Asunto(s)

Quimiocinas/fisiología , Neuroinmunomodulación/fisiología , Receptores de Quimiocina/fisiología , Receptores Opioides/fisiología , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Humanos , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Péptidos Opioides/fisiología , Receptor Cross-Talk , Receptores Opioides/agonistas

RESUMEN

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)

Asunto(s)

Proteínas Quimioatrayentes de Monocitos/farmacología , Oligopéptidos/farmacología , Receptores CCR5/efectos de los fármacos , Receptores CXCR4/efectos de los fármacos , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/fisiología , Receptores de Lipoxina , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/fisiología , Antivirales/farmacología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Macrófagos/virología , Osteosarcoma , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Receptores de Formil Péptido , Receptores del VIH/genética , Receptores Inmunológicos/genética , Receptores de Péptidos/genética , Transfección , Células Tumorales Cultivadas

RESUMEN

Chemokine receptors are subjected to heterologous desensitization by activation of formyl peptide receptors. We investigated the cross-talk between formyl peptide receptors and the chemokine receptor CCR5 in human monocyte-differentiated immature dendritic cells (iDC). Monocytes cultured with GM-CSF and IL-4 for 4 days exhibit markers characteristic of iDC and maintain the expression of both formyl peptide receptors FPR and FPRL1, as well as CCR5. Pretreatment of iDC with W peptide (WKYMVm), a potent agonist for FPR and FPRL1 but with preference for FPRL1, resulted in down-regulation of CCR5 from the cell surface and reduced cell response to the CCR5 ligands through a PKC-dependent pathway. Furthermore, W peptide induced a PKC-dependent phosphorylation of CCR5 and inhibited infection of iDC by R5 HIV-1. Our results indicate that the expression and functions of CCR5 in iDC can be attenuated by W peptide, which activates formyl peptide receptors, and suggest an approach to the design of novel anti-HIV-1 agents.

Asunto(s)

Células Dendríticas/fisiología , Receptores CCR5/fisiología , Receptores Inmunológicos/fisiología , Receptores de Lipoxina , Receptores de Péptidos/fisiología , Diferenciación Celular , Quimiocina CCL4 , Quimiocina CCL5/farmacología , Células Dendríticas/virología , Regulación hacia Abajo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Proteínas Inflamatorias de Macrófagos/farmacología , Fosforilación , Receptores de Formil Péptido

RESUMEN

Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Asunto(s)

Quimiocina CCL2/biosíntesis , Quimiocina CCL5/biosíntesis , Quimiocinas CXC/biosíntesis , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Receptores Opioides mu/fisiología , Células Cultivadas , Quimiocina CCL2/sangre , Quimiocina CCL2/genética , Quimiocina CCL5/sangre , Quimiocina CCL5/genética , Quimiocina CXCL10 , Quimiocinas CXC/sangre , Quimiocinas CXC/genética , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Humanos , Interferón gamma/fisiología , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Péptidos/farmacología , Fitohemaglutininas/farmacología , ARN Mensajero/biosíntesis , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/sangre , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología

RESUMEN

Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

Asunto(s)

Regulación hacia Abajo/efectos de los fármacos , Monocitos/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Receptores CCR5/biosíntesis , Receptores Inmunológicos/efectos de los fármacos , Receptores de Péptidos/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Fusión Celular , Células Cultivadas , Efecto Citopatogénico Viral , Diseño de Fármacos , Productos del Gen env/fisiología , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Monocitos/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Receptores CCR5/genética , Receptores de Formil Péptido , Receptores Inmunológicos/fisiología , Receptores de Péptidos/fisiología , Transfección

