Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B.

Nuclear factor-kappa B (NF-kappa B) has been shown to play a central role in stimulating human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed viral gene expression. We have previously described a cell line (TE671/RD) that fails to respond to phorbol myristate acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha) in terms of amplifying HIV-1 LTR-driven gene expression unless it is concurrently treated with sodium butyrate. It was not determined whether this lack of response stemmed from an inability of these cells to produce free NF-kappa B or from ineffectual interaction of this sequence-specific transcriptional factor with its target. We now show that these cells are in fact capable of inducing a free nuclear NF-kappa B-binding activity when stimulated with PMA but not when treated with sodium butyrate alone. Furthermore, we show that sodium butyrate alone is equally potent in stimulating HIV-1 LTR-directed gene expression in latently infected U1 and ACH-2 cells in the absence of induction of nuclear NF-kappa B, as compared with PMA, which induces NF-kappa B activation in these cells. We also show that stimulation of HIV-1 expression in U1 cells with sodium butyrate is not blocked by N-acetylcysteine, whereas that of PMA stimulation is blocked. These observations are discussed in the context of a model where chromatin structure participates in the maintenance of restricted HIV-1 viral gene expression in these cells.

A CNS-enriched factor that binds to NF-kappa B and is required for interaction with HIV-1 tat.

The Human Immunodeficiency Virus type 1 (HIV-1) Tat protein is a potent activator of transcription directed by the long terminal repeat (LTR), an essential step in the life-cycle of HIV-1. While interaction of Tat with an RNA element encoded by downstream LTR sequences (termed TAR) is commonly considered essential to activation, numerous recent reports have implicated upstream transcription elements within the LTR as participants in mediating this activation. We have recently demonstrated that Tat activation occurs independent of the TAR element in certain cells derived from the central nervous system (CNS), and that this activation is mediated by the kappa B domain of the LTR. Further, CNS-derived cells were found to contain kappa B-binding activity capable of both interacting with Tat and activating LTR transcription in vitro. The present study demonstrates that the kappa B-binding transcription factor derived from CNS cells consists of a component indistinguishable from prototypical Nuclear Factor-kappa B (NF-kappa B) (in size, mobility on native gel, kinetics of activation and cognate binding sequence) as well as a supershifting component that is dissociable under certain conditions. The supershifting activity is found to stabilize binding of the presumed NF-kappa B to DNA. Further, only the form of NF-kappa B which is associated with this supershifting activity is capable of binding Tat. We hypothesize a model in which Tat utilizes this interaction to activate HIV-1 through the NF-kappa B domain of the LTR in circumstances where TAR is absent. This model has implications with respect to the ability of Tat to alter cellular gene expression and perhaps contribute to the array of problems seen in HIV-1 infection such as altered immune status, CNS toxicity, and the formation of tumors.

Detection of HIV-1 proviral DNA in sperm from HIV-1-infected men.

OBJECTIVE: Sexual transmission is a major mode of the spread of HIV-1, although the cellular and molecular mechanisms are poorly defined. In this study, we sought to assess the cellular reservoirs of HIV-1 proviral DNA in the semen of HIV-1-infected men. DESIGN AND METHODS: An in situ polymerase chain reaction (IS-PCR), which amplifies specific genes within intact cells, was used to evaluate levels of HIV-1 provirus in seminal cells from HIV-1-infected men in various stages of clinical disease. RESULTS: Initial studies demonstrated HIV-1 provirus in relatively low numbers (1:100 to 1:6000) of both the seminal mononuclear cells and sperm from certain HIV-1-infected men. To extend these findings, 94 seminal samples from HIV-1-infected men were evaluated. HIV-1 proviral DNA was detected in seminal cells of a significant percentage of HIV-1-infected men (45%) at all stages of clinical immunodeficiency. Both seminal mononuclear cells and sperm (35 and 33% of samples studied, respectively) harbored HIV-1 proviral sequences. HIV-1-harboring sperm are shown to stain positively for HIV-1 in the mid-pieces of these cells, with rarer staining of the sperm heads. CONCLUSIONS: HIV-1 proviral DNA can be demonstrated by IS-PCR in seminal mononuclear cells and sperm from certain HIV-1-infected men. The role played by proviral DNA in these cells in the sexual transmission of this retroviral agent will require further study.

