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P M Pitha

Publications and source records attributed to P M Pitha.

At least 91 records · Page 5Linked to original sources
PubMed1992

Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence.

Herpes simplex virus type 1 (HSV-1) infection induces expression of the human immunodeficiency virus type 1 (HIV-1) provirus in the chronically infected T-cell line ACH-2. The HSV-1-mediated induction correlates with the appearance of two NF-kappa B-specific proteins of 55 and 85 kDa in the nucleus and with the binding of 50-kDa nuclear protein to the LBP-1 binding site of the untranslated leader sequence of the HIV-1 long terminal repeat. The HSV-1-induced LBP-1 binding protein, designated HLP-1, is present exclusively in HSV-1-infected, but not in phorbol-12-myristate-13-acetate- or tumor necrosis factor alpha-treated ACH-2 cells. Both the NF-kappa B and LBP-1 target sequences, when inserted either alone or together 5' of a heterologous minimal promoter (thymidine kinase), confer inducibility by HSV-1 infection in a transient transfection assay. Thus, it appears that the HSV-1-mediated activation of HIV-1 provirus is brought about by the binding of both NF-kappa B and HLP-1 specific proteins to two distinct regions of HIV-1 long terminal repeat.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent.

Chloramphenicol O-Acetyltransferase↗
PubMed1991

Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus.

The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.

Cells, Cultured↗
PubMed1991

Identification of a novel virus-responsive sequence in the promoter of murine interferon-alpha genes.

We have previously shown that the infection of mouse L-cells with Newcastle disease virus activates transcription of the alpha 4 but not the alpha 6 interferon gene and that the induction is mediated by a 35-base pair inducible element (IE) found in the alpha 4 promoter (-109 to -75). In the present study, we show that the inactivity of the alpha 6 promoter can be mapped to 2 out of 6 nucleotides in which the alpha 6 differs from alpha 4 IE. The symmetrical sequence, GTAAAGAAAGT (-103 to -93), present in the alpha 4 IE is essential for its inducibility and binding of nuclear protein(s) to the alpha 4 IE.

Animals↗
PubMed1991

Aberrant expression of cytokine genes in peritoneal macrophages from mice infected with LP-BM5 MuLV, a murine model of AIDS.

Mice infected with LP-BM5 murine leukemia virus (MuLV) develop a syndrome denoted as murine AIDS. Macrophages harvested from the peritoneal cavities of these mice at 4 or 9 wk postinoculation with LP-BM5 MuLV were analyzed by Northern hybridization for the presence of the defective LP-BM5 virus and their ability to synthesize various cytokines upon induction with Newcastle disease virus (NDV) or (LPS). Neither IFN-alpha or IFN-beta was found to be constitutively expressed in LP-BM5-infected macrophages and in NDV induction studies, and the levels of biologically active IFN-alpha and its mRNA were found to be lower in LP-BM5 MuLV-infected macrophages than in the macrophages from uninfected controls. Similarly, after NDV or LPS induction, the levels of TNF mRNA and TNF protein were significantly lower in LP-BM5-infected macrophages than in macrophages from uninfected mice. The LP-BM5 MuLV-infected macrophages constitutively expressed low levels of IL-1 beta, and when induced with LPS, the relative levels of IL-1 beta were significantly higher in infected than in uninfected macrophages. Although no constitutive expression of IL-6 was detected, the levels of IL-6 mRNA induced with NDV were higher in LP-BM5 MuLV-infected macrophages than in controls. Thus, we found alterations in the expression of selected cytokines in macrophages from mice inoculated with LP-BM5 MuLV rather than a general deregulation of all cytokine expression. These results show that macrophages infected with the defective LP-BM5 virus respond differently to NDV- or LPS-stimulation and suggest that aberrant expression of certain cytokine genes may play a role in the immunopathologic condition in mice with murine AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Relative antiviral activity of in vitro-synthesized murine interferon-alpha 4 and -alpha 1.