RESUMEN

Human peripheral blood T lymphocytes are transformed in vitro to continuous proliferation by Herpesvirus saimiri subgroup C strains. It has been previously shown that H. saimiri-transformed human T cell lines are a permissive system for HIV-1 and 2 replication and are highly susceptible to infection by HIV-1 and 2. Two open reading frames of H. saimiri, StpC and Tip, are required for T cell transformation and are unique to this herpesvirus. The successful transduction of human T cells with retroviral vectors expressing H. saimiri proteins StpC and Tip has allowed us to extend the previously mentioned observations and investigate the role of StpC and Tip in replication of HIV-1 T-tropic strains (IIIB, MN, and RF) in human T cell lines. StpC expression in Molt4 dramatically enhanced HIV-1 replication as measured by Tat protein expression, syncytia formation, and accumulation of reverse transcriptase activity. In contrast, Tip expression in Molt4 cells inhibited HIV-1 replication and cytopathic effects relative to Molt4 cells transduced with the empty vector alone. The StpC-induced phenotype dominated in Molt4 cells transduced to express both StpC and Tip, suggesting that StpC is responsible for facilitating HIV-1 replication in H. saimiri-transformed T cells. Colony-forming ability of Tip-expressing Molt4 cells following HIV-1 infection was greatly enhanced over Molt4 cells expressing either StpC or no H. saimiri proteins at all. HIV-1 proviral DNA could be detected by PCR in surviving Molt4 cells expressing StpC or Tip, indicating that a persistent infection was established. A better understanding of the effects of Tip and StpC proteins on the biology of human hemopoietic stem cells may lead to novel therapeutic interventions for the treatment of AIDS.

Asunto(s)

VIH-1/fisiología , Herpesvirus Saimiriino 2/genética , Fosfoproteínas/metabolismo , Linfocitos T/virología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Bases , Línea Celular , ADN Viral/genética , Genes env , Genes pol , Células Gigantes , Duplicado del Terminal Largo de VIH , VIH-1/genética , VIH-2/fisiología , Humanos , Sistemas de Lectura Abierta , Fosfoproteínas/genética , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Transducción Genética , Transfección , Proteínas Virales/genética

RESUMEN

Some new 2',5'-oligonucleotide trimers containing 5'-terminal 5'-amino-5'-deoxy- and 5'-amino-3',5'-dideoxyadenosine derivatives have been synthesized. Some of the trimers showed biological inhibitions of HIV-1 reverse transcriptase (RT), HIV-1 induced syncytia formation and PCR amplification.

Asunto(s)

Nucleótidos de Adenina/síntesis química , Didesoxiadenosina/química , Oligorribonucleótidos/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Nucleótidos de Adenina/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Biopolímeros , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Oligorribonucleótidos/farmacología , Reacción en Cadena de la Polimerasa , Inhibidores de la Transcriptasa Inversa/farmacología

RESUMEN

We have previously demonstrated that Epstein-Barr virus (EBV) strain B95.8 can infect and initiate a partial transcriptional program in both CD4+ and CD8+ lymphocytes (Guan, M. X., R.-D. Zhang, B. Wu, and E. E. Henderson. 1996. J. Virol. 70:7341-7346). Experiments were undertaken to determine whether EBV infection can alter the growth potential of T lymphocytes. Peripheral blood lymphocytes were separated into populations consisting of 99.8% CD4+ and 98.6% CD8+ T lymphocytes by FACS. Infection of these populations with EBV resulted in blastogenesis in both CD4+ and CD8+ populations. Clones were established from the CD4+ population in the presence of interleukin-2. Two of these clones expressed the T cell surface markers CD3 and CD4 and carried the EBV genome. One lymphoblastoid cell line (LCL) had a mixture of CD4+ and CD19+ cells. The T-LCLs harboring the EBV genome in circular form and transcribed mRNA transcripts corresponding to BZLF1, BRLF1, BMLF1, and EBER-1 and -2. Immunofluorescence demonstrated EBNA in the nucleus of T rosette-positive lymphoblasts with an absence of viral capsid antigen expression. In situ hybridization for EBER showed nuclear and cytoplasmic staining in B95.8 cells, whereas EBV-carrying T-LCLs only showed nuclear staining. These results demonstrate that EBV can both infect and induce growth transformation of T lymphocytes, supporting a direct role for EBV in AIDS-related, EBV-associated T cell lymphomas. A better understanding of the EBV transcriptional program during EBV-induced T cell transformation could directly lead to adoptive T cell therapeutic strategies and/or more effective antiviral chemotherapy for EBV-associated T cell lymphoma.

