Selective Killing of Human Immunodeficiency Virus-Infected Cells by Targeted Gene Transfer and Inducible Gene Expression Using a Recombinant Human Immunodeficiency Virus Vector

ABSTRACT The expression levels of ∼4,600 cellular RNA transcripts were assessed in CD4+-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1BRU) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1BRU infection, consistent with the G2 arrest of HIV-1-infected cells induced by Vpr. These included genes involved in cell division and transcription, a family of DEAD-box proteins (RNA helicases), and all genes involved in translation and splicing. However, the overall level of cell activation and signaling was increased in infected cells, consistent with strong virus production. These included a subgroup of transcription factors, including EGR1 and JUN, suggesting they play a specific role in the HIV-1 life cycle. Some regulatory changes were cell line specific; however, the majority, including enzymes involved in cholesterol biosynthesis, of changes were regulated in most infected cell lines. Compendium analysis comparing gene expression profiles of our HIV-1 infection experiments to those of cells exposed to heat shock, interferon, or influenza A virus indicated that HIV-1 infection largely induced specific changes rather than simply activating stress response or cytokine response pathways. Thus, microarray analysis confirmed several known HIV-1 host cell interactions and permitted identification of specific cellular pathways not previously implicated in HIV-1 infection. Continuing analyses are expected to suggest strategies for impacting HIV-1 replication in vivo by targeting these pathways.

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Long-Term Inducible Gene Expression in the Eye via Adeno-Associated Virus Gene Transfer in Nonhuman Primates

Human Gene Therapy ◽

10.1089/hum.2005.16.178 ◽

2005 ◽

Vol 16 (2) ◽

pp. 178-186 ◽

Cited By ~ 41

Author(s):

Corinna Lebherz ◽

Alberto Auricchio ◽

Albert M. Maguire ◽

Victor M. Rivera ◽

Waixing Tang ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Nonhuman Primates ◽

Inducible Gene Expression ◽

Adeno Associated Virus ◽

Virus Gene ◽

Inducible Gene

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CAF-Mediated Human Immunodeficiency Virus (HIV) Type 1 Transcriptional Inhibition Is Distinct from α-Defensin-1 HIV Inhibition

Journal of Virology ◽

10.1128/jvi.77.12.6777-6784.2003 ◽

2003 ◽

Vol 77 (12) ◽

pp. 6777-6784 ◽

Cited By ~ 70

Author(s):

Theresa Li-Yun Chang ◽

Fleur François ◽

Arevik Mosoian ◽

Mary E. Klotman

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

Viral Entry ◽

Neutralizing Antibody ◽

Inhibitory Effect ◽

Transcriptional Inhibition ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Hiv 1

ABSTRACT CD8+ T lymphocytes can inhibit human immunodeficiency virus type 1 (HIV-1) replication by secreting a soluble factor(s) known as CD8+ T-lymphocyte antiviral factor (CAF). One site of CAF action is inhibition of HIV-1 RNA transcription, particularly at the step of long terminal repeat (LTR)-driven gene expression. The inhibitory effect of CAF on HIV-1 LTR activation is mediated through STAT1 activation. A recent study reports that α-defensins 1 to 3 account for CAF activity against HIV-1. Here, we address whether α-defensins, particularly α-defensin-1, contribute to CAF-mediated inhibition of HIV-1 transcription. Both recombinant α-defensin-1 and CAF derived from herpesvirus saimiri (HVS)-transformed CD8+ cells inhibited HIV-1 infection and gene expression. For both factors, the inhibition of HIV-1 infection did not occur at the level of viral entry. Pretreatment of cells with α-defensin-1 followed by a washing out prior to infection blocked infection by HIV-1, indicating that direct inactivation of virions was not required for its inhibitory effect. In contrast to CAF, α-defensin-1 did not inhibit phorbol myristate acetate- or Tat-mediated HIV-1 LTR activation in a transient transfection system, nor did it activate STAT1 tyrosine phosphorylation. Furthermore, α-defensins 1 to 3 were below the level of detection in a panel of HVS-transformed CD8+ cells with potent HIV-1 inhibitory activity and a neutralizing antibody against α-defensins 1 to 3 did not reverse the inhibitory effect of CAF on HIV-1 gene expression in infected cells and on HIV-1 LTR activation in transfected cells. Taken together, our results suggest that α-defensin-1 inhibits HIV-1 infection following viral entry but that α-defensins 1 to 3 are not responsible for the HIV-1 transcriptional inhibition by CAF.

