scholarly journals Multiple Blocks to Human Immunodeficiency Virus Type 1 Replication in Rodent Cells

2000 ◽  
Vol 74 (21) ◽  
pp. 9868-9877 ◽  
Author(s):  
Paul D. Bieniasz ◽  
Bryan R. Cullen
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Infectious Virus ◽  
Human Gene ◽  
Human Cells ◽  
Gene Products ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.

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 Cyclin T1 Expression Is Mediated by a Complex and Constitutively Active Promoter and Does Not Limit Human Immunodeficiency Virus Type 1 Tat Function in Unstimulated Primary Lymphocytes

2002 ◽  
Vol 76 (1) ◽  
pp. 208-219 ◽  
Author(s):  
Juan Martin-Serrano ◽  
Kelvin Li ◽  
Paul D. Bieniasz
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Expression Levels ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Immortalized Cell ◽  
Hiv 1

ABSTRACT Cyclin T1 (CycT1), a component of positive-transcription-elongation factor-b (P-TEFb), is an essential cofactor for transcriptional activation by lentivirus Tat proteins. It is thought that low CycT1 expression levels restrict human immunodeficiency virus type 1 (HIV-1) expression levels and replication in resting CD4+ lymphocytes. In this study, we undertook a functional analysis of the cycT1 promoter to determine which, if any, promoter elements might be responsible for cellular activation state-dependent CycT1 expression. The cycT1 gene contains a complex promoter that exhibits an extreme degree of functional redundancy: five nonoverlapping fragments were found to exhibit significant promoter activity in immortalized cell lines, and these elements could interact in a synergistic or redundant manner to mediate cycT1 transcription. Reporter gene expression, mediated by the cycT1 promoter, was detectable in unstimulated transfected primary lymphocytes and multiple sites within the promoter could serve to initiate transcription. While utilization of these start sites was significantly altered by the application of exogenous stimuli to primary lymphocytes and two distinct promoter elements exhibited enhanced activity in the presence of phorbol ester, overall cycT1 transcription was only modestly enhanced in response to cell activation. These observations prompted a reexamination of CycT1 protein expression in primary lymphocytes. In fact, steady-state CycT1 expression is only slightly lower in unstimulated lymphocytes compared to phorbol ester-treated cells or a panel of immortalized cell lines. Importantly, CycT1 is expressed at sufficient levels in unstimulated primary cells to support robust Tat activity. These results strongly suggest that CycT1 expression levels in unstimulated primary lymphocytes do not profoundly limit HIV-1 gene expression or provide an adequate mechanistic explanation for proviral latency in vivo.

scholarly journals Diminished Rev-Mediated Stimulation of Human Immunodeficiency Virus Type 1 Protein Synthesis Is a Hallmark of Human Astrocytes

1999 ◽  
Vol 73 (10) ◽  
pp. 8279-8289 ◽  
Author(s):  
Eva Ludwig ◽  
Francesca Ceccherini- Silberstein ◽  
Jutta van Empel ◽  
Volker Erfle ◽  
Markus Neumann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Human Astrocytes ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Activation Capacity

ABSTRACT Astrocytes are target cells for human immunodeficiency virus type 1 (HIV-1) in the central nervous system with attenuated virus replication in vivo and in vitro. In infected astrocytes, viral gene expression is restricted mainly to nonstructural (early) viral components like Nef, suggesting inhibition of Rev-dependent posttranscriptional processes in these cells. Because of the heterogeneity of astrocytic cells, the objective of this study was to determine whether restriction of HIV-1 Rev-associated activities is a common property of human astrocytes. To this end, we compared the trans activation capacity and intracellular distribution of Rev in four astrocytoma cell lines previously shown to be infectible by HIV-1 and in primary human fetal astrocytes from different sources with Rev-permissive nonglial control cell lines. In all astrocytic cell cultures, the Rev response was reduced to about 10% of that of Rev-permissive control cells. Rev was apparent both in cytoplasmic and in nuclear compartments of living astrocytes, in contrast to the typical nuclear and/or nucleolar localization of Rev in permissive control cells. Nuclear accumulation of Rev in astrocytes was restored by blocking export of Rev. Thetrans activation capacity and nuclear localization of Tat were not affected in astrocytes. These results demonstrate that inhibition of Rev-dependent posttranscriptional regulation of HIV-1 is a hallmark of human astrocytes and may contribute to suppression of HIV-1 production in these HIV-1 reservoirs. Astrocytes constitute the first example of a human cell type showing an impaired Rev response, indicating that posttranscriptional control of HIV-1 gene expression can be modulated in a cell-dependent manner.

scholarly journals Mouse-Human Heterokaryons Support Efficient Human Immunodeficiency Virus Type 1 Assembly

2001 ◽  
Vol 75 (7) ◽  
pp. 3141-3151 ◽  
Author(s):  
Roberto Mariani ◽  
Beth A. Rasala ◽  
Gabriel Rutter ◽  
Klaus Wiegers ◽  
Stephanie M. Brandt ◽  
...  
Keyword(s):  
Cell Lines ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Fold Increase ◽  
Human Cells ◽  
Virus Release ◽  
Cyclin T1 ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

scholarly journals Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription

1999 ◽  
Vol 73 (3) ◽  
pp. 2499-2508 ◽  
Author(s):  
Catherine Ulich ◽  
Amanda Dunne ◽  
Emma Parry ◽  
C. William Hooker ◽  
Richard B. Gaynor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with atat gene deletion (Δtat). Cell lines that contained Δtat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3 Lys were seen in the HIV-1 Δtat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functionaltat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.

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.

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.

Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level

1991 ◽  
Vol 11 (7) ◽  
pp. 3522-3527
Author(s):  
S Yamagoe ◽  
T Kohda ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Specific Gene ◽  
Adp Ribosylation ◽  
Immunodeficiency Virus ◽  
Posttranscriptional Level ◽  
Hiv 1

Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed.

scholarly journals Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

2004 ◽  
Vol 78 (20) ◽  
pp. 11263-11271 ◽  
Author(s):  
Audrey Brussel ◽  
Pierre Sonigo
Keyword(s):  
Gene Expression ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Gene ◽  
Viral Expression ◽  
Molecular Stability ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication.

scholarly journals A concurrent human herpesvirus-6 infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to human immunodeficiency virus type-1

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4737-4745 ◽  
Author(s):  
G Furlini ◽  
M Vignoli ◽  
E Ramazzotti ◽  
MC Re ◽  
G Visani ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Human Herpesvirus ◽  
Human Herpesvirus 6 ◽  
Hematopoietic Progenitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection.

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