Molecular and virological effects of intracellular anti-Rev single-chain variable fragments on the expression of various human immunodeficiency virus-1 strains.

A variety of genetic therapies or intracellular immunization techniques hold promise as modalities to inhibit human immunodeficiency virus type 1 (HIV-1) replication in vivo. We have recently demonstrated that a single-chain variable fragment (SFv) construct, derived from a monoclonal antibody that binds to the HIV-1 regulatory protein Rev, can be expressed intracellularly and potently inhibits HIV-1 replication. This single-chain intracellular antibody, which avidly binds to the effector domain of Rev, is now demonstrated to dramatically inhibit various diverse laboratory and primary clinical strains of HIV-1. Potent suppression of HIV-1 replication by this modality is maintained over several months in long-term cultures. As well, the intracellular expression of anti-Rev SFv is shown to alter HIV-1 replication by specifically affecting Rev function. Importantly, no alterations in HIV-1 internalization, reverse transcription, or initial transcription of multiply spliced viral mRNAs are demonstrated in SFv-immunized cells, as compared to controls. Thus, these studies extend the understanding of the molecular mechanisms involved in the inhibition of lentivirus replication, by these intracellular antibody constructs.

Association of alterations in NF-kappa B moieties with HIV type 1 proviral latency in certain monocytic cells.

Human immunodeficiency virus type 1 (HIV-1) replication is controlled by a complex array of virally encoded and cellular proteins. A wide spectrum of levels of HIV-1 expression have been demonstrated in various cells, both in cell culture and in vivo. Molecular mechanisms leading to restricted HIV-1 replication may differ between certain cell types. It is now demonstrated that HIV-1 proviral latency in the monocytic cell line U1, in which only extremely low levels of HIV-1 expression are detected in the baseline unstimulated state, is associated with alterations in nuclear factor-kappa B (NF-kappa B) moieties demonstrated in these cells by electrophoretic mobility shift assays (EMSAs) and in situ UV cross-linking studies. A predominance of p50 NF-kappa B moieties and possibly p50 homodimers or closely related species, rather than the p50-p56 heterodimer of NF-kappa B that is the predominant NF-kappa B species in most T lymphocytic and monocytic cells, is demonstrated in the nuclei of U1 cells. This pattern of NF-kappa B-related moieties differs from the latently infected T lymphocytic cell line ACH-2, and from the U937 monocytic line, the parental cell line of the U1 cellular clone. As such, these data suggest that different proximal mechanisms may lead to restricted HIV-1 replication in various cell types.

The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication.

The p53 tumor suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act as a transcriptional activator and repressor both in vitro and in vivo. Consistent with its role in regulating transcription are recent observations that the N-terminal acidic domain of p53 binds directly to the TATA box-binding protein subunit of the general transcription factor, TF IID. It is now demonstrated that wild-type p53 (wt-p53) inhibits human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed chloramphenicol acetyltransferase activity in a cotransfection assay system. Importantly, this effect of wt-p53 on the HIV-1 LTR was also demonstrated by in vitro transcription assays. In addition, the Sp1 sites and the TATA box of the HIV-1 LTR are demonstrated to be the primary sites involved with p53-induced effects on this viral promoter. The upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) binding sites, decrease the p53-induced inhibitory effects on viral transcription. In the presence of the HIV-1 TAR sequence and Tat protein, the HIV-1 LTR also becomes less sensitive to wt-p53-induced inhibition. By using a retroviral vector delivery system, mutant forms of p53 genes were expressed in two HIV-1 latently infected cell lines, ACH-2 and U1. In the ACH-2 cell line, which is now demonstrated to contain an endogenous mutant form of p53 (amino acid 248, Arg to Gln), additional mutant p53 proteins did not alter HIV-1 replication. In U1 cells, which completely lack endogenous p53, overexpression of mutant p53 led to an increase in HIV-1 replication. Thus, these data indicate a possible functional role for wt-p53 and mutant p53 proteins in the control of HIV-1 replication patterns and proviral latency.

Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

Tat and rev differentially affect restricted replication of human immunodeficiency virus type 1 in various cells.

