scholarly journals Nonproductive Human Immunodeficiency Virus Type 1 Infection in Nucleoside-Treated G0 Lymphocytes

1999 ◽  
Vol 73 (8) ◽  
pp. 6526-6532 ◽  
Author(s):  
Yael D. Korin ◽  
Jerome A. Zack
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Productive Infection ◽  
Transcription Process ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Productive infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. We have previously identified G1b as the cell cycle stage required for the optimal completion of the reverse transcription process in T lymphocytes. However, the mechanism(s) involved in the blockage of reverse transcription remains undefined. In this study we investigated whether nucleotide levels influence viral reverse transcription in G0 cells. For this purpose the role of the enzyme ribonucleotide reductase was bypassed, by adding exogenous deoxyribonucleosides to highly purified T cells in the G0 or the G1a phase of the cell cycle. Our data showed a significant increase in the efficiency of the reverse transcription process following the addition of the deoxyribonucleosides. To define the stability and functionality of these full reverse transcripts, we used an HIV-1 reporter virus that expresses the murine heat-stable antigen on the surfaces of infected cells. Following activation of infected quiescent cells treated with exogenous nucleosides, no increased rescue of productive infection was seen. Thus, in addition to failure to complete reverse transcription, there was an additional nonreversible blockage of productive infection in quiescent T cells. These experiments have important relevance in the gene therapy arena, in terms of improving the ability of lentivirus vectors to enter metabolically inactive cells, such as hematopoietic stem cells.

scholarly journals Kinetics of Human Immunodeficiency Virus Type 1 Decay following Entry into Resting CD4+ T Cells

2005 ◽  
Vol 79 (4) ◽  
pp. 2199-2210 ◽  
Author(s):  
Yan Zhou ◽  
Haili Zhang ◽  
Janet D. Siliciano ◽  
Robert F. Siliciano
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Half Life ◽  
Immunodeficiency Virus ◽  
Kinetics Of ◽  
Hiv 1

ABSTRACT In untreated human immunodeficiency virus type 1 (HIV-1) infection, most viral genomes in resting CD4+ T cells are not integrated into host chromosomes. This unintegrated virus provides an inducible latent reservoir because cellular activation permits integration, virus gene expression, and virus production. It remains controversial whether HIV-1 is stable in this preintegration state. Here, we monitored the fate of HIV-1 in resting CD4+ cells by using a green fluorescent protein (GFP) reporter virus carrying an X4 envelope. After virus entry into resting CD4+ T cells, both rescuable virus gene expression, visualized with GFP, and rescuable virion production, assessed by p24 release, decayed with a half-life of 2 days. In these cells, reverse transcription goes to completion over 2 to 3 days, and 50% of the viruses that have entered undergo functional decay before reverse transcription is complete. We distinguished two distinct but closely related factors contributing to loss of rescuable virus. First, some host cells undergo virus-induced apoptosis upon viral entry, thereby reducing the amount of rescuable virus. Second, decay processes directly affecting the virus both before and after the completion of reverse transcription contribute to the loss of rescuable virus. The functional half-life of full-length, integration-competent reverse transcripts is only 1 day. We propose that rapid intracellular decay processes compete with early steps in viral replication in infected CD4+ T cells. Decay processes dominate in resting CD4+ T cells as a result of the slow kinetics of reverse transcription and blocks at subsequent steps. Therefore, the reservoir of unintegrated HIV-1 in recently infected resting CD4+ T cells is highly labile.

scholarly journals Immature Dendritic Cells Selectively Replicate Macrophagetropic (M-Tropic) Human Immunodeficiency Virus Type 1, while Mature Cells Efficiently Transmit both M- and T-Tropic Virus to T Cells

1998 ◽  
Vol 72 (4) ◽  
pp. 2733-2737 ◽  
Author(s):  
Angela Granelli-Piperno ◽  
Elena Delgado ◽  
Victoria Finkel ◽  
William Paxton ◽  
Ralph M. Steinman
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Dendritic Cells ◽  
Human Immunodeficiency Virus Type ◽  
Reverse Transcription ◽  
Blood Monocytes ◽  
Gm Csf ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Dendritic cells (DCs) can develop from CD14+ peripheral blood monocytes cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). By 6 days in culture, the cells have the characteristics of immature DCs and can be further induced to mature by inflammatory stimuli or by monocyte-conditioned medium. After infection with macrophagetropic (M-tropic) human immunodeficiency virus type 1 (HIV-1), monocytes and mature DCs show a block in reverse transcription and only form early transcripts that can be amplified with primers for the R/U5 region. In contrast, immature DCs cultured for 6 or 11 days in GM-CSF and IL-4 complete reverse transcription and show a strong signal when LTR/gag primers are used. Blood monocytes and mature DCs do not replicate HIV-1, whereas immature DCs can be productively infected, but only with M-tropic HIV-1. The virus produced by immature DCs readily infects activated T cells. Although mature DCs do not produce virus, these cells transmit both M- and T-tropic virus to T cells. In the cocultures, both DCs and T cells must express functional chemokine coreceptors for viral replication to occur. Therefore, the developmental stage of DCs can influence the interaction of these cells with HIV-1 and influence the extent to which M-tropic and T-tropic virus can replicate.