Asunto(s)

Linfocitos T CD4-Positivos/citología , División Celular/fisiología , Herpesvirus Humano 4/fisiología , Secuencia de Bases , Linfocitos T CD4-Positivos/virología , Transformación Celular Viral , Cartilla de ADN , Genes Inmediatos-Precoces , Herpesvirus Humano 4/genética , Humanos , Hibridación Fluorescente in Situ , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

AIDS-related Epstein-Barr virus (EBV)-associated T cell lymphomas are emerging as a new, distinct histopathological entity. The pathway whereby EBV infects T cells as well as the initial EBV transcriptional program in T cells has not been established. In order to shed light on the early events of the EBV infection of T cells, we have used in situ reverse transcription based polymerase chain reaction (RT-PCR) to study the initial EBV transcriptional program in homogeneous CD4+ and CD8+ lymphocytes. Following EBV infection, Epstein-Barr nuclear antigen (EBNA) expression could be detected in T rosetting CD4+ and CD8+ T lymphocytes. Only a few cells showed viral capsid antigen (VCA). EBV immediate-early gene transcripts (BZLF1, BRLF1, and BMLF1) encoded in the BamHI Z, R, and M fragments could be detected by in situ RT-PCR in the EBV producer cell line B95.8. Both BZLF1 and BRLF1 immediate-early transcripts, but not BMLF1 transcript, could be detected in individual CD4+ and CD8+ T cells infected with EBV. Demonstration of EBV mRNA transcripts encoding immediate-early transcriptional transactivators in EBV-infected T cells provides the first evidence for a possible mechanism whereby EBV could contribute to T cell proliferation and EBV-associated T cell malignancies.

Asunto(s)

Antígenos Virales/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales , Linfoma de Burkitt/virología , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

CD4+ lymphocytes constitute one of the major cell targets for human immunodeficiency virus type 1 (HIV-1) infection. The eventual loss of CD4+ lymphocytes contributes substantially to the pathogenesis of HIV-1 and development of acquired immunodeficiency syndrome (AIDS). CD4+ lymphocytes consist of the subgroups Th1, Th2, and Th0, which differ in their cytokine profile. Th1 cells produce cytokines that favor cell-mediated immune responses, whereas Th2 cells produce cytokines that favor humoral immunity. Th0 cells are precursors to the Th1 and Th2 subsets. A shift from a Th1 to a Th2 response has been reported for HIV-1-infected patients (Kannagi et al. 1990. J. Virol. 64, 3399-3406; Walker et al. 1986. Science 234, 1563-1566; Walker et al. 1991. J. Virol. 65, 5921-5927). For this reason, the potential role of cytokines in the development of AIDS has received a great deal of attention. Interleukin (IL)-12 is a disulfide-linked, 70-kDa heterodimeric cytokine produced by antigen-presenting cells. IL-12 has a central role in the development of the Th1-type immune responses. Therefore, we investigated the ability of T-tropic HIV-1 IIIB to replicate in Th1, Th2, and Th0 T cell clones and studied the effects of IL-12 on HIV-1 replication in these cells types. These studies demonstrate several points. (1) Th1, Th2, and Th0 T cell clones support HIV-1 IIIB replication nearly equally well, and it is, therefore, unlikely that differences in ability to support HIV-1 replication can explain changes in Th1, Th2, or Th0 subtype 1 following HIV-1 infection. (2) Using this model, we show that IL-12 can inhibit HIV-1 replication, consistent with a role for IL-12 in HIV-1 replication in T cells. (3) HIV-1 can form a persistent infection in T cell clones, providing a reservoir model for study of viral sanctuary and persistence in a system closely approximating the in vivo situation.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/sangre , VIH-1 , Interleucina-12/uso terapéutico , Subgrupos de Linfocitos T/virología , Línea Celular , Epítopos , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/virología , Células Th2/virología , Replicación Viral

