A Genome-wide Short Hairpin RNA Screening of Jurkat T-cells for Human Proteins Contributing to Productive HIV-1 Replication

ABSTRACT We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

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Efficient Gene Transfer of HIV-1-Specific Short Hairpin RNA into Human Lymphocytic Cells Using Recombinant Adeno-associated Virus Vectors

Molecular Therapy ◽

10.1016/j.ymthe.2003.11.025 ◽

2004 ◽

Vol 9 (3) ◽

pp. 396-402 ◽

Cited By ~ 33

Author(s):

Daniel Boden ◽

Oliver Pusch ◽

Fred Lee ◽

Lynne Tucker ◽

Bharat Ramratnam

Keyword(s):

Gene Transfer ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Adeno Associated Virus ◽

Virus Vectors ◽

Efficient Gene ◽

Short Hairpin ◽

Hiv 1

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Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA

Nucleic Acids Research ◽

10.1093/nar/gkg876 ◽

2003 ◽

Vol 31 (22) ◽

pp. 6444-6449 ◽

Cited By ~ 50

Author(s):

O. Pusch

Keyword(s):

Nucleotide Sequence ◽

Sequence Homology ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Nucleotide Sequence Homology ◽

Short Hairpin ◽

Hiv 1

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Inhibition of HIV-1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction

The Journal of Gene Medicine ◽

10.1002/jgm.1440 ◽

2010 ◽

Vol 12 (3) ◽

pp. 255-265 ◽

Cited By ~ 36

Author(s):

Min Liang ◽

Masakazu Kamata ◽

Kevin N. Chen ◽

Nonia Pariente ◽

Dong Sung An ◽

...

Keyword(s):

Chemokine Receptor ◽

Progenitor Cell ◽

Short Hairpin Rna ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Hairpin Rna ◽

Short Hairpin ◽

Hiv 1 ◽

Chemokine Receptor 5

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IRF7 and RNH1 are modifying factors of HIV-1 reservoirs: a genome-wide association analysis

BMC Medicine ◽

10.1186/s12916-021-02156-5 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zhenhua Zhang ◽

Wim Trypsteen ◽

Marc Blaauw ◽

Xiaojing Chu ◽

Sofie Rutsaert ◽

...

Keyword(s):

T Cells ◽

Association Analysis ◽

Antiretroviral Treatment ◽

Genome Wide Association ◽

Viral Reservoirs ◽

Genome Wide Association Analysis ◽

Genome Wide ◽

A Genome ◽

Rna:Dna Ratio ◽

Hiv 1

Abstract Background Combination antiretroviral treatment (cART) cannot eradicate HIV-1 from the body due to the establishment of persisting viral reservoirs which are not affected by therapy and reinitiate new rounds of HIV-1 replication after treatment interruption. These HIV-1 reservoirs mainly comprise long-lived resting memory CD4+ T cells and are established early after infection. There is a high variation in the size of these viral reservoirs among virally suppressed individuals. Identification of host factors that contribute to or can explain this observed variation could open avenues for new HIV-1 treatment strategies. Methods In this study, we conducted a genome-wide quantitative trait locus (QTL) analysis to probe functionally relevant genetic variants linked to levels of cell-associated (CA) HIV-1 DNA, CA HIV-1 RNA, and RNA:DNA ratio in CD4+ T cells isolated from blood from a cohort of 207 (Caucasian) people living with HIV-1 (PLHIV) on long-term suppressive antiretroviral treatment (median = 6.6 years). CA HIV-1 DNA and CA HIV-1 RNA levels were measured with corresponding droplet digital PCR (ddPCR) assays, and genotype information of 522,455 single-nucleotide variants was retrieved via the Infinium Global Screening array platform. Results The analysis resulted in one significant association with CA HIV-1 DNA (rs2613996, P < 5 × 10−8) and two suggestive associations with RNA:DNA ratio (rs7113204 and rs7817589, P < 5 × 10−7). Then, we prioritized PTDSS2, IRF7, RNH1, and DEAF1 as potential HIV-1 reservoir modifiers and validated that higher expressions of IRF7 and RNH1 were accompanied by rs7113204-G. Moreover, RNA:DNA ratio, indicating relative HIV-1 transcription activity, was lower in PLHIV carrying this variant. Conclusions The presented data suggests that the amount of CA HIV-1 DNA and RNA:DNA ratio can be influenced through PTDSS2, RNH1, and IRF7 that were anchored by our genome-wide association analysis. Further, these observations reveal potential host genetic factors affecting the size and transcriptional activity of HIV-1 reservoirs and could indicate new targets for HIV-1 therapeutic strategies.

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A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model

Blood ◽

10.1182/blood-2009-04-215855 ◽

2010 ◽

Vol 115 (8) ◽

pp. 1534-1544 ◽

Cited By ~ 105

Author(s):

Saki Shimizu ◽

Patrick Hong ◽

Balamurugan Arumugam ◽

Lauren Pokomo ◽

Joshua Boyer ◽

...

Keyword(s):

T Cell ◽

Mouse Model ◽

Lentiviral Vector ◽

Short Hairpin Rna ◽

Lymphoid Organs ◽

Great Promise ◽

Ccr5 Expression ◽

Hairpin Rna ◽

Short Hairpin ◽

Hiv 1

AbstractInhibiting the expression of the HIV-1 coreceptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report stable knockdown of human CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/liver/thymus (BLT) mouse model. We delivered a potent shRNA against CCR5 into human fetal liver-derived CD34+ hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced HPSCs solidified with Matrigel and a thymus segment under the mouse kidney capsule. Vector-transduced autologous CD34+ cells were subsequently injected in the irradiated mouse, intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues, including gut-associated lymphoid tissue, the major site of HIV-1 replication. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T-cell development as assessed by polyclonal T-cell receptor Vβ family development and naive/memory T-cell differentiation. CCR5 knockdown in the secondary transplanted mice suggested the potential of long-term hematopoietic reconstitution by the shRNA-transduced HPSCs. CCR5 tropic HIV-1 infection was effectively inhibited in mouse-derived human splenocytes ex vivo. These results demonstrate that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 in systemic lymphoid organs in vivo.

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