scholarly journals Molecular Characterization of Preintegration Latency in Human Immunodeficiency Virus Type 1 Infection

2002 ◽  
Vol 76 (17) ◽  
pp. 8518-8531 ◽  
Author(s):  
Theodore C. Pierson ◽  
Yan Zhou ◽  
Tara L. Kieffer ◽  
Christian T. Ruff ◽  
Christopher Buck ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Viral Genome ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Most current evidence suggests that the infection of resting CD4+ T cells by human immunodeficiency virus type 1 (HIV-1) is not productive due to partial or complete blocks in the viral life cycle at steps prior to integration of the viral genome into the host cell chromosome. However, stimulation of an infected resting T cell by antigen, cytokines, or microenvironmental factors can overcome these blocks and allow for the production of progeny virions. In this study, we sought to understand the structure and fate of the virus in unstimulated resting CD4+ T cells. Using a novel linker-mediated PCR assay designed to detect and characterize linear unintegrated forms of the HIV-1 genome, we demonstrate that reverse transcription can proceed to completion following the infection of resting T cells, generating the substrate for the retroviral integration reaction. However, reverse transcription in resting T cells is far slower than in activated T cells, requiring 2 to 3 days to complete. The delay in completing reverse transcription may make the viral DNA genome more susceptible to competing decay processes. To explore the relationship between the formation of the linear viral genome and the stability of the preintegration state, we employed a recombinant HIV-1 virus expressing the enhanced green fluorescent protein to measure the rate at which HIV-1 decays in the preintegration state. Our results demonstrate that the preintegration state is labile and decays rapidly (half-life = 1 day) following the entry of HIV-1 into a resting T cell, with significant decay occurring during the slow process of reverse transcription.

scholarly journals Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages

1998 ◽  
Vol 72 (8) ◽  
pp. 6671-6677 ◽  
Author(s):  
Laco Kacani ◽  
Ines Frank ◽  
Martin Spruth ◽  
Michael G. Schwendinger ◽  
Brigitte Müllauer ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Dendritic Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Productive Infection ◽  
Lower Amount ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4+ T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4+ cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83− DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83+DC (maDC); this finding is in contrast to data previously obtained with CD4+ T cells. Third, maturation from imDC to maDC efficiently silenced expression of β2-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.

scholarly journals Preferential Cytolysis of Peripheral Memory CD4+ T Cells by In Vitro X4-Tropic Human Immunodeficiency Virus Type 1 Infection before the Completion of Reverse Transcription

2008 ◽  
Vol 82 (18) ◽  
pp. 9154-9163 ◽  
Author(s):  
Yan Zhou ◽  
Lin Shen ◽  
Hung-Chih Yang ◽  
Robert F. Siliciano
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Death ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Host Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT CD4+ T-cell depletion is the hallmark of AIDS pathogenesis. Multiple mechanisms may contribute to the death of productively infected CD4+ T cells and innocent-bystander cells. In this study, we characterize a novel mechanism in which human immunodeficiency virus type 1 (HIV-1) infection preferentially depletes peripheral memory CD4+ T cells before the completion of reverse transcription. Using a recombinant HIV-1 carrying the green fluorescent protein reporter gene, we demonstrate that memory CD4+ T cells were susceptible to infection-induced cell death at a low multiplicity of infection. Infected memory CD4+ T cells underwent rapid necrotic cell death. Killing of host cells was dependent on X4 envelope-mediated viral fusion, but not on virion-associated Vpr or Nef. In contrast to peripheral resting CD4+ T cells, CD4+ T cells stimulated by mitogen or certain cytokines were resistant to HIV-1-induced early cell death. These results demonstrate that early steps in HIV-1 infection have a detrimental effect on certain subsets of CD4+ T cells. The early cell death may serve as a selective disadvantage for X4-tropic HIV-1 in acute infection but may play a role in accelerated disease progression, which is associated with the emergence of X4-tropic HIV-1 in the late stage of AIDS.

scholarly journals Progression to the G1b Phase of the Cell Cycle Is Required for Completion of Human Immunodeficiency Virus Type 1 Reverse Transcription in T Cells

