A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

AbstractGenome-wide CRISPR/Cas9 knock-out genetic screens are powerful approaches to unravel new regulators of viral infections. Here, we took advantage of the ability of interferon (IFN) to restrict HIV-1 infection, in order to create an environment hostile to replication and reveal new inhibitors through a CRISPR screen. This approach led to the identification of the RNA helicase DDX42 as an intrinsic inhibitor of HIV-1. Depletion of endogenous DDX42 increased HIV-1 DNA accumulation and infection in cell lines and primary cells, irrespectively of IFN treatment. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant of DDX42 increased infection. Importantly, DDX42 impacted retrotransposition of long interspersed elements-1 (LINE-1), infection with other retroviruses and positive-strand RNA viruses, including Chikungunya virus (CHIKV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, DDX42 did not inhibit infection with negative-strand RNA viruses such as influenza A virus (IAV), arguing against a general, unspecific effect on target cells. Proximity ligation assays showed DDX42 in the vicinity of viral elements during infection, and RNA immunoprecipitation confirmed DDX42 interaction with LINE-1 RNAs. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results identify DDX42 as a new, broadly active intrinsic antiviral inhibitor.

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BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.063 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi17-vi18

Author(s):

Crismita Dmello ◽

Aarón Sonabend ◽

Víctor Arrieta ◽

Daniel Zhang ◽

Deepak Kanojia ◽

...

Keyword(s):

Breast Cancer ◽

Endoplasmic Reticulum ◽

Glioma Cells ◽

Predictive Biomarkers ◽

Predictive Biomarker ◽

Precision Oncology ◽

Patient Response ◽

Knock Out ◽

Genome Wide ◽

A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

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What do we learn from a Genome Wide Association Study performed on HIV-1 infected Long Term Non Progressors individuals?

Retrovirology ◽

10.1186/1742-4690-6-s2-o27 ◽

2009 ◽

Vol 6 (S2) ◽

Author(s):

Julien Guergnon ◽

◽

Cyril Dalmasso ◽

Ioannis Theodorou ◽

Agostino Riva

Keyword(s):

Association Study ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Genome Wide ◽

A Genome ◽

Hiv 1

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Genome-Wide Analysis of Chromosomal Features Repressing Human Immunodeficiency Virus Transcription

Journal of Virology ◽

10.1128/jvi.79.11.6610-6619.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 6610-6619 ◽

Cited By ~ 193

Author(s):

M. K. Lewinski ◽

D. Bisgrove ◽

P. Shinn ◽

H. Chen ◽

C. Hoffmann ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Fluorescent Protein ◽

Centromeric Heterochromatin ◽

Regulatory Sequences ◽

Hiv Transcription ◽

Genome Wide ◽

A Genome ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

In Cells

ABSTRACT We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

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Double-Stranded RNA Is Produced by Positive-Strand RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Strand RNA Viruses

Journal of Virology ◽

10.1128/jvi.80.10.5059-5064.2006 ◽

2006 ◽

Vol 80 (10) ◽

pp. 5059-5064 ◽

Cited By ~ 578

Author(s):

Friedemann Weber ◽

Valentina Wagner ◽

Simon B. Rasmussen ◽

Rune Hartmann ◽

Søren R. Paludan

Keyword(s):

Viral Infections ◽

Rna Viruses ◽

Innate Immune ◽

Genome Replication ◽

Dna Viruses ◽

Double Stranded Rna ◽

Positive Strand ◽

Negative Strand ◽

A Genome ◽

Positive Strand Rna

ABSTRACT Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

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Correction: Novel Genetic Locus Implicated for HIV-1 Acquisition with Putative Regulatory Links to HIV Replication and Infectivity: A Genome-Wide Association Study

PLoS ONE ◽

10.1371/journal.pone.0129671 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0129671

Author(s):

Eric O. Johnson ◽

Dana B. Hancock ◽

Nathan C. Gaddis ◽

Joshua L. Levy ◽

Grier Page ◽

...

Keyword(s):

Association Study ◽

Genome Wide Association Study ◽

Genetic Locus ◽

Genome Wide Association ◽

Hiv Replication ◽

Genome Wide ◽

A Genome ◽

Hiv 1

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P08-06 LB. A genome-wide association study of host genetic determinants of T cell responses to the MRKAd5 HIV-1 gag/pol/nef vaccine in the STEP trial

Retrovirology ◽

10.1186/1742-4690-6-s3-p398 ◽

2009 ◽

Vol 6 (S3) ◽

Cited By ~ 3

Author(s):

J Fellay ◽

KV Shianna ◽

ET Cirulli ◽

J McElrath ◽

DR Casimiro ◽

...

Keyword(s):

T Cell ◽

Association Study ◽

Genome Wide Association Study ◽

Genome Wide Association ◽

Genetic Determinants ◽

Cell Responses ◽

Genome Wide ◽

A Genome ◽

Host Genetic ◽

Hiv 1

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Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Journal of Virology ◽

10.1128/jvi.01606-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 8

Author(s):

Teng-Feng Li ◽

Myra Hosmillo ◽

Hella Schwanke ◽

Ting Shu ◽

Zhaowei Wang ◽

...

Keyword(s):

Rna Synthesis ◽

Rna Viruses ◽

Rna Helicase ◽

Diverse Group ◽

Rna Helicases ◽

Human Norovirus ◽

Content Type ◽

Rna Remodeling ◽

Positive Strand Rna

ABSTRACTRNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, theirin vitroactivities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the familyCaliciviridaeand constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesisin vitroby norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded byCaliciviridaeand highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCENoroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitatein vitroviral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

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Site-Specific Evolutionary Rate Shifts in HIV-1 and SIV

Viruses ◽

10.3390/v12111312 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1312

Author(s):

Maoz Gelbart ◽

Adi Stern

Keyword(s):

Evolutionary Rate ◽

Species Barrier ◽

Epistatic Interactions ◽

Site Specific ◽

Genome Wide ◽

Rate Acceleration ◽

A Genome ◽

Rate Deceleration ◽

A Site ◽

Hiv 1

Site-specific evolutionary rate shifts are defined as protein sites, where the rate of substitution has changed dramatically across the phylogeny. With respect to a given clade, sites may either undergo a rate acceleration or a rate deceleration, reflecting a site that was conserved and became variable, or vice-versa, respectively. Sites displaying such a dramatic evolutionary change may point to a loss or gain of function at the protein site, reflecting adaptation, or they may indicate epistatic interactions among sites. Here, we analyzed full genomes of HIV and SIV-1 and identified 271 rate-shifting sites along the HIV-1/SIV phylogeny. The majority of rate shifts occurred at long branches, often corresponding to cross-species transmission branches. We noted that in most proteins, the number of rate accelerations and decelerations was equal, and we suggest that this reflects epistatic interactions among sites. However, several accessory proteins were enriched for either accelerations or decelerations, and we suggest that this may be a signature of adaptation to new hosts. Interestingly, the non-pandemic HIV-1 group O clade exhibited a substantially higher number of rate-shift events than the pandemic group M clade. We propose that this may be a reflection of the height of the species barrier between gorillas and humans versus chimpanzees and humans. Our results provide a genome-wide view of the constraints operating on proteins of HIV-1 and SIV.

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