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1.
Curr HIV Res ; 11(3): 198-209, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23746211

ABSTRACT

The HIV-1 Nef protein brings about increased T cell activity and viral titers through mechanisms that are poorly understood. Nef activity has been described as an enhancer, but not an inducer, of certain signaling pathways that lead to T cell activation and viral production, particularly from resting T cells. The protein has also been found to associate with and promote autophosphorylation of a serine kinase, Pak2, but the Nef-associated kinase level is very low and difficult to study. Here we demonstrate that Nef expression mediates phosphorylation of Mek1 serine298 in T cell lines as well as primary human T cells, thus directly affecting the Erk cascade. This phosphorylation is through a Pak and Rac activity. We also find that Pak2 in Nef expressing cells is phosphorylated on serine192/197, the first biochemical description of the Nef-mediated activation state for this kinase.


Subject(s)
HIV-1/physiology , Host-Pathogen Interactions , MAP Kinase Kinase 1/metabolism , nef Gene Products, Human Immunodeficiency Virus/metabolism , p21-Activated Kinases/metabolism , Cells, Cultured , Humans , Phosphorylation , Protein Processing, Post-Translational , T-Lymphocytes/enzymology , T-Lymphocytes/virology
2.
Anesthesiology ; 118(3): 587-601, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23254148

ABSTRACT

BACKGROUND: Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and the authors hypothesized that it has a central role in anesthesia. METHODS: Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca imaging and patch clamp electrophysiology. RESULTS: Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in the mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca flux that is altered in dRyr mutants and correlates with strong hyperpolarization. CONCLUSIONS: In Drosophila, neurally expressed dRyr mediates a substantial proportion of the anesthetic effects of halothane in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. The authors' results provide support for Ryr as an important mediator of immobilization by volatile anesthetics.


Subject(s)
Anesthesia, General , Anesthetics, Inhalation/pharmacology , Halothane/pharmacology , Ryanodine Receptor Calcium Release Channel/physiology , Amino Acid Sequence , Animals , Cell Line , Drosophila melanogaster , Immobilization/methods , Male , Molecular Sequence Data , Point Mutation/drug effects , Point Mutation/physiology , Ryanodine Receptor Calcium Release Channel/biosynthesis , Ryanodine Receptor Calcium Release Channel/genetics
3.
Cell ; 134(5): 782-92, 2008 Sep 05.
Article in English | MEDLINE | ID: mdl-18775311

ABSTRACT

Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here, we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction and that HIV utilizes the Galphai-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin-depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a postentry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.


Subject(s)
Actins/metabolism , CD4-Positive T-Lymphocytes/virology , Cofilin 1/metabolism , HIV Envelope Protein gp120/metabolism , Receptors, CXCR4/metabolism , Amino Acid Sequence , CD4 Antigens , Cells, Cultured , Cofilin 1/chemistry , HIV , HIV Infections , Humans , Molecular Sequence Data , Signal Transduction
4.
Virology ; 372(2): 300-12, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18054979

ABSTRACT

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.


Subject(s)
DNA, Viral/genetics , Gene Expression Regulation, Viral , HIV-1/genetics , Macrophages/virology , Transcription, Genetic , Cells, Cultured , DNA, Viral/metabolism , Gene Deletion , HIV-1/physiology , Human Immunodeficiency Virus Proteins/genetics , Human Immunodeficiency Virus Proteins/metabolism , Humans , RNA, Viral/genetics , RNA, Viral/metabolism , Virus Replication
5.
Curr HIV Res ; 5(4): 394-402, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17627502

ABSTRACT

Measuring virion infectivity is critical for studying and monitoring the process of HIV-1 infection. The easiest and the most common method utilizes reporter cell lines based on the HIV LTR promoter. The early HIV gene product Tat amplifies expression from the LTR; however, there is a background transcriptional activity that is independent of Tat. Furthermore, LTR activity can be influenced by cellular activation states. We have recently constructed a Rev-dependent expression vector, and as a test of this construct's functionality, we have integrated this vector into a continuous T cell line. This novel indicator cell has no measurable background signal, is not affected by elevated metabolic states, and yet responds robustly to the presence of HIV. The line is able to complete TCID50 assays in 3-5 days, and appears sensitive to both CCR5- and CXCR4-utilizing viruses.