1998 ◽  
Vol 72 (4) ◽  
pp. 3161-3168 ◽  
Author(s):  
Yael D. Korin ◽  
Jerome A. Zack
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Reverse Transcription ◽  
Cell Cycle Progression ◽  
Target Cells ◽  
Cycle Progression ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Successful infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. In the present study, we isolated highly purified quiescent T cells and utilized the CD3/CD28 activation pathways as well as cell cycle inhibitors to further define the role of costimulation and cell cycle progression in HIV-1 reverse transcription. Activation with αCD3 alone resulted in cell cycle progression into only G1a and incomplete HIV-1 reverse transcription. Costimulation through the CD28 receptor and transition into G1b was required to efficiently complete the reverse transcription process. These findings have relevance to immune activation in vivo, since lymphocytes rendered anergic by a single activation signal would be nonpermissive for productive infection with HIV-1. Importantly, these data also suggest that HIV vector-based genetic transduction strategies might be successful only in target cells that transition into the G1b phase of the cell cycle.

scholarly journals Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription

2008 ◽  
Vol 82 (19) ◽  
pp. 9318-9328 ◽  
Author(s):  
James A. Thomas ◽  
William J. Bosche ◽  
Teresa L. Shatzer ◽  
Donald G. Johnson ◽  
Robert J. Gorelick
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Nucleocapsid Protein ◽  
Productive Infection ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP(−) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55Gag processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

scholarly journals Diminished Human Immunodeficiency Virus Type 1 Reverse Transcription and Nuclear Transport in Primary Macrophages Arrested in Early G1 Phase of the Cell Cycle

2000 ◽  
Vol 74 (4) ◽  
pp. 1712-1717 ◽  
Author(s):  
Neeltje A. Kootstra ◽  
Bianca M. Zwart ◽  
Hanneke Schuitemaker
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Proviral Integration ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Previously, we and others have demonstrated that the process of reverse transcription of human immunodeficiency virus type 1 (HIV-1) is disturbed in nondividing macrophages and quiescent T lymphocytes. Here we analyzed which phase of the cell cycle in macrophages is crucial for early steps in the HIV-1 replication cycle. HIV-1 Ba-L-inoculated macrophages arrested early in the G1 phase byn-butyrate contained incomplete products of reverse transcription. In gamma-irradiated macrophages, reverse transcription was successfully completed but proviral integration could not be detected. In these cells, nuclear import was disturbed as reflected by the absence of two-long-terminal-repeat circles. In macrophages arrested late in G1 phase by aphidicolin or 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), reverse transcription was unaffected. Proviral integration occurred efficiently in DRB-treated macrophages, whereas integrated proviral DNA could not be detected after aphidicolin treatment. Arrest at G2 phase of the cell cycle by nocodazole did not affect reverse transcription or proviral integration. Treatment of macrophages with hydroxyurea (HU), which reduces the intracellular deoxynucleoside triphosphate (dNTP) pool by blocking the de novo synthesis of dNTP, resulted in a dose-dependent inhibition of HIV-1 reverse transcription. This could partially be restored by the addition of nucleoside precursors. Addition of nucleoside precursors enhanced both reverse transcription and cell proliferation. However, the disturbed reverse transcription observed in the nonproliferating andn-butyrate-treated macrophages could not be restored by addition of nucleoside precursors. Similar to observations in quiescent T lymphocytes, incomplete proviral DNA species were arrested in the cytoplasm of the macrophages. Our results indicate that also in primary macrophages the intracellular nucleotide pools and other cellular factors that coincide with late G1 phase of the cell cycle may contribute to efficient reverse transcription and nuclear localization.

scholarly journals Cell Cycle- and Vpr-Mediated Regulation of Human Immunodeficiency Virus Type 1 Expression in Primary and Transformed T-Cell Lines

1999 ◽  
Vol 73 (7) ◽  
pp. 5422-5430 ◽  
Author(s):  
Suryaram Gummuluru ◽  
Michael Emerman
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytotoxic Effects ◽  
Immunodeficiency Virus ◽  
T Cell Lines ◽  
Hiv 1

ABSTRACT Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) transiently arrests cells in the G2 phase of the cell cycle and is a weak transcriptional transactivator. We found that Vpr increased HIV-1 long terminal repeat (LTR) activity in all cells examined but, when expressed at high levels, decreased HIV-1 LTR expression due to cytotoxic effects. Moreover, Vpr-mediated enhancement of HIV-1 LTR-driven transcription was observed in cycling primary human CD4+ T cells but not in terminally differentiated, noncycling primary human macrophages. In single-round infection experiments using primary human CD4+ T cells, proviral clones expressing either wild-type Vpr or Vpr mutants that retained the ability to cause a G2 arrest replicated to higher levels than proviruses lacking Vpr or expressing mutants of Vpr that did not cause an arrest. In support of the hypothesis that enhancement of HIV-1 LTR transcription by Vpr is an indirect effect of the ability of Vpr to delay cells in G2, counterflow centrifugal elutriation of cells into different phases of the cell cycle demonstrated that HIV-1 LTR expression was highest in G2. Finally, the ability of Vpr to upregulate viral transcription was dependent on a minimal promoter containing a functional TATA box and an enhancer.

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