scholarly journals Simian Virus 40-Based Replication of Catalytically Inactive Human Immunodeficiency Virus Type 1 Integrase Mutants in Nonpermissive T Cells and Monocyte-Derived Macrophages

2004 ◽  
Vol 78 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Richard Lu ◽  
Noriko Nakajima ◽  
Wolfgang Hofmann ◽  
Monsef Benkirane ◽  
Kuan Teh-Jeang ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Integrase function is required for retroviral replication in most instances. Although certain permissive T-cell lines support human immunodeficiency virus type 1 (HIV-1) replication in the absence of functional integrase, most cell lines and primary human cells are nonpermissive for integrase mutant growth. Since unintegrated retroviral DNA is lost from cells following cell division, we investigated whether incorporating a functional origin of DNA replication into integrase mutant HIV-1 might overcome the block to efficient gene expression and replication in nonpermissive T-cell lines and primary cells. Whereas the Epstein-Barr virus (EBV) origin (oriP) did little to augment expression from an integrase mutant reporter virus in EBV nuclear antigen 1-expressing cells, simian virus 40 (SV40) oriT dramatically enhanced integrase mutant infectivity in T-antigen (Tag)-expressing cells. Incorporating oriT into the nef position of a full-length, integrase-defective virus strain yielded efficient replication in Tag-expressing nonpermissive Jurkat T cells without reversion to an integration-competent genotype. Adding Tag to integrase mutant-oriT viruses yielded 11.3-kb SV40-HIV chimeras that replicated in Jurkat cells and primary monocyte-derived macrophages. Real-time quantitative PCR analyses of Jurkat cell infections revealed that amplified copies of unintegrated DNA likely contributed to SV40-HIV integrase mutant replication. SV40-based HIV-1 integrase mutant replication in otherwise nonpermissive cells suggests alternative approaches to standard integrase-mediated retroviral gene transfer strategies.

scholarly journals Death of CD4+ T-Cell Lines Caused by Human Immunodeficiency Virus Type 1 Does Not Depend on Caspases or Apoptosis

2002 ◽  
Vol 76 (10) ◽  
pp. 5094-5107 ◽  
Author(s):  
Diane L. Bolton ◽  
Beom-Ik Hahn ◽  
Eugenia A. Park ◽  
Laura L. Lehnhoff ◽  
Felicita Hornung ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Lines ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which human immunodeficiency virus type 1 (HIV-1) induces death in CD4+ T lymphocytes. A better understanding of the death process occurring in infected cells may provide valuable insight into the viral component responsible for cytopathicity. This would aid the design of preventive treatments against the rapid decline of CD4+ T cells that results in AIDS. Previously, apoptotic cell death has been reported in HIV-1 infections in cultured T cells, and it has been suggested that this could affect both infected and uninfected cells. To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting several T-cell lines and assessing the level of apoptosis by using various biochemical and flow cytometric assays. Contrary to the prevailing view that apoptosis plays a prominent role in HIV-1-mediated T-cell death, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. Among the parameters investigated, Annexin V display, caspase activity and cleavage of caspase substrates, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) signal, and APO2.7 display were detected at low to negligible levels. Neither peptide caspase inhibitors nor the antiapoptotic proteins Bcl-xL or v-FLIP could prevent cell death in HIV-1-infected cultures. Furthermore, Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity. These results suggest that the primary mode of cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis. Rather, cell death occurs most likely via a necrotic or lytic form of death independent of caspase activation in directly infected cells.

scholarly journals Murine T Cells Potently Restrict Human Immunodeficiency Virus Infection

2004 ◽  
Vol 78 (22) ◽  
pp. 12537-12547 ◽  
Author(s):  
Jörg G. Baumann ◽  
Derya Unutmaz ◽  
Michael D. Miller ◽  
Sabine K. J. Breun ◽  
Stacy M. Grill ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Viral Gene ◽  
Human T Cell ◽  
Virus Dose ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Development of a mouse model for human immunodeficiency virus type 1 (HIV-1) infection has advanced through the progressive identification of host cell factors required for HIV-1 replication. Murine cells lack HIV-1 receptor molecules, do not support efficient viral gene expression, and lack factors necessary for the assembly and release of virions. Many of these blocks have been described using mouse fibroblast cell lines. Here we identify a postentry block to HIV-1 infection in mouse T-cell lines that has not been detected in mouse fibroblasts. While murine fibroblastic lines are comparable to human T-cell lines in permissivity to HIV-1 transduction, infection of murine T cells is 100-fold less efficient. Virus entry occurs efficiently in murine T cells. However, reduced efficiency of the completion of reverse transcription and nuclear transfer of the viral preintegration complex are observed. Although this block has similarities to the restriction of murine retroviruses by Fv1, there is no correlation of HIV-1 susceptibility with cellular Fv1 genotypes. In addition, the block to HIV-1 infection in murine T-cell lines cannot be saturated by a high virus dose. Further studies of this newly identified block may lend insight into the early events of retroviral replication and reveal new targets for antiretroviral interventions.

