IL-8 mRNA expression that was upregulated by IFN-gamma in a dose- and time-dependent manner and via a protein-synthesis-dependent process in the human monocytic cell line U937. IL-8 protein secretion was also stimulated by IFN-gamma. Nuclear run-on experiments showed that the IL-8 gene was transcriptionally active in control cells and that IFN-gamma did not enhance the transcriptional activity. The increase in IL-8 mRNA by IFN-gamma was concomitant with the stabilization of the mRNA and, therefore, controlled primarily at a posttranscriptional level. These results represent the first evidence that IFN-gamma upregulates IL-8 gene expression in cells of the monocytic lineage, and show the involvement of posttranscriptional mechanisms in the induction of IL-8 mRNA.

IL-2 up-regulates but IFN-gamma suppresses IL-8 expression in human monocytes.

IL-2 has pleiotropic properties and is a potent activator of monocytic functions. Since monocytes are an important source of the chemoattractant cytokine IL-8, we studied the effects of IL-2 on the expression of IL-8 in human monocytes. IL-8 mRNA expression was detectable in resting human monocytes. Treatment of monocytes with IL-2 increased IL-8 mRNA expression by a protein synthesis-independent process. The augmentation of IL-8 mRNA by IL-2 was associated with an increase in IL-8 secretion. The expression of IL-8 mRNA was not a nonspecific response to any stimulus of monocyte activation. In fact, IFN-gamma, which is also a potent monocyte activator, not only failed to induce IL-8 expression but inhibited the stimulation of IL-8 by IL-2. Nuclear run-on experiments demonstrated that both the enhancement of IL-8 mRNA expression and its down-regulation by IFN-gamma occurred at the transcriptional level. These results show for the first time that in fresh human monocytes, IL-8 expression is differentially regulated by IL-2 and IFN-gamma and suggest that the interactions among IL-2, IL-8, and IFN-gamma may be important for the development and control of the inflammatory response.

Interleukin 1 induction of the c-jun promoter.

Interleukin 1 (IL-1) induces pleiotropic effects in many cell types during inflammation and immunity. We have recently shown how the IL-1 signal is transmitted to the nucleus: In T cells and in pituitary cells, IL-1 induced genes via activation of the nuclear factor AP-1. We now demonstrate how IL-1 activates the AP-1 factor in liver cells, which are a major target for IL-1 during the acute phase response in vivo. IL-1 induced gene transcription of both AP-1 components, c-jun and c-fos. IL-1 also increased the stability of c-jun mRNA. We define two enhancer sites in the jun promoter that are required for induction by IL-1. Although the binding sites share some similarity with the AP-1 binding site, the nuclear factors binding the jun motifs are not composed of Jun or Fos proteins. Thus these data identify two binding proteins that serve as one of the first nuclear targets for IL-1 signal transduction.

Selective transformation of host lymphocytes in vivo by retrovirus-producing macrophages.

Macrophage-mediated retroviral transformation of host cells was studied in vivo utilizing the cloned murine macrophage-line GG2EE, generated by in vitro infection of bone marrow cells from C3H/HeJ mice (H-2k) with the acute transforming retrovirus J2 bearing the v-myc and v-raf oncogenes. Because GG2EE macrophages produce the J2 retrovirus, the development of secondary, J2 virus-induced tumors after the injection of the cell line into several strains of mice was evaluated. GG2EE cells proliferated and gave rise to histiocytic tumors in syngeneic mice and in allogeneic athymic Swiss mice. The inoculum of GG2EE cells in allogeneic DBA/2 mice (H-2d) and, to a lesser extent, in BALB/c (H-2d) and BALB/k (H-2k) mice gave rise to a small, solid mass at the injection site. Although the initial tumor was slowly rejected, secondary lymphomas belonging to the B or T cell lineage developed, leading to mouse death. Extensive phenotypic, functional, and chromosomal analyses proved that lymphomas were derived from host T and B cell transformation. Southern and Northern blot studies showed that J2 virus was integrated and expressed in lymphoma cells, demonstrating that the virus was transmitted to the host lymphocytes and suggesting that it was causal in lymphoma development. The existence of close and protracted interactions between GG2EE macrophages and allogeneic host lymphocytes and the presence of viral particles in the area of macrophage-lymphocyte contact were demonstrated by histologic and ultrastructural analysis. Rejection of J2 virus-infected lymphocytes in allogeneic mice suggested that host lymphocyte transformation was dependent upon the macrophage cell type. These results demonstrate that macrophage-derived J2 retrovirus transforms host lymphocytes in vivo in allogeneic mice and that a condition of host alloreactivity is critical for such event.

Regulation of IL-2 receptor subunit genes in human monocytes. Differential effects of IL-2 and IFN-gamma.