RESUMEN

Herpesvirus saimiri is a virus capable of inducing oncogenic transformation of T lymphocytes of New World primates and immortalizing human T cells in vitro. T lymphocytes immortalized by H. saimiri demonstrate functional biological responses to their antigens. Therefore, H. saimiri-induced transformation of T cells emerges as a very powerful tool of T-cell biology. Although the mechanism of this transformation remains to be understood, it is thought that H. saimiri proteins Tip and StpC play important roles. To facilitate functional studies of Tip and StpC, we retrovirally transduced human MOLT4, Jurkat and JCaM1 T-cell lines to express these H. saimiri proteins, using a three-plasmid system allowing for rapid and efficient production of high-titer retroviral stocks. Several cell lines expressing Tip and/or StpC in a stable fashion were obtained and characterized.

Asunto(s)

Herpesvirus Saimiriino 2/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Linfocitos T/metabolismo , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Células 3T3 , Animales , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , Vectores Genéticos , Herpesvirus Saimiriino 2/metabolismo , Humanos , Células Jurkat , Leucemia de Células T , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Retroviridae , Linfocitos T/virología , Transfección , Células Tumorales Cultivadas

RESUMEN

Cyclin T1 has been identified recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is sufficient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-specific signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the first time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.

Asunto(s)

Ciclinas/metabolismo , Activación de Linfocitos , Proteínas Quinasas/metabolismo , Linfocitos T/metabolismo , Regulación hacia Arriba , Ciclo Celular , Células Cultivadas , Ciclina T , Quinasa 9 Dependiente de la Ciclina , VIH-1 , Células HeLa , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Mitógenos/farmacología , Fosforilación , Fitohemaglutininas/farmacología , ARN Polimerasa II/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

A panel of human hematopoietic cell lines was genetically engineered to express recombinant complement receptor 2 (CR2 or CD21), which is also the Epstein-Barr virus (EBV) receptor. The panel was composed of SupT1, J1.1, and U1.HIV cells. The latter is a promonocytic cell line, whereas the other two are T lymphocytic cell lines. J1.1 and U1.HIV cells are latently infected by human immunodeficiency virus type 1 (HIV-1). These three cell lines were transduced with a murine leukemia virus (MLV)-based retroviral vector system. CR2 was efficiently and consistently expressed on the cell membranes, conferring enhanced susceptibility to EBV infection. The efficient expression of recombinant CR2 in cell lines of hematopoietic origin allowed for study of the interaction between EBV infection and HIV-1 gene regulation in suitable cell-culture models. The effects of EBV and HIV-1 coinfection results were cell-type dependent. In the two T lymphocytic cell lines, HIV-1 expression was rapidly and persistently down-regulated by EBV. Conversely, in the promonocytic cell line U1.HIV-CR2, HIV-1 expression was transiently enhanced by EBV. The EBV and HIV-1 coinfection result in U1.HIV-CR2 cells is potentially important, as the activation of HIV-1 gene expression in monocyte-like cells may play a crucial role in the mechanism of CD4+ T cell depletion by apoptosis. Therefore, the U1.HIV-CR2 cell line may represent a useful cell-culture system to study the synergism between EBV and HIV-1 in inducing apoptosis in primary CD4+ T cells.