Human immunodeficiency virus type 1 (HIV-1) can infect various cell lines in culture and be maintained in a chronic state of restricted replication. These states of proviral latency are characterized by a predominance of spliced compared to unspliced viral RNA species. The proximate molecular mechanisms leading to restricted HIV-1 replication may differ in various cell lines. Importantly, recent studies have demonstrated that the site of integration is the critical parameter leading to proviral latency in ACH-2 cells. Utilizing murine retroviral shuttle vectors, the HIV-1 Tat protein was demonstrated to dramatically increase HIV-1 expression in the restrictively infected U1 monocytic cell line but not in the ACH-2 T-lymphocytic line. The HIV-1 Rev protein only modestly increased viral expression in both of these cell types. Thus, these data support the hypothesis that the mechanisms which initiate and/or maintain restricted HIV-1 expression may differ in various cell types in cell culture, and possibly in vivo.

High percentages of CD4-positive lymphocytes harbor the HIV-1 provirus in the blood of certain infected individuals.

OBJECTIVES: HIV-1 infection of humans leads to states of immunosuppression. Therefore, we sought to determine precise levels of HIV-1 infection of cells in vivo, as these data may assist in the understanding of the pathogenetic processes involved in HIV infection. DESIGN AND METHODS: We have developed an in situ polymerase chain reaction (IS-PCR), which allows amplification of various genetic elements within intact cells. Initial studies using this technique have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells (PBMC) of infected individuals than have been demonstrated in many previous studies using standard PCR techniques. This study describes a combined protocol in which an immunomagnetic bead separation technique is used with IS-PCR to specifically determine cellular reservoirs for HIV-1 and levels of infected cell types in the peripheral blood. RESULTS: CD4-positive lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected patients evaluated. The percentages of HIV-1-infected CD4-positive lymphocytes increased significantly with advancing stages of disease. These procedures also demonstrated that, with the exception of small percentages of infected peripheral blood monocytes, the CD4-positive lymphocyte is clearly the major cellular reservoir for HIV-1 in the peripheral blood. CONCLUSIONS: These data suggest that, in certain infected individuals, high levels of CD4-positive lymphocytes may harbor the HIV-1 provirus. Thus, the levels of infected lymphocytes are consistent with possible direct effects of HIV-1 on lymphocyte depletion in vivo.

Frequency of cells positive for HIV-1 sequences assessed by in situ polymerase chain reaction.

OBJECTIVE: HIV-1 infection of humans causes immunosuppression. The determination of precise levels of HIV-1 infection of cells in vivo may assist in the understanding of the pathogenetic processes involved in clinical infection with this virus. Studies using polymerase chain reaction have demonstrated higher levels of HIV-1 provirus in unfractionated peripheral blood mononuclear cells of infected subjects than many previous studies. METHODS: We developed a new, highly sensitive, polymerase chain reaction method that amplifies selected genetic regions within intact single cells. We used this technique before and after immunomagnetic bead separation to determine the proportion of unfractionated peripheral blood mononuclear cells and CD4+ lymphocytes carrying the HIV-1 provirus in infected subjects in different stages of disease. RESULTS: None of the peripheral blood mononuclear cells from 11 HIV-1-seronegative subjects were positive for HIV-1 provirus by the in situ polymerase chain reaction method. In 56 subjects infected with HIV-1, the percentage of peripheral blood mononuclear cells with HIV-1 ranged from 0.1 to 13.5%. CD4+ lymphocytes infected with HIV-1 ranged from 0.2 to 69% in the 42 HIV-1-infected subjects evaluated. The percentages of HIV-1-infected CD4+ lymphocytes increased significantly with an advancing stage of disease. The proportion of peripheral blood mononuclear cells infected with HIV-1 appeared to be at least 10 times higher than previously described. CONCLUSION: These data suggest that in certain infected patients, high levels of infected lymphocytes may harbor the HIV-1 provirus.

A quantitative reverse transcriptase-polymerase chain reaction for HIV-1-specific RNA species.

The ability to evaluate the patterns and levels of human immunodeficiency virus type I (HIV-1)-specific RNA in latently and productively-infected cell lines, and primary human cells, is critical to the understanding of HIV-1 expression in cell cultures and possibly in vivo. We have developed a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing in vitro transcribed RNA standards, to evaluate the copy number per cell and per microgram of total cellular RNA of multiply-spliced, unspliced and total HIV-1-specific RNA species. The latently-infected monocytic and T-lymphocyte cell lines, U1 and ACH-2 respectively, are shown to express between 10(4) to 10(6) copies of total HIV-1-specific RNA per cell, based on the state of cellular stimulation. A dramatic increase of unspliced HIV-1-specific RNA in both the U1 cell line and the ACH-2 cell line is demonstrated by this quantitative RT-PCR, 24 h after stimulation with phorbol esters. These data suggest that a single integrated HIV-1 provirus can rapidly express large quantities of HIV-1-specific RNA. Quantitative RT-PCR, for HIV-1-specific transcripts, should prove extremely useful in evaluating retroviral load and pathogenesis in cell cultures and in vivo.

