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Biomedical subjects

P M Pitha

Publications and source records attributed to P M Pitha.

At least 55 records · Page 3Linked to original sources
PubMed1996

Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus.

We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.7 macrophage cell line and peritoneal macrophages of LPS-responsive C3HeB/FeJ mice. In LPS-hyporesponsive C3H/HeJ mice, only NDV induces this chemokine gene, indicating that the pathways of transcriptional activation by NDV and LPS are not identical. Using a transient transfection assay, the minimal virus-responsive element (VRE) was localized between nt -175 and -116. The VRE contains previously defined LRE motif 1 (TCAYRCTT) and motif 3 ((T/A)GRTTTCA(G/C)TTT), which were shown to also be important for initiation of transcription by virus. NDV-stimulated nuclear extracts were tested for trans-activating factors able to bind the VRE. The chromosomal protein HMG-I(C) was shown to bind the 3'-A.T-rich domains of the VRE, and the presence of HMG-I(C) was demonstrated in the VRE-protein complex formed with nuclear extracts from NDV-stimulated, but not unstimulated cells. These findings demonstrate the role of HMG-I(C) in activation of MuRantes promoter by NDV.

Animals↗
PubMed1996

Binding of a protein to an AU-rich domain of tumour necrosis factor alpha mRNA as a 35 kDa complex and its regulation in primary rat astrocytes.

Newcastle disease virus (NDV) induces tumour necrosis factor alpha (TNF alpha) gene transcription and increases the mRNA stability. NDV stabilizes TNF alpha mRNA by preventing poly(A) shortening in a protein kinase C-dependent manner. TNF alpha 3'-untranslated region (UTR) contains an AU-rich domain (ARD) with seven AUUUA pentamers, a motif implicated in poly(A) removal and mRNA degradation. In this report, protein binding to TNF alpha ARD and the effects of NDV and kinases on ARD-binding activity were investigated in primary rat astrocytes. Both nuclear and cytoplasmic extracts contained proteins binding to centrally located 27 nt AUUUAUUAUUUAUUUAUUAUUUAUUUA, within TNF alpha ARD. Portions of ARD with a single AUUUA did not show ARD-binding activity. The ARD-protein complexes migrated as two bands on electrophoretic mobility-shift assay. The slower moving complexes appeared either as a broader band or doublets. The UV cross-linked ARD-protein complexes, however, migrated as a single 35 kDa band on SDS/PAGE. In cytoplasmic extracts treated with alkaline phosphatase there was a decrease in the faster moving complex and an increase in the slower moving complex, whereas NDV infection produced the reverse effect. In addition, the faster moving complex was decreased when cytoplasmic extracts from NDV-infected cells were treated with protein phosphatase 1 or 2A. Neither NDV infection nor phosphatase treatment affected the mobility pattern of nuclear extracts. The data indicate that a protein of molecular mass less than 35 kDa binds to a segment of TNF alpha ARD containing primarily UUAUUUAUU motifs, and the ARD-binding activity in cytoplasmic compartment is post-transcriptionally modified.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Role of interferon-stimulated gene ISG-15 in the interferon-omega-mediated inhibition of human immunodeficiency virus replication.

Interferon-alpha (IFN-alpha) inhibits human immunodeficiency virus (HIV) replication in vivo and in vitro. In this study, we show that IFN-omega (IFN-omega) is also a potent inhibitor of HIV replication in vitro and that both laboratory and primary isolates of HIV-1 are more sensitive to IFN-omega than to IFN-alpha 2. Like IFN-alpha 2, IFN-omega inhibited proviral synthesis in acutely infected cells, but in contrast to IFN-alpha 2, IFN-omega did not alter the levels of HIV-1 unspliced messages. Yet, inhibition of HIV protein synthesis was greater in IFN-omega-treated than in IFN-alpha 2-treated cells. Whereas expression of IFN-stimulated genes was transient in IFN-alpha 2-treated cells, their expression was sustained in IFN-omega-treated cells. Expression of ISG-15 in particular was higher on treatment with IFN-omega than with IFN-alpha 2. Overexpression of ISG-15 in IFN-alpha 2-treated cells mimicked the effects of IFN-omega. In untreated cells, it resulted in the trapping of HIV unspliced RNA in the nucleus and a decrease in cytoplasmic HIV transcripts and HIV protein synthesis. These findings suggest that the sustained induction of IFN-stimulated genes by IFN-omega and that of ISG-15 in particular may confer a higher therapeutic index to IFN-omega in controlling HIV infection.

Antiviral Agents↗
PubMed1996

Cooperation between herpes simplex virus type 1-encoded ICP0 and Tat to support transcription of human immunodeficiency virus type 1 long terminal repeat in vivo can occur in the absence of the TAR binding site.