Asunto(s)

Regulación Viral de la Expresión Génica , VIH-1/fisiología , Herpesvirus Humano 4/fisiología , Monocitos/virología , Receptores de Complemento 3d/fisiología , Linfocitos T/virología , Línea Celular , Infecciones por VIH/virología , Humanos , Monocitos/inmunología , Transducción de Señal , Linfocitos T/inmunología , Transfección , Replicación Viral

RESUMEN

The Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) can coinfect resting B cells, leading to EBV-carrying lymphoblastoid cell lines (LCLs) persistently infected with HIV-1. LCLs established from coinfected peripheral blood lymphocytes (PBLs) differed from LCLs derived from HIV-1-infected cell lines, in that the majority if not all of the cells expressed gp120 and a high percentage produced infectious HIV-1 after continuous passage for 6-9 months. Restriction analysis of the putative HIV-1 provirus revealed that persistently infected LCLs carried variable copies of primarily unintegrated circular and/or linear forms of HIV-1 DNA. This extrachromosomal location is strikingly different from that of the one to three copies of integrated proviral DNA deleted in persistently infected T cell and monocytic cell lines. Anti-gp120 monoclonal antibody and 3'-azido-3'-deoxythymidine (AZT) inhibited HIV-1 expression and reduced HIV-1 DNA copy number in persistently infected LCLs, supporting the hypothesis that unintegrated HIV-1 DNA accumulates primarily as a result of superinfection. We propose that the extrachromosomal location of the HIV-1 DNA contributes to the semipermissive nature of B cell infection by HIV-1.

Asunto(s)

Linfocitos B/virología , ADN Viral/química , VIH-1/genética , Aciclovir/farmacología , Fármacos Anti-VIH/farmacología , Línea Celular , Células Cultivadas , ADN Viral/aislamiento & purificación , Genoma Viral , Células Gigantes/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/fisiología , Herpesvirus Humano 4/fisiología , Humanos , Provirus/genética , Provirus/fisiología , Mapeo Restrictivo , Zidovudina/farmacología

RESUMEN

CR2 (CD21), the EBV receptor, was detected on three of four CD4-positive cell lines by indirect fluorescent labeling, and its corresponding mRNA was found by use of the reverse transcription-based polymerase chain reaction. To determine whether CR2 on CD4-positive cells was functional, their ability to be infected by EBV was analyzed. EBV DNA, EBV nuclear antigen 2 (EBNA-2A), and EBV-encoded small RNA (EBER1) transcripts could be detected in CR2-expressing CD4-positive cells following infection by the B95.8 strain of EBV. Analysis of the terminal region showed the EBV genome remained linear following infection, and copy number decreased with time. Since CD4-positive cell lines are targets for HIV-1 infection, the effects of EBV infection on HIV-1 expression were analyzed. HIV-1 replication was upregulated when CD4-positive cells were coinfected with EBV strain B95.8 but not P3HR-1K. These results suggested that EBNA-2 is involved in upregulation of HIV-1 expression in T lymphoblastoid cell lines. To test this hypothesis an EBNA-2-expression vector was transfected into T lymphoblastoid cell lines and HIV-1 expression measured. First, trans-activation of HIV-1 long terminal repeat (LTR) by Tat was enhanced by EBNA-2 type 1 expression. trans-Activation of the HIV-1 LTR by Tat was also enhanced when CD4-positive cells were infected by EBV (strain B95.8) encoding an intact EBNA-2, but not by P3HR-1K with a deleted EBNA-2. In addition, CD4-positive cell clones stably expressing EBNA-2 supported enhanced HIV-1 replication as measured by accumulation of reverse transcriptase activity and syncytium induction. This provides direct evidence that EBV infection can enhance HIV-1 replication in T cells. Whether this in vitro phenomenon contributes to disease progression in vivo remains to be determined.

