Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Brass, Abraham L.; Dykxhoorn, Derek M.; Benita, Yair; Yan, Nan; Engelman, Alan; Xavier, Ramnik J.; Lieberman, Judy; Elledge, Stephen J.

doi:10.1126/science.1152725

Cited by 1,315 publications

(1,446 citation statements)

References 35 publications

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“…[26][27][28][29][30][31][32][33][34][35] Multiple screens for HIV-1 inhibitors relied on in vitro assays, which used viral proteins or their fragments, or on HIV-1 infections/replication as a readout. In vitro screening has identified competitive inhibitors of assembly of the gp41 HR1-and HR2-derived peptides into the 6HB.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Marin

Giroud

et al. 2015

ASSAY and Drug Development Technologies

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HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors. Here, we adapted and optimized an enzymatic HIV-cell fusion assay, which reports the transfer of virus-encapsulated b-lactamase into the cytoplasm, to high-throughput screening (HTS) with a 384-well format. The assay was robustly performed in HTS format and was validated by the pilot screen of a small library of pharmacologically active compounds. Several hits identified by screening included a prominent cluster of purinergic receptor antagonists. Functional studies demonstrated that P2X1 receptor antagonists selectively inhibited HIV-1 fusion without affecting the fusion activity of an unrelated virus that enters cells through an endocytic route. The inhibition of HIV-cell fusion by P2X1 antagonists was not through downmodulation of the cell surface expression of CD4 or coreceptors, thus implicating P2X1 receptor in the HIV-1 fusion step. The ability of these antagonists to inhibit viruses regardless of their coreceptor (CXCR4 or CCR5) preference indicates that fusion is blocked at a late step downstream of coreceptor binding. A future large-scale screening campaign for HIV-1 fusion inhibitors, using the above functional readout, will likely reveal novel classes of inhibitors and suggest potential targets for antiviral therapy.

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Section: Introductionmentioning

confidence: 99%

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Marin

Giroud

et al. 2015

ASSAY and Drug Development Technologies

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“…This is also incorporated into our database, allowing us to see which HIV proteins are known to be associated with any of our screening hits, together with the nature of the interaction and the evidence for it. Similarly, the collections of host factors described in previous published HIV screens [4][5][6] have been mined from the papers and added to the database, allowing direct comparison of the degree of overlap and gene composition of the various studies with each other and with our screens.…”

Section: Interaction Networkmentioning

confidence: 99%

“…Since the rate of mutations of cellular genes is substantially lower than for viral genomes, the particular benefit of targeting host factors is that it may provide a higher barrier to the generation of anti-drug resistance. A most powerful and versatile approach to identify such potential cellular interaction partners of HIV-1 are RNAi-based loss-of-function screens, as suggested by very recent reports [4][5][6].…”

mentioning

confidence: 99%

From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV‐1 replication

Börner

Hermle

Sommer

et al. 2010

Biotechnology Journal

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RNA interference (RNAi) has emerged as a powerful technique for studying loss‐of‐function phenotypes by specific down‐regulation of gene expression, allowing the investigation of virus‐host interactions by large‐scale high‐throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single‐cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two‐stage procedure in which potential host factors are first identified in a primary screen and afterwards re‐tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus‐1 (HIV‐1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus‐host interactions. The ViroQuant‐CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.

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“…The first screen to be published was performed in cultured human HeLa cells expressing CD4 as the essential receptor for HIV-1 entry [58]. The goal was to identify host proteins involved in early events of infection such as virus entry, uncoating, reverse transcription, integration and viral gene expression as well as factors needed for late stages in infection such as viral assembly and release.…”

Section: Rnai Screens To Identify Host Factors That Influence Viral Imentioning

confidence: 99%

“…The goal was to identify host proteins involved in early events of infection such as virus entry, uncoating, reverse transcription, integration and viral gene expression as well as factors needed for late stages in infection such as viral assembly and release. Considering more than 21 000 genes, Brass et al [58] identified 273 host factors required for HIV-1 replication. Preliminary characterization of three of the genes revealed previously unknown functions: Rab6, which is involved in the retrograde transport from endosomes to the Golgi and from the Golgi to the ER, was shown to be important for virus entry.…”

Section: Rnai Screens To Identify Host Factors That Influence Viral Imentioning

confidence: 99%

Illuminating the host – How RNAi screens shed light on host‐pathogen interactions

Prudêncio

Lehmann

2009

Biotechnology Journal

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Over millions of years pathogens have coevolved with their respective hosts utilizing host cell functions for survival and replication. Despite remarkable progress in developing antibiotics and vaccination strategies in the last century, infectious diseases still remain a severe threat to human health. Meanwhile, genomic research offers a new era of data‐generating platforms that will dramatically enhance our knowledge of pathogens and the diseases they cause. Improvements in gene knockdown studies by RNA interference (RNAi) combined with recent developments in instrumentation and image analysis enable the use of high‐throughput screening approaches to elucidate host gene functions exploited by pathogens. Although only a few RNAi‐based screens focusing on host genes have been reported so far, these studies have already uncovered hundreds of genes not previously known to be involved in pathogen infection. This review describes recent progress in RNAi screening approaches, highlighting both the limitations and the tremendous potential of RNAi‐based screens for the identification of essential host cell factors during infection.

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Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Cited by 1,315 publications

References 35 publications

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV‐1 replication

Illuminating the host – How RNAi screens shed light on host‐pathogen interactions

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