Expression of human immunodeficiency virus type 1 (HIV-1) provirus can be stimulated by herpes simplex virus type 1 (HSV-1) infection; the stimulation occurs at the level of transcriptional activation of the HIV long terminal repeat (LTR) and is mediated by both cellular and HSV-1-encoded transactivators. We have shown in this study that HSV-1 immediate-early gene ICP0 cooperates effectively with the HIV-1-encoded transactivator, Tat, in the stimulation of HIV-1 LTR-directed transcription. The cooperation between ICP0 and Tat is specific for the HIV-1 LTR and was not observed with other promoters (e.g., ICP0) that can be transactivated by ICP0 but not by Tat. Analyses of HIV-1 LTR deletion mutants have shown that ICP0 not only transactivates an HIV-1 LTR mutant that is unresponsive to NF-kappaB and Tat-mediated transactivation, such as the HIV-1 LTR with the enhancer deleted (-83 LTR) and TAR deleted (+20 to +81), but also restores responsiveness to Tat. ICP0 also showed cooperation with Gal4-Tat fusion protein-mediated transactivation of Gal4-HIV-1 LTR with TAR deleted. Enhancement of the transcriptional activation of ICP0 by Tat requires both the cysteine-rich and core domains of Tat and is inhibited by RO5-3335. ICP0 stimulates transcription of not only the HIV-1 LTR but also the TAR-defective HIV-1 provirus. We suggest that ICP0 can (i) recruit Tat to the vicinity of the HIV-1 promoter, thereby providing an alternative binding site for Tat, and (ii) substitute for the enhancer-binding proteins that are required for efficient Tat transactivation in T cells.

Binding Sites↗
PubMed1996

Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha2-integrin expression in human mammary epithelial cells.

The human alpha2-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha2-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions --64 to +1) of the alpha2-integrin promoter plays an essential role in the alpha2-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha2-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha2-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha2-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of (alpha)2(beta)1-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.

Antigens, CD↗
PubMed1996

Binding of human immunodeficiency virus type 1 to CD4 induces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.

We have analyzed CD4-mediated signaling during the early stages of human immunodeficiency virus type 1 (HIV-1) infection. Binding of purified HIV-1 virions or recombinant HIV-1 glycoprotein gp120 to CD4 receptors resulted in association and tyrosine phosphorylation and activation of tyrosine kinase Lck and serine/threonine kinase Raf-1. The association between Lck and Raf-1 was mediated by stimulation of the CD4 receptors, since it was abolished by preincubation of the virus with soluble CD4 and was not detected in CD4-negative A201 T cells. However, the Lck-Raf-1 association was restored in A201 cells permanently transfected with human CD4 cDNA and stimulated with anti-CD4 antibodies. In addition, a catalytically active Lck was required for the association of Lck and Raf-1. Surprisingly, the CD4-mediated signaling, induced by the HIV-1 binding, did not result in stimulation of the Ras GTP-binding activity or its association with Raf-1, indicating that the signaling pathway generated by the HIV-1 binding is not identical to the classical Ras/Raf-1 pathway. Furthermore, overexpression of activated Raf-1 in Jurkat T cells stimulated the HIV long terminal repeat promoter activity and significantly enhanced HIV-1 replication. This suggests that the Lck-Raf-1 pathway, rapidly stimulated by the binding of HIV-1 or gp120 to CD4 receptors, may play an essential role in the transcriptional activation of the integrated HIV-1 provirus as well as in its pathogenicity.

Antibodies↗
PubMed1995

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.

Amino Acid Sequence↗
PubMed1995

E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.

Expression of the Ca(2+)-dependent, homotypic cell:cell adhesion molecule, E-cadherin (E-cad), suppresses tumor cell invasion and metastasis in experimental tumor models. Decreased E-cad expression is common in poorly differentiated, advanced-stage carcinomas. These data implicate E-cad as an "invasion suppressor" gene. The mechanism by which E-cad is silenced in advanced stage carcinomas is unclear. In this report, we show that: (a) the 5' CpG island of E-cad is densely methylated in E-cad-negative breast and prostate carcinoma cell lines and primary breast carcinoma tissue but is unmethylated in normal breast tissue; (b) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, partially restores E-cad RNA and protein levels in E-cad-negative breast and prostate carcinoma cell lines; and (c) and E-cad promoter/CAT construct is expressed in both E-cad-positive and -negative breast and prostate carcinoma cell lines, indicating that these cells have the active transcriptional machinery necessary for E-cad expression. Our data demonstrate that frequent loss of E-cad expression in human breast and prostate carcinomas results from hypermethylation of the E-cad promoter region.

Base Sequence↗
PubMed1995

Microsatellite instability in primary neoplasms from HIV + patients.