Asunto(s)

Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Herpesvirus Humano 4/fisiología , Línea Celular Transformada , ADN Viral/análisis , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Regulación Viral de la Expresión Génica , Productos del Gen tat , Duplicado del Terminal Largo de VIH , VIH-1/genética , Herpesvirus Humano 4/genética , Humanos , ARN Mensajero , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/inmunología , Receptores de Complemento 3d/fisiología , Receptores Virales/genética , Receptores Virales/inmunología , Receptores Virales/fisiología , Activación Transcripcional , Transfección , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

CD4+ and CD8+ T lymphocytes purified from normal adult donors by flow cytometry could be infected with Epstein-Barr virus (EBV) as measured by the accumulation of components of the EBV replicative cycle, viral DNA and viral transcripts encoding EBER1 and BRLF1. EBV infection resulted in enhanced replication of human immunodeficiency virus type 1 (HIV-1) IIIB in CD4+ lymphocytes as measured by accumulation of reverse transcriptase and formation of syncytia. Furthermore, a small percentage of CD8+ T cells became permissive after infection with EBV. Inactivation of transforming functions by irradiation with UV light greatly reduced the ability of EBV to enhance HIV-1 replication in T4+ T cell, suggesting that live virus is needed for enhancement. These results demonstrate a direct synergy between EBV and HIV-1 during coinfection of T cells in vitro and may explain the beneficial effect of acyclovir in combination with antiretroviral chemotherapy as well as the increased incidence of T-cell lymphomas associated with EBV in patients with AIDS.

Asunto(s)

Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , VIH-1 , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4 , Infecciones Tumorales por Virus/virología , Adulto , Células Cultivadas , Citometría de Flujo , Humanos , Replicación Viral

RESUMEN

Cells from persons with Bloom syndrome feature an elevated rate of sister-chromatid exchange (SCE). However, in some affected persons a minority of blood lymphocytes have a normal SCE rate. Persons who inherit the Bloom syndrome gene BLM identical by descent from a common ancestor very rarely exhibit this high-SCE/low-SCE mosaicism; conversely, mosaicism arises predominantly in persons who do not share a common ancestor. These population data suggested that most persons with Bloom syndrome in whom the exceptional low-SCE cells arise are not homozygous for a mutation at BLM but instead are compound heterozygotes. Following this clue, we carried out a genotype analysis of loci syntenic with BLM in 11 persons who exhibited mosaicism. In five of them, polymorphic loci distal to BLM that were heterozygous in their high-SCE cells had become homozygous in their low-SCE cells, whereas heterozygous loci proximal to BLM remained heterozygous. These observations are interpreted to mean that intragenic recombination between paternally derived and maternally derived mutated sites within BLM can generate a functionally wild-type gene and that low-SCE lymphocytes are progeny of a somatic cell in which such intragenic recombination had occurred.

Asunto(s)

Síndrome de Bloom/genética , Recombinación Genética , Intercambio de Cromátides Hermanas , Deleción Cromosómica , Mapeo Cromosómico , ADN/análisis , Humanos , Fenotipo

RESUMEN

2',5'-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway. Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses. Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication. The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(Rp)ApA, A(Sp)ApA and ApA(Rp)A are agonists (IC50 = 2 x 10(-7), 2 x 10(-6) and 8 x 10(-6) M); ApA(Sp)A is an antagonist. The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(Rp)A, ApA(Rp)ApA, and ApApA(Rp)A are agonists (IC50 = 5 x 10(-7) M); ApA(Sp)A, ApA(Sp)ApA, and ApApA(Sp)A are antagonists. Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2',3'-terminus. ApA(Rp)A, ApA(Sp)A, ApApA(Rp)A, and ApApA(Sp)A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control). HIV-1 infection results in enhanced uptake and accumulation of ApA(Rp)A and ApA(Sp)A (7- and 10-fold, respectively). These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy.

Asunto(s)

Nucleótidos de Adenina/síntesis química , Nucleótidos de Adenina/farmacología , Antivirales/farmacología , Endorribonucleasas/metabolismo , VIH-1/fisiología , Replicación Viral/efectos de los fármacos , Nucleótidos de Adenina/metabolismo , Animales , Antivirales/síntesis química , Secuencia de Bases , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN , Endorribonucleasas/biosíntesis , Activación Enzimática , Escherichia coli , Células Gigantes/efectos de los fármacos , VIH-1/efectos de los fármacos , Humanos , Indicadores y Reactivos , Cinética , Células L , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Relación Estructura-Actividad