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Comparison of Gene Transfer Efficiencies and Gene Expression Levels Achieved with Equine Infectious Anemia Virus- and Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors

Journal of Virology ◽

10.1128/jvi.76.3.1510-1515.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1510-1515 ◽

Cited By ~ 33

Author(s):

J. P. O’Rourke ◽

G. C. Newbound ◽

D. B. Kohn ◽

J. C. Olsen ◽

B. A. Bunnell

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

Gene Transfer ◽

Equine Infectious Anemia Virus ◽

Fluorescent Protein ◽

Human Cells ◽

Immunodeficiency Virus ◽

Equine Infectious Anemia ◽

Anemia Virus

ABSTRACT This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

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A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6141 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6141-6151 ◽

Cited By ~ 27

Author(s):

M C Hu ◽

N Davidson

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Lac Repressor ◽

Tata Box ◽

Inducible Gene Expression ◽

Lac Operator ◽

Positive Induction ◽

Responsive Element ◽

High Level ◽

Inducible Gene

We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.

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A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6141-6151.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6141-6151

Author(s):

M C Hu ◽

N Davidson

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Lac Repressor ◽

Tata Box ◽

Inducible Gene Expression ◽

Lac Operator ◽

Positive Induction ◽

Responsive Element ◽

High Level ◽

Inducible Gene

We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.

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Human immunodeficiency virus-associated systemic lymphomas may be subdivided into two main groups according to Epstein-Barr viral latent gene expression.

Journal of Clinical Oncology ◽

10.1200/jco.1993.11.9.1674 ◽

1993 ◽

Vol 11 (9) ◽

pp. 1674-1681 ◽

Cited By ~ 84

Author(s):

A Carbone ◽

U Tirelli ◽

A Gloghini ◽

R Volpe ◽

M Boiocchi

Keyword(s):

Gene Expression ◽

Human Immunodeficiency Virus ◽

B Cell ◽

Cell Morphology ◽

Large Cell ◽

Barr Virus ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Epstein Barr

PURPOSE We report a pathologic characterization of human immunodeficiency virus (HIV)-associated systemic lymphomas, including the association of Epstein-Barr virus (EBV) in different categories. PATIENTS AND METHODS Eighty-seven HIV-associated non-Hodgkin's lymphoma (NHL) were classified according to classic NHL classification and a recent description of morphologic variants of high-grade B-cell NHL. Seventy-one cases were immunophenotypically-genotypically characterized, whereas, in 49 representative cases, the association of EBV was assessed by nonisotopic in situ hybridization (ISH) and the immunohistochemical demonstration of latent membrane protein-1 (LMP-1). In addition, 14 Hodgkin's disease (HD) cases, occurring in patients with HIV infection, were investigated for the frequency of LMP-1 expression. RESULTS Most lymphomas were of B-cell derivation and showed a blastic cell morphology, with (1) small noncleaved cells (SNCCs; 36 cases), (2) large noncleaved cells (10 cases), and (3) immunoblasts, usually polymorphic (12 cases). Moreover, 12 cases were classified as anaplastic large-cell (ALC) Ki-1-positive (Ki-1+) lymphoma. Combined ISH studies (for viral DNA and EBV RNA [EBER]) and immunohistologic demonstration of LMP-1 suggested that there were differences in viral latent gene expression between ALC Ki-1+ or immunoblastic lymphomas (usually EBV+, LMP-1+), and EBV-infected cells of SNCC lymphomas, which did not show LMP-1 expression. A high proportion (10 of 14) of LMP-1+ HD cases was found. CONCLUSION Differences in EBV association and LMP-1 expression were found between a major group of HIV-associated systemic NHL with blastic cell morphology, including SNCC lymphoma and its variants, and anaplastic cell lymphomas. A proportion of immunoblastic (polymorphic) lymphomas was different in viral latent gene expression from other blastic cell systemic lymphomas. It is concluded that only a group of these lymphomas (most ALC Ki-1+ and HD cases, along with a nonnegligible fraction of immunoblastic lymphomas) seems to be linked etiopathologically to EBV.

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