Meningocele manqué: radiologic findings with clinical correlation.

PURPOSE: To determine whether meningocele manqué can be detected by neuroimaging techniques in dysraphic patients. METHODS: We reviewed the records and imaging studies of 16 patients with surgically proved meningocele manqué seen at our institution between 1989 and 1990. Both CT and MR imaging techniques were used. CT of the spine was performed immediately following contrast myelography. RESULTS: Nine of 16 patients (CT, four; and MR, five) showed evidence of meningocele manqué which corresponded to intraoperative findings. Fourteen of 16 patients were found to have diastematomyelia, eight with medium septum and six without a septum. Associated findings included syrinx (six), lipoma (five), dermoid cyst (one), and neuroenteric cyst (one). After completing this review, we were able to prospectively diagnose dorsal bands in two new patients; these bands were confirmed at surgery. CONCLUSION: Dorsal bands can be detected in dysraphic patients with CT or MR using operative findings as a road map.

CD14 is involved in control of human immunodeficiency virus type 1 expression in latently infected cells by lipopolysaccharide.

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in transfected monocyte-macrophage cell lines and dramatically increases HIV-1 production in the latently infected monocyte-macrophage-like cell line U1. This response to LPS, however, can only be observed after pretreatment of the U1 cells with granulocyte-macrophage colony-stimulating factor (GM-CSF). CD14, the differentiation antigen that acts as a receptor for complexes of LPS and LPS-binding protein, is now demonstrated to be involved in LPS-induced stimulation of HIV-1 replication. CD14 is shown to be expressed on a subpopulation of U1 cells only after treatment with GM-CSF and correlates with HIV-1 production stimulated by LPS. Importantly, only those U1 cells that express CD14 can be induced by LPS to upregulate HIV-1 production. In addition, a monoclonal antibody directed against CD14 can block LPS-induced stimulation of HIV-1 production from these latently infected cells.

Meningioangiomatosis: CT and MR findings.

Meningioangiomatosis (MA) is a rare hamartomatous lesion of the cerebral cortex; to date only 18 cases with imaging findings have been reported in the English literature. The origin of MA is probably malformative, with possible association with neurofibromatosis. These lesions frequently cause seizures in young patients. We report two new cases seen at our institution and present their CT and MR findings clearly illustrating MA cortex infiltration. Gd-DTPA used in one of the two cases failed to cause enhancement.

Potentiation in the intact rat of the hepatotoxicity of acetaminophen by 1,3-bis(2-chloroethyl)-1-nitrosourea.

Studies of the killing of cultured hepatocytes by acetaminophen indicate that the cells are injured by an oxidative stress that accompanies the metabolism of the toxin (J. L. Farber et al. (1988) Arch. Biochem. Biophys. 267, 640-650). The present report documents that the essential features of the killing of cultured hepatocytes by acetaminophen are reproduced in the intact animal. Male rats had no evidence of liver necrosis 24 h after administration of up to 1000 mg/kg of acetaminophen. Induction of mixed function oxidase activity by 3-methylcholanthrene increased the hepatotoxicity of acetaminophen. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the hepatotoxicity of acetaminophen in male rats induced with 3-methylcholanthrene. Whereas the pretreatment with BCNU reduced the GSH content by 40%, a comparable depletion of GSH by diethylmaleate did not potentiate the toxicity of acetaminophen. The antioxidant diphenylphenylenediamine (25 mg/kg) and the ferric iron chelator deferoxamine (1000 mg/kg) prevented the liver necrosis produced by 500 mg/kg acetaminophen in rats pretreated with BCNU. Neither protective agent prevented the fall in GSH produced by acetaminophen. It is concluded the conditions of the irreversible injury of cultured hepatocytes by acetaminophen previously reported are not necessarily different from those that obtain in the intact rat with this toxin.