Murine interferon-alpha 4 (MuIFN-alpha 4) is notable among the MuIFN-alpha subtypes because it lacks 5 amino acids corresponding to positions 103-107 of the other subtypes, yet is the most highly expressed subtype. Site-directed in vitro mutagenesis has been used to modify the genes coding for MuIFN-alpha 4 and MuIFN-alpha 1. The modifications have allowed (i) the in vitro expression of the mature form of each MuIFN-alpha subtype and (ii) the insertion of five amino acids, corresponding to amino acid positions 103-107 of MuIFN-alpha 1, into the MuIFN-alpha 4 sequence. In contrast to previously published data MuIFN-alpha 4 and MuIFN-alpha 1 show only a twofold difference in antiviral activity, with MuIFN-alpha 4 being the more active subtype. In keeping with this observation, it was also found that insertion of the five "missing" amino acids into MuIFN-alpha 4 resulted in an analogue MuIFN-alpha with antiviral activity equivalent to MuIFN-alpha 1. It may be inferred from this work that the deletion of amino acids 103-107 (QVGVQ) is solely responsible for the difference in antiviral activity between MuIFN-alpha 4 and MuIFN-alpha 1.

Animals↗
PubMed1991

Expression of defective virus and cytokine genes in murine AIDS.

A syndrome characterized by severe immunodeficiency and lymphoproliferation develops in susceptible strains of mice infected with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV. The etiologic agent in this mixture has been shown to be a replication-defective virus (BM5d) with a 4.8-kb genome that required replication-competent helper viruses, primarily ecotropic (BM5e), for cell-to-cell spread in the host. In the present study, we studied the expression of BM5d and BM5e in tissues of infected mice at various times after inoculation in relation to the expression of cytokine genes that may contribute to the pathogenesis of this disorder. Northern (RNA) analysis of total RNA showed that BM5d was expressed at significant levels in lymphoid tissues within 1 week of infection and that the levels of expression increased with time after inoculation. By 16 weeks postinfection, BM5d was expressed in all tissues examined. Expression of BM5e was relatively more restricted to lymphoid tissues and was detected at lower levels than expression of BM5d at early times after infection, but this virus was expressed in all tissues by 16 weeks. Infection with the virus mixture was associated with constitutive expression of tumor necrosis factor in all tissues examined and of interleukin-1 (IL-1) in lymphoid tissues within 1 week of infection, and at later times with widespread expression of these cytokines and gamma interferon. Also, the levels of interferon regulatory factor 1 mRNA were significantly increased in all infected tissues during the infection. In contrast, expression of IL-3, IL-4, IL-5, and IL-6 was not detectable by Northern analysis of the respective mRNAs in any infected tissue at early or late times postinfection.

Animals↗
PubMed1990

Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes.

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency.

Infection of cells by HIV can result in a period of quiescence or latency which may be obviated by treatment with inducing agents such as 5-azacytidine. Evidence from these experiments demonstrate the existence of two CpG sites in the HIV LTR which can silence transcription of both reporter genes (CAT) and infectious proviral DNA when enzymatically methylated. This transcriptional block was consistently overcome by the presence of the trans-activator tat without significant demethylation of the HIV LTR. These results suggest that DNA hypermethylation of the HIV LTR may change the binding characteristics between LTR sequences and cellular proteins, thereby suppressing HIV LTR transcription and modulating viral expression.

Animals↗
PubMed1990

Retrovirus vector-targeted inducible expression of human beta-interferon gene to B-cells.

We have introduced the human beta-interferon gene with its promoter region into murine B-cell and fibroblast cell lines via a Moloney murine leukemia virus (M-MuLV) vector and have studied the inducible expression of the beta-interferon gene as a function of the various retroviral vector designs. By deleting the enhancer within the 3' viral long terminal repeat (LTR), inserting the human beta-interferon gene, and varying placement of the immunoglobulin heavy chain enhancer, we were able to construct vectors which yielded proviruses with various cell type-specific regulation. One of the vectors (pT154) led to a greater than 21-fold increase in beta-interferon protein synthesis after viral infection in the two B-cell lines analyzed, while no inducibility was seen in the fibroblast cells. The data show that inducible beta-interferon expression within a MuLV vector was highly dependent on the absence of the viral enhancer region in the long terminal repeat and the orientation of the beta-interferon gene within the proviral transcriptional unit; the insertion of the immunoglobulin enhancer elevated both constitutive and (or) inducible expression of beta-interferon in B-cells but inhibited constitutive expression of this gene in fibroblasts.