AIDS is associated with a high risk of certain malignancies, notably Kaposi's sarcoma (KS) and B-cell non-Hodgkin's lymphoma (NHL). The pathogenesis of these malignancies is not fully understood. One mechanism of malignant transformation recently described in colon tumorigenesis results from defects in DNA mismatch repair, manifest as widespread microsatellite instability. We demonstrate a high rate of microsatellite instability in KS and aggressive lymphomas obtained from HIV-infected patients, whereas there is no evidence of instability in similar lesions from HIV-negative patients. Further elucidation of the underlying mechanisms responsible for HIV-associated instability in primary tumours may provide insight into the pathogenesis of these AIDS-related neoplasms.

DNA Repair↗
PubMed1995

Identification of human immunodeficiency virus primary isolates resistant to interferon-alpha and correlation of prevalence to disease progression.

Human immunodeficiency virus (HIV) primary isolates, derived from donors at various stages of HIV infection, were assayed for their sensitivity to interferon (IFN)-alpha 2 in vitro. These isolates displayed a broad range of sensitivity to IFN-alpha 2. The prevalence of IFN-alpha 2 resistance was low in the absence of AIDS but dramatically increased once HIV infection progressed to AIDS. Although there was no linear correlation between the percentage of IFN-alpha 2 inhibition in vitro and the CD4 cell number in vivo or the level of endogenous IFN-alpha, serum IFN-alpha levels were higher in donors with AIDS and were associated with low CD4 cell numbers. Thus, circulating IFN-alpha appeared to either promote resistance or favor survival of IFN-alpha resistant variants. IFN-alpha 2 resistance was neither limited to a particular cell tropism nor enhanced by therapy with zidovudine. Sequential analysis indicated that reversion to IFN-alpha 2 sensitivity could occur during the course of infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells.

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.

Animals↗
PubMed1995

Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways.

The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5-c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN-alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN-alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity.

Aminoquinolines↗
PubMed1995

Antiviral activity of herpes simplex virus vectors expressing murine alpha 1-interferon.

Mutant herpes simplex virus type I (HSV-1) vectors were engineered to express murine alpha 1 interferon (IFN) and assessed for their ability to inhibit the replication of challenge viruses in infection of monolayer cell cultures. The alpha 1 IFN gene was placed under control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) region in the thymidine kinase (tk) locus of both the wild-type HSV-1 strain KOS and the replication-defective KOS mutant dl20, in which both copies of the ICP4 immediate-early (IE) gene are deleted. To evaluate IFN expression, vector-infected cell culture media from epithelial, fibroblast and neuronal cells were assayed for alpha IFN release. These cells did not secrete detectable levels of alpha IFN when infected with control KOS or d120-based viruses. Cells infected at a multiplicity of infection (MOI) of 10 with either RSV-LTR-IFN vector produced from approximately 12 to 100 U of alpha 1-IFN per millilitre of culture fluid in 24 h, as determined by a standard vesicular stomatitis virus (VSV) replication inhibition assay. Vector-derived alpha 1-IFN expression did not inhibit the growth of the KOS-RSV-IFN virus in IFN sensitive cell lines. However, pretreatment of murine L cells with IFN produced from the RSV-IFN vectors blocked the replication of HSV-1. Additionally, L cells infected with d120-RSV-IFN (at an MOI of 0.5 to 0.06) were protected from superinfection with VSV. Thus, pre-infection of a small percentage of cells with a nonreplicating HSV-IFN expression vector provided complete protection of tissue culture cell monolayers from the cytopathic effect of a challenge virus infection. These vectors should be useful for in vivo analysis of the antiviral potential of IFN expression from within the nervous system.

Animals↗
PubMed1994

Evidence that levels of the dimeric cellular transcription factor CP2 play little role in the activation of the HIV-1 long terminal repeat in vivo or following superinfection with herpes simplex virus type 1.

The dimeric transcription factor CP2 binds a sequence element found near the transcription start site of the human immunodeficiency virus (HIV-1) long terminal repeat. Several groups have suggested that cellular factors binding this element might play a role in modulating HIV-1 promoter activity in vivo. For example, induction of latent HIV-1 gene expression in response to superinfection by herpes simplex virus type 1 (HSV-1) or cytomegalovirus is thought to be mediated, in part, by factors binding the CP2 site. In this report we began to examine directly the relationship between CP2 and expression of the HIV-1 promoter. First, we tested what effect HSV-1 infection of T cells had on the cellular levels of CP2. The results showed that HSV-1 infection led to a significant reduction in the level of CP2 DNA binding activity and protein within 20 h. Next, we tested the effect of overexpressing either the wild-type factor or a dominant negative variant of CP2 on HIV-1 promoter activity in vivo. The results showed that CP2 had little effect or slightly repressed HIV-1 promoter activity in vivo. In addition, these expression constructs had little effect on the induction of HIV-1 promoter activity elicited by HSV-1 infection.