Subject(s)
Genes, Reporter/physiology , Genes, env/physiology , HIV-1/physiology , T-Lymphocytes/virology , Cell Line , Gene Products, rev , Genetic Engineering , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HIV-1/genetics , Humans , rev Gene Products, Human Immunodeficiency Virus
6.
Retrovirology ; 4: 12, 2007 Feb 07.
Article in English | MEDLINE | ID: mdl-17286866

ABSTRACT

BACKGROUND: HIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells. RESULTS: We have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency. CONCLUSION: As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.


Subject(s)
Gene Expression , Gene Products, rev/metabolism , Genes, env/genetics , Genetic Vectors , HIV/genetics , HIV/physiology , Lentivirus/genetics , Cells, Cultured , DNA, Viral/genetics , Fluorescence , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HIV Long Terminal Repeat/genetics , Humans , Polymerase Chain Reaction , RNA Splicing/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , rev Gene Products, Human Immunodeficiency Virus
7.
J Virol ; 77(19): 10376-82, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12970422

ABSTRACT

Replication of human immunodeficiency virus (HIV) involves obligatory sequential processes. Following viral entry and reverse transcription, the newly synthesized viral DNA integrates into the host chromatin. Integration is mandatory for viral production, yet HIV infection of CD4 T cells in vivo results in high levels of nonintegrated DNA. The biological potential of nonintegrated HIV DNA is unclear; however, prior work has demonstrated a limited transcription of the nef gene by nonintegrated HIV in infected quiescent T-cell populations. In a kinetic analysis of HIV infection of metabolically active transformed and primary CD4 T cells, we find an unexpected transient expression of both early and late message by nonintegrated HIV DNA. However, only the early multiply spliced transcript was measurably translated. This restriction of protein expression was due in part to inadequate Rev function, since expression of Rev in trans resulted in the expression of the late structural gene gag by nonintegrated HIV DNA.


Subject(s)
DNA, Viral/biosynthesis , HIV Infections/virology , HIV/genetics , Transcription, Genetic , Virus Integration , CD4-Positive T-Lymphocytes/virology , Gene Products, gag/biosynthesis , Gene Products, rev/physiology , Humans , Polymerase Chain Reaction , Protein Biosynthesis , Protein Precursors/biosynthesis , Virus Replication , rev Gene Products, Human Immunodeficiency Virus
8.
Microbes Infect ; 5(11): 1023-7, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12941394

ABSTRACT

Transcription from the HIV genome is regulated by the 5' long-terminal-repeat viral promoter as well as regulatory proteins, especially Tat and Rev. Both the promoter activity and the function of regulatory proteins require the activity of cellular components, thus the virus remains highly dependent on the metabolic state of the cell. HIV also possesses the unusual capacity to transcribe from non-integrated DNA.


Subject(s)
HIV Infections/genetics , HIV-1/genetics , Transcription, Genetic , Gene Expression Regulation, Viral , Gene Products, tat/physiology , HIV Long Terminal Repeat , HIV-1/metabolism , Humans , tat Gene Products, Human Immunodeficiency Virus
9.
J Biol Chem ; 277(8): 6137-42, 2002 Feb 22.
Article in English | MEDLINE | ID: mdl-11726657

ABSTRACT

The human immunodeficiency regulatory protein Nef enhances viral replication and is central to viral pathogenesis. Although Nef has displayed a capacity to associate with a diverse assortment of cellular molecules and to increase T cell activity, the biochemical activity of Nef in T cells remains poorly defined. In this report we examine the bioactivity of Nef in primary CD4 T cells and, in particular, focus on the biochemical pathways known to be central to T cell activity. The extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway was dramatically affected by Nef expression with increases in ERK, MEK, and Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase known to affect T cell activity, viral replication, and viral infectivity.


Subject(s)
CD4-Positive T-Lymphocytes/physiology , HIV/genetics , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Nitrogen Fixation/genetics , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Enzyme Activation , Gene Expression Regulation, Viral , Humans , JNK Mitogen-Activated Protein Kinases , Kinetics , MAP Kinase Kinase 1 , MAP Kinase Kinase 2 , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism
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