Animals↗
PubMed1989

Virus infection and interferon can activate gene expression through a single synthetic element, but endogenous genes show distinct regulation.

Virus inducible elements (IE) in promoters of mouse alpha-interferon and human beta 1-interferon genes contain multiple copies of the hexanucleotide sequence AGT-GAA or its variants which are also found in the interferon-stimulated response element of genes transcriptionally induced by interferon. We have examined the similarities between virus and interferon induction of gene expression and the role of AGTGAA and AAT-GAA hexamers in these responses. Hybrid plasmids were constructed by inserting the IE region, the alpha 4 promoter, or the multiple copies of AGTGAA or AAT-GAA 5' to the inactive-45 human immunodeficiency-chloramphenicol acetyltransferase hybrid gene, and their inducible expression was studied in a transient expression assay. In L-cells, multiple hexamers were efficiently induced both by infection with Newcastle disease virus and by interferon treatment; while the alpha 4 promoter and the IE inducible region were induced predominantly by virus rather than by interferon. In order to dissociate the effect of virus and endogenous interferon on the induction process, we examined the gene expression in Vero cells, which have undergone homozygous deletion of type 1 interferon genes, and in VNPT-159 cells, which were derived from Vero cells by insertion of an inducible human interferon beta 1 gene. The results show that while the alpha 4 promoter was efficiently induced only by virus in both cell types, the constructs containing shorter segments of the IE were induced by both virus and interferon in Vero cells. However, the inducibility by interferon was not detected in VNPT-159 cells, suggesting that the presence of endogenous interferon suppresses interferon-induced expression of hexanucleotide repeats and the short inducible region. In contrast, virus inducibility of endogenous interferon-stimulated genes, ISG-15 and ISG-54, was about 100-fold more efficient in VNPT-159 cells than in Vero cells, suggesting that this induction is largely mediated through synthesis of endogenous interferon. Hence, endogenous interferon may play a role in the autoregulation of both interferon genes and interferon-stimulated genes.

Animals↗
PubMed1989

Upstream regulatory elements of murine alpha 4-interferon gene confer inducibility and cell type-restricted expression.

We have identified and functionally characterized DNA sequences that are required for the inducible and cell-restricted expression of the murine alpha 4-interferon gene. Hybrid plasmids in which the alpha 4 promoter region or its 5' deletions were inserted upstream of the CAT gene were constructed, and the expression of these hybrid genes was studied in mouse L-cells both in permanent and transient assays with comparable results. Inducible expression was not affected by deletions up to -109; however, when the deletion was extended to -96, inducibility by Newcastle disease virus was abolished; however, this hybrid plasmid was expressed constitutively. Further deletion to -88 did not permit either constitutive or inducible expression. Insertion of the 35-base pair-long sequence (-109 to -75 base pairs) from the alpha 4 promoter region 5' of the minimal alpha 4 or human immunodeficiency virus promoter region, conferred inducibility to these two inactive promoters. The 5' deleted hybrids or plasmids containing the inducible element were induced only at low levels in transfected NIH/3T3 cells that do not express endogenous alpha 4 gene efficiently, indicating that the inducible region also determines the cell-specific expression. A tandem repeat of AGTGAA, which is present in the -109 to -88 region of alpha 4 in two copies, showed both basal levels of expression and inducibility in L-cells, while its analogue AATGAA was highly inducible but was not expressed constitutively. The inducibility of the synthetic hexamer repeats did not show cell type-restricted expression, suggesting that their response does not fully reflect the range of expression observed for the inducible region and the endogenous alpha genes.