RESUMEN

In vitro-synthesized human T-cell leukemia virus type 1 (HTLV-I) Rex response element (Rex-RE) activates the interferon-induced 2',5'-oligoadenylate synthetase (2-50AS) in a dose-dependent manner. In addition, Rex-RE at 1 microgram/ml activates a second interferon-induced enzyme, p68 kinase (PKR); however, at 50 micrograms/ml, Rex-RE inhibits PKR activity. Poly(rl)-poly(rC) (10 micrograms/ml) dissociates the ribonucleoprotein complexes, Rex-RE/2-5OAS, or Rex-RE/PKR, whereas poly(rC) (100 micrograms/ml) does not, indicating the presence of high affinity interactions between Rex-RE and these two enzymes. To further characterize the interaction of Rex-RE with 2-5OAS and PKR, [32P]Rex-RE was uv-cross-linked to 2-5OAS and PKR present in interferon-treated HeLa cell extracts. The affinity of Rex-RE to highly purified 40-kDa human recombinant 2-5OAS was determined to be Kd = 4.7 nM. The relevance of these results to the pathogenesis of HTLV-I-associated adult T-cell leukemia/lymphoma is discussed.

Asunto(s)

2',5'-Oligoadenilato Sintetasa/biosíntesis , Virus Linfotrópico T Tipo 1 Humano/fisiología , Interferón-alfa/farmacología , Proteínas Serina-Treonina Quinasas/biosíntesis , ARN Viral/metabolismo , 2',5'-Oligoadenilato Sintetasa/aislamiento & purificación , Reactivos de Enlaces Cruzados , Relación Dosis-Respuesta a Droga , Endorribonucleasas , Activación Enzimática , Inducción Enzimática , Células HeLa , Humanos , Cinética , Poli C/farmacología , Poli I-C/farmacología , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Ribonucleasa III , Rayos Ultravioleta , eIF-2 Quinasa

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) isolated from patients with acquired immunodeficiency syndrome (AIDS) shows resistance to 3'azido-3'deoxythymidine (AZT) after one or two years of treatment. AZT also has significant toxic side effects, further limiting its use in the therapy of HIV-1-infected individuals. Dehydroepiandrosterone (DHEA) has been shown to have a broad spectrum of biological functions, to be bioavailable orally and to be relatively nontoxic. Epidemiological studies provide evidence that reduced serum levels of DHEA are related to the progression of AIDS in HIV-1 infection. DHEA has also been shown to inhibit HIV-1 replication in vitro and block HIV-1 reactivation from chronically infected cell lines. However, there have been no reports on the ability of DHEA to inhibit the replication of AZT-resistant strains of HIV-1. We investigated whether DHEA treatment could inhibit replication of AZT-resistant strains of HIV-1. Addition of DHEA to MT-2 cell cultures infected with either AZT-sensitive or AZT-resistant isolates of HIV-1 resulted in dose-dependent inhibition of HIV-1-induced cytopathic effect and suppression of HIV-1 replication as measured by accumulation of reverse transcriptase activity. At a concentration as low as 50 microM, DHEA reduced AZT-resistant HIV-1 replication over 50 percent as measured by cytopathic effect and accumulation of reverse transcriptase activity. This study provides evidence that DHEA can inhibit the replication of AZT-resistant as well as wild-type HIV-1. Since the main targets for DHEA are metabolic and cellular signaling pathways leading to HIV-1 replication-activation, DHEA should be effective against multidrug-resistant strains of HIV-1. Combined with recently discovered immunoregulatory properties, the finding that DHEA is able to inhibit replication of both wild-type and AZT-resistant HIV-1 suggests that in vivo DHEA may have a much broader spectrum of action than originally anticipated.

Asunto(s)

Deshidroepiandrosterona/farmacología , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Zidovudina/farmacología , Efecto Citopatogénico Viral , Farmacorresistencia Microbiana , VIH-1/crecimiento & desarrollo , Humanos , Técnicas In Vitro , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacos