Transient gene expression optimization and expression vector comparison to improve HIV-1 VLP production in HEK293 cell lines

2017 ◽  
Vol 102 (1) ◽  
pp. 165-174 ◽  
Author(s):  
Javier Fuenmayor ◽  
Laura Cervera ◽  
Sonia Gutiérrez-Granados ◽  
Francesc Gòdia
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Expression Vector ◽  
Hek293 Cell ◽  
Transient Gene Expression ◽  
Expression Optimization ◽  
Hiv 1

Production and characterization of HIV-1 virus-like particles using transient gene expression in mammalian cells

2014 ◽  
Vol 31 ◽  
pp. S42
Author(s):  
Sonia Gutiérrez-Granados ◽  
Laura Cervera ◽  
Segura Maria de las Mercedes ◽  
Francesc Gòdia
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Transient Gene Expression ◽  
Virus Like Particles ◽  
Expression In Mammalian Cells ◽  
Hiv 1

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.

Serial analysis of gene expression in HIV-1-infected T cell lines

FEBS Letters ◽  
1999 ◽  
Vol 462 (1-2) ◽  
pp. 182-186 ◽  
Author(s):  
Akihide Ryo ◽  
Youichi Suzuki ◽  
Kouji Ichiyama ◽  
Toru Wakatsuki ◽  
Nobuo Kondoh ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Lines ◽  
Hiv 1

scholarly journals Extensive proteomic and transcriptomic changes quench the TCR/CD3 activation signal of latently HIV-1 infected T cells

2021 ◽  
Vol 17 (1) ◽  
pp. e1008748
Author(s):  
Eric Carlin ◽  
Braxton Greer ◽  
Kelsey Lowman ◽  
Alexandra Duverger ◽  
Frederic Wagner ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Network Analysis ◽  
Cell Lines ◽  
Data Set ◽  
Latently Infected ◽  
Latent Hiv ◽  
T Cell Lines ◽  
Hiv 1

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.

Creation of CD63-deficient HEK293 cell lines using a polycistronic CAS9/EGFP/HSVtk/PuroR expression vector

Matters ◽  
2016 ◽  
Author(s):  
Francis Fordjour ◽  
Shawn Owiredu ◽  
Hayley Muendlein ◽  
Jerry Plange ◽  
James Morrell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Expression Vector ◽  
Hek293 Cell

scholarly journals Development of HIV-1 dependent gene expression vector with the Cre/IoxP (ON) system

2002 ◽  
Vol 2 (1) ◽  
pp. 295-296
Author(s):  
T. Nagawa ◽  
Y. Habu ◽  
N. Matsumoto ◽  
H. Takeuchi ◽  
N. Kurosaki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Hiv 1

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.

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