Animals↗
PubMed1989

Inhibition of human immunodeficiency virus (HIV) replication by HIV-trans-activated alpha 2-interferon.

We have prepared stable cell lines, derived from Vero cells and A3.01 cells, that express a hybrid human alpha 2-interferon gene under control of the human immunodeficiency virus (HIV) long terminal repeat. These cells constitutively produced low levels (50-150 units/ml) of alpha 2-interferon. However, high levels of interferon (10(3) units/ml) could be induced upon trans-activation by the product of the tat gene (pIIIextatIII), and de novo infection by HIV resulted in a moderate increase (400 units/ml) in alpha 2-interferon synthesis. In contrast to the fully permissive HIV replication, in transfected Vero cells or infected A3.01 cells, the transcription and replication of HIV in Vero or A3.01 cells containing the HIV long terminal repeat--alpha 2-interferon hybrid gene (VN89 and A3N89 cells, respectively) was completely inhibited. These data suggest that virus-trans-activated alpha 2-interferon synthesis can be used as a selective inhibitor of HIV replication.

Animals↗
PubMed1989

Production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus.

Rat astrocytes, immunologically competent glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharide-stimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and alpha- and beta-interferons. Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems.

Animals↗
PubMed1988

Abnormal regulation of IFN-alpha, -beta, and -gamma expression in MAIDS, a murine retrovirus-induced immunodeficiency syndrome.

Mice infected with LP-BM5 murine leukemia viruses (MuLV) develop a syndrome with many features in common with AIDS including lymphadenopathy and profound immunodeficiency associated with enhanced susceptibility to infection and terminal B cell lymphomas. To evaluate cellular defects that may predispose infected mice to these sequelae, we studied the regulation of IFN gene expression. Spleen cells from mice infected with LP-BM5 MuLV expressed high levels of IFN-gamma mRNA by 1 wk post-inoculation and throughout the course of disease. By comparison, transcripts of IFN-alpha/beta genes were not detected in spleen cells at any time after infection. In uninfected mice, expression of IFN-alpha/beta genes is induced rapidly after infection with New-castle disease virus, but mice inoculated with LP-BM5 MuLV were unable to induce these genes by 4 wk after retroviral infection. Inhibition of IFN-alpha/beta induction due to LP-BM5 MuLV infection also occurred in nude mice, indicating this effect was not mediated by activated T cells. Furthermore, low levels of IFN-gamma transcripts were detected in spleens of nude infected mice, suggesting that cells other than T cells can express this gene. These results suggest that the normal contributions of IFN to control of microbial spread, immune surveillance, and lymphoid interactions are disrupted by infection with LP-BM5 MuLV.

Animals↗
PubMed1988

Differential and cell type specific expression of murine alpha-interferon genes is regulated on the transcriptional level.

In mouse cells induced with virus infection or dsRNA, the relative levels of alpha-4 interferon mRNA were higher than the levels of alpha-1 and alpha-6 mRNAs; the ratio between relative levels of alpha-4 and alpha-1 or alpha-6 mRNA was, however, dependent on the cell type. Recombinant plasmids, in which the expression of chloramphenicol acetyltransferase (CAT) gene was directed by the promoter regions of alpha-1, alpha-4 or alpha-6 interferon genes were constructed and their inducible expression was studied either in transient assay or in permanently transfected mouse cells. The highest levels of CAT activity and CAT mRNA were observed with alpha-4 CAT plasmid, while the expression of alpha-1 CAT was consistently higher than that coded by alpha-6 CAT plasmid; the ratio between CAT activities coded by alpha-4 CAT and alpha-1 CAT was dependent on cell type. However, in heterologous Vero cells, the transfected alpha-1 and alpha-4 genes were expressed constitutively, and the levels of mRNAs were comparable. These results show that the difference in the relative levels of individual alpha-1 and alpha-4 mRNAs reflects the transcriptional inducibility of the respective promoter regions.

Animals↗