Amino Acid Sequence↗
PubMed1994

Differential effect of tumor necrosis factor-alpha and herpes simplex virus type 1 on the Tat-targeted inhibition of human immunodeficiency virus type 1 replication.

In this study, we have examined whether the Tat antagonist can inhibit human immunodeficiency virus type 1 (HIV-1) replication in the presence of cofactors that can activate transcription of HIV-1 provirus by an NF-kappa B-mediated mechanism, such as tumor necrosis factor-alpha (TNF-alpha) or herpes simplex virus type 1 (HSV-1) infection. As a prototype, we have chosen a low-molecular-weight Tat inhibitor, Ro5-3335, and analyzed its effect on HIV-1 replication in the presence of TNF-alpha and HSV-1 infection in acutely infected peripheral blood lymphocytes (PBLs) and T cells. Ro5-3335 inhibited HIV-1 replication both in CEM-174 cells and in PBLs, but the magnitude of the inhibition was inversely related to viral inoculum and the inhibition was only temporary; viral replication resumed at later times postinfection in spite of the continuous presence of the drug. In contrast, Ro5-3335 suppressed TNF-alpha-induced activation of HIV-1 replication in chronically infected T cells and monocytes that both expressed only low levels of HIV-1 constitutively, while its effect in high-expressing OM-10.1 cells was negligible in the presence of TNF-alpha. The inhibition of HIV-1 replication by Ro5-3335 was specific for the Tat-mediated effect and this drug was not able to inhibit the TNF-alpha-induced expression of the tat-defective HIV-1 provirus. In contrast to TNF-alpha, HSV-1-stimulated HIV-1 expression in the ACH-2 cells was effectively inhibited in the presence of Ro5-3335. These results demonstrate that Tat plays an essential role in HSV-1-mediated activation of HIV-1 provirus, while the TNF-alpha complementation of Tat shows cell-type specificity. These observations suggest that inhibition of the Tat function alone may not be sufficient for an effective anti-HIV-1 inhibition.

Antiviral Agents↗
PubMed1994

Modulation of interferon-mediated inhibition of human immunodeficiency virus type 1 by Tat.

Recently, we have shown that in acutely infected T cells interferons (IFNs) effectively inhibit the human immunodeficiency type 1 (HIV-1) proviral DNA synthesis during a single replication cycle. In the present study, we have evaluated the relative effectiveness of IFNs in restricting HIV-1 expression at post-transcriptional level. Treatment of HeLa cells with IFNs A* and B (up to 1,000 U/ml) did not result in a reduction in HIV-1 RNA and protein synthesis encoded by the transfected HIV-1 proviral clone. Interestingly, IFN treatment reduced significantly the HIV-1 mRNA levels encoded by the transfected tat-defective HIV-1 provirus, and this inhibition could be overcome by transfection with Tat- and Rev-expressing plasmids. These results suggest that HIV-1-encoded Tat and Rev can overcome the inhibitory effects of IFNs on HIV-1 replication.

Gene Products, tat↗
PubMed1994

The presence of tat protein or tumor necrosis factor alpha is critical for herpes simplex virus type 1-induced expression of human immunodeficiency virus type 1.

Tat-independent transcription of human immunodeficiency virus type 1 (HIV-1) plays an important role in virus life cycle before biologically significant levels of Tat protein have been accumulated. Using a latently infected T-cell line containing an integrated Tat-defective HIV-1 provirus, we examined whether factors known to up-regulate the HIV-1 expression in vitro can replace the requirement for a functional Tat protein and induce the expression of the Tat-defective HIV-1 provirus. Both tumor necrosis factor alpha (TNF-alpha) and herpes simplex virus type 1 (HSV-1) infection stimulated transcription of the Tat-defective HIV-1 provirus to comparable levels, but in HSV-1-infected cells, the cytoplasmic HIV-1 transcripts were not efficiently translated in the absence of Tat protein and were excluded from the large polysomes. However, HSV-1 infection did not affect the distribution of cellular gamma-actin RNA or 28S RNA in the polysomal fractions. The translational block of HIV-1 RNA was not mediated by the virion-associated host cell shutoff protein (vhs); dissociation of HIV-1 transcripts from the polysomes and inefficient translation was also observed in cells infected with the vhs-defective mutant of HSV-1 (vhs-1). Overexpression of Rev protein did not rescue the synthesis of HIV-1 proteins in these cells; however, the observed inhibition of HIV-1 RNA translation was efficiently overcome in the presence of Tat protein or TNF-alpha. These findings suggest that, in contrast to TNF-alpha, HSV-1 infection is not able to induce a full cycle of HIV-1 replication and that cytokines and Tat have a critical role in the activation of HIV-1 provirus by HSV-1.

Cell Compartmentation↗