scholarly journals Cellular Gene Expression upon Human Immunodeficiency Virus Type 1 Infection of CD4+-T-Cell Lines

2003 ◽  
Vol 77 (2) ◽  
pp. 1392-1402 ◽  
Author(s):  
Angélique B. van 't Wout ◽  
Ginger K. Lehrman ◽  
Svetlana A. Mikheeva ◽  
Gemma C. O'Keeffe ◽  
Michael G. Katze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv 1

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.

scholarly journals Expanded Tropism of Primary Human Immunodeficiency Virus Type 1 R5 Strains to CD4+ T-Cell Lines Determined by the Capacity To Exploit Low Concentrations of CCR5

1999 ◽  
Vol 73 (9) ◽  
pp. 7842-7847 ◽  
Author(s):  
Nathalie Dejucq ◽  
Graham Simmons ◽  
Paul R. Clapham
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) non-syncytium-inducing (NSI) strains predominantly use the chemokine receptor CCR5, while syncytium-inducing (SI) strains use CXCR4. In vitro, SI isolates infect and replicate in a range of CD4+ CXCR4+ T-cell lines, whereas NSI isolates usually do not. Here we describe three NSI strains that are able to infect two CD4+ T-cell lines, Molt4 and SupT1. For one strain, a variant of JRCSF selected in vitro, replication on Molt4 was previously shown to be conferred by a single amino-acid change in the V1 loop (M.T. Boyd et al., J. Virol. 67:3649–3652, 1993). On CD4+ cell lines expressing different coreceptors, these strains use CCR5 predominantly and do not replicate in CCR5-negative peripheral blood mononuclear cells derived from individuals homozygous for Δ32 CCR5. Furthermore, infection of Molt4 and SupT1 by each of these three strains is potently inhibited by ligands for CCR5, including 2D7, a monoclonal antibody specific for CCR5. CCR5 mRNA was present in both Molt4 and SupT1 by reverse transcription-PCR, although CCR5 protein could not be detected either on the cell surface or in intracellular vesicles. The expanded tropism of the three strains shown here is therefore not due to adaptation to a new coreceptor but due to the capacity to exploit extremely low levels of CCR5 on Molt4 and SupT1 cells. This novel tropism observed for a subset of primary HIV-1 isolates may represent an extended tropism to new CD4+ cell types in vivo.

scholarly journals High-Throughput Human Immunodeficiency Virus Type 1 (HIV-1) Full Replication Assay That Includes HIV-1 Vif as an Antiviral Target

2005 ◽  
Vol 49 (9) ◽  
pp. 3833-3841 ◽  
Author(s):  
Joan Cao ◽  
Jason Isaacson ◽  
Amy K. Patick ◽  
Wade S. Blair
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
High Throughput ◽  
Antiviral Compounds ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable Z′ value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >106 compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.

scholarly journals Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism

1999 ◽  
Vol 73 (9) ◽  
pp. 7117-7125 ◽  
Author(s):  
Yanjie Yi ◽  
Stuart N. Isaacs ◽  
Darlisha A. Williams ◽  
Ian Frank ◽  
Dominique Schols ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4).

scholarly journals Novel Pathway for Induction of Latent Virus from Resting CD4+ T Cells in the Simian Immunodeficiency Virus/Macaque Model of Human Immunodeficiency Virus Type 1 Latency

2006 ◽  
Vol 81 (4) ◽  
pp. 1660-1670 ◽  
Author(s):  
Anding Shen ◽  
Hung-Chih Yang ◽  
Yan Zhou ◽  
Amanda J. Chase ◽  
Jean D. Boyer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Simian Immunodeficiency Virus ◽  
Cell Lines ◽  
Macaque Model ◽  
Immunodeficiency Virus ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACT Although combination therapy allows the suppression of human immunodeficiency virus type 1 (HIV-1) viremia to undetectable levels, eradication has not been achieved because the virus persists in cellular reservoirs, particularly the latent reservoir in resting CD4+ T lymphocytes. We previously established a simian immunodeficiency virus (SIV)/macaque model to study latency. We describe here a novel mechanism for the induction of SIV from latently infected resting CD4+ T cells. Several human cell lines including CEMx174 and Epstein-Barr virus-transformed human B-lymphoblastoid cell lines mediated contact-dependent activation of resting macaque T cells and induction of latent SIV. Antibody-blocking assays showed that interactions between the costimulatory molecule CD2 and its ligand CD58 were involved, whereas soluble factors and interactions between T-cell receptors and major histocompatibility complex class II were not. Combinations of specific antibodies to CD2 also induced T-cell activation and virus induction in human resting CD4+ T cells carrying latent HIV-1. This is the first demonstration that costimulatory signals can induce latent virus without the coengagement of the T-cell receptor, and this study might provide insights into potential pathways to target latent HIV-1.

scholarly journals Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

2001 ◽  
Vol 75 (17) ◽  
pp. 7944-7955 ◽  
Author(s):  
Noriko Nakajima ◽  
Richard Lu ◽  
Alan Engelman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Dna Recombination ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

Sign in / Sign up

Export Citation Format

Share Document