We investigated the effects of IFN-gamma and IL-2 on IL-2R alpha and beta mRNA expression in human monocytes. Low basal expression of IL-2R beta mRNA was detected in fresh monocytes. Stimulation of monocytes with IL-2 induced a significant increase of IL-2R beta mRNA, but did not induce IL-2R alpha mRNA. In contrast, stimulation of monocytes with IFN-gamma-induced IL-2R alpha mRNA, but did not modify IL-2R beta mRNA. Five U/ml of IFN-gamma induced IL-2R alpha mRNA and 2.2 nM of IL-2 induced IL-2R beta mRNA, both within 3 h. Nuclear run-on experiments demonstrated that the induction of IL-2R alpha mRNA by IFN-gamma is controlled, at least in part, at the transcriptional level. In contrast, the enhancement of IL-2R beta mRNA by IL-2 is controlled at a posttranscriptional level and is associated with an increase in the half-life of IL-2R beta mRNA. The results of studies on the cytotoxic activity and on the expression of c-fms mRNA of monocytes activated by the combination of IFN-gamma and IL-2 show that pretreatment with IFN-gamma renders monocytes more sensitive to activation by IL-2. These results demonstrate that the IL-2R alpha and IL-2R beta subunits are induced by different lymphokines through distinct mechanisms and that both receptor subunits can influence the response of monocytes to IL-2.

IL-2 induces IL-6 production in human monocytes.

IL-2 is a potent activator of effector and secretory activities of human monocytes. Since monocytes are an important source of IL-6, we investigated whether IL-2 can induce IL-6 production and whether regulatory circuits can modulate this process. We found that stimulation of monocytes with IL-2 induced expression of IL-6 mRNA and bioactivity in a dose-dependent manner. Production of IL-6 in monocytes can be induced by other cytokines such as IL-1 beta. By using mAb alpha-IL-1 beta we showed that IL-2-induced IL-6 production is not mediated by the autocrine stimulation of IL-1 beta elicited by IL-2. IL-6 induction by monocytes is not a common response to activating signals because IFN-gamma did not induce IL-6 expression under conditions in which it elicits tumoricidal activity. In contrast, IFN-gamma could completely abrogate the induction of IL-6 expression by IL-1 beta but did not affect the levels of mRNA and the secretion of IL-2-elicited IL-6. We have previously reported that transforming growth factor-beta inhibits IL-6 production in response to IL-1 beta. Studies on the inhibitory activity of transforming growth factor-beta demonstrated that this cytokine differs from IFN-gamma because it inhibited both IL-1- and IL-2-induced IL-6 expression. These data demonstrate that, in human monocytes, both IL-1 and IL-2 stimulate IL-6 expression by independent mechanisms that can be dissociated by the susceptibility to the inhibitory effect of IFN-gamma. IL-6 production is also down-regulated by TGF-beta, whose inhibitory activity is stimulus-unrelated.

Human Harvey-ras is biochemically different from Kirsten- or N-ras.

The biochemical effects of the human H-, N- and K-ras oncogenes were studied. We analysed the induction of c-fos mRNA and protein by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in exponentially growing NIH3T3 fibroblasts transformed by transfection with ras oncogenes. We found that H-ras has the unique ability to inhibit c-fos induction by TPA. In contrast, normal c-fos expression was induced by TPA in fibroblasts transformed by N- or K-ras or by the ras-unrelated oncogenes dbl and trk. The inhibition of c-fos induction by H-ras was not due to alteration in the binding of TPA to the transformed cells or to the selection of idiosyncratic clones. These results provide clear evidence that H-ras is functionally different from K- or N-ras.

Dexamethasone inhibits the induction of monocyte chemotactic-activating factor production by IL-1 or tumor necrosis factor.

Recently purified and molecularly cloned monocyte chemotactic and activating factor (MCAF) may play a major role in recruiting and activating monocytes in the inflammatory process. We examined the effects of a potent anti-inflammatory agent, dexamethasone (DXS), on the production of this factor. Over a wide range of concentrations (10(-5) to 10(-8) M), DXS inhibited the production of MCAF at the mRNA and protein level in a human fibrosarcoma cell line, which was stimulated with either IL-1 or TNF-alpha. We examined the turn-over of synthesized MCAF mRNA that showed DXS decreased the stability of MCAF mRNA. Furthermore, the addition of actinomycin D and cycloheximide abolished this effect of DXS, indicating that de novo mRNA and protein synthesis were required for this process. In addition, a nuclear run-off analysis revealed that DXS also inhibited the transcription of IL-1- or TNF-activated MCAF genes. Therefore, both the destabilization of MCAF mRNA and the inhibition of transcription of the gene contribute to the decrease in the MCAF mRNA steady state level by DXS.