Publications by authors named "Aharon Nachshon"

21 Publications

  • Page 1 of 1

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

Nature 2021 06 12;594(7862):240-245. Epub 2021 May 12.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.
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http://dx.doi.org/10.1038/s41586-021-03610-3DOI Listing
June 2021

Bromodomain proteins regulate human cytomegalovirus latency and reactivation allowing epigenetic therapeutic intervention.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Cambridge Institute of Therapeutic Immunology and Infectious Disease, Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, CB2 0QQ, United Kingdom;

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.
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http://dx.doi.org/10.1073/pnas.2023025118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936348PMC
March 2021

Profiling the Blood Compartment of Hematopoietic Stem Cell Transplant Patients During Human Cytomegalovirus Reactivation.

Front Cell Infect Microbiol 2020 8;10:607470. Epub 2021 Jan 8.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.
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http://dx.doi.org/10.3389/fcimb.2020.607470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820775PMC
June 2021

SARS-CoV-2 infected cells present HLA-I peptides from canonical and out-of-frame ORFs.

bioRxiv 2020 Oct 2. Epub 2020 Oct 2.

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.
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http://dx.doi.org/10.1101/2020.10.02.324145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536868PMC
October 2020

Context-dependent functional compensation between Ythdf mA reader proteins.

Genes Dev 2020 10 17;34(19-20):1373-1391. Epub 2020 Sep 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.

The N6-methyladenosine (mA) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous mA-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.
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http://dx.doi.org/10.1101/gad.340695.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528697PMC
October 2020

The coding capacity of SARS-CoV-2.

Nature 2021 01 9;589(7840):125-130. Epub 2020 Sep 9.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.
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http://dx.doi.org/10.1038/s41586-020-2739-1DOI Listing
January 2021

Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication.

PLoS Pathog 2020 04 15;16(4):e1008390. Epub 2020 Apr 15.

Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel.

Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5' end, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.
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http://dx.doi.org/10.1371/journal.ppat.1008390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185721PMC
April 2020

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity.

Cell Rep 2020 02;30(7):2248-2260.e5

Institute for Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany. Electronic address:

Human cytomegalovirus (HCMV) causes diseases in individuals with immature or compromised immunity. To evade immune control, HCMV evolved numerous antagonists targeting the interferon system at multiple levels. By comparative analysis of naturally arising variants of the most widely studied HCMV strain, AD169, and a panel of targeted mutants, we uncover the UL145 gene as indispensable for STAT2 downregulation. Ribosome profiling confirms the translation of the canonical pUL145 protein (pUL145-Long) and newly identifies a shorter isoform (pUL145-Short). Both isoforms recruit DDB1-containing ubiquitin ligases to induce proteasomal degradation of STAT2. An alanine-scanning mutagenesis discloses the DDB1 interaction motif of pUL145 that resembles the DDB1-binding interface of cellular substrate receptors of DDB1-containing ubiquitin ligases. Thus, pUL145 constitutes a viral DDB1-cullin-associated factor (vDCAF), which mimics cellular DCAFs to exploit the ubiquitin-proteasome system to impede antiviral immunity. Notably, the viral exploitation of the cullins can be targeted to restore the efficacy of the host immune response.
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http://dx.doi.org/10.1016/j.celrep.2020.01.070DOI Listing
February 2020

Single cell analysis reveals human cytomegalovirus drives latently infected cells towards an anergic-like monocyte state.

Elife 2020 01 22;9. Epub 2020 Jan 22.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate.
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http://dx.doi.org/10.7554/eLife.52168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039680PMC
January 2020

Comprehensive annotations of human herpesvirus 6A and 6B genomes reveal novel and conserved genomic features.

Elife 2020 01 16;9. Epub 2020 Jan 16.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.
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http://dx.doi.org/10.7554/eLife.50960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964970PMC
January 2020

Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.

Nat Commun 2020 01 9;11(1):164. Epub 2020 Jan 9.

Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA, 02142, USA.

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.
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http://dx.doi.org/10.1038/s41467-019-13965-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952391PMC
January 2020

An iPSC-Derived Myeloid Lineage Model of Herpes Virus Latency and Reactivation.

Front Microbiol 2019 9;10:2233. Epub 2019 Oct 9.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Herpesviruses undergo life-long latent infection which can be life-threatening in the immunocompromised. Models of latency and reactivation of human cytomegalovirus (HCMV) include primary myeloid cells, cells known to be important for HCMV latent carriage and reactivation . However, primary cells are limited in availability, and difficult to culture and to genetically modify; all of which have hampered our ability to fully understand virus/host interactions of this persistent human pathogen. We have now used iPSCs to develop a model cell system to study HCMV latency and reactivation in different cell types after their differentiation down the myeloid lineage. Our results show that iPSCs can effectively mimic HCMV latency/reactivation in primary myeloid cells, allowing molecular interrogations of the viral latent/lytic switch. This model may also be suitable for analysis of other viruses, such as HIV and Zika, which also infect cells of the myeloid lineage.
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http://dx.doi.org/10.3389/fmicb.2019.02233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795026PMC
October 2019

Rho-Associated Coiled-Coil Kinase 1 Translocates to the Nucleus and Inhibits Human Cytomegalovirus Propagation.

J Virol 2019 10 12;93(19). Epub 2019 Sep 12.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus. ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCK's antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus.
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http://dx.doi.org/10.1128/JVI.00453-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744247PMC
October 2019

Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02;20(2):243

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.
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http://dx.doi.org/10.1038/s41590-019-0314-4DOI Listing
February 2019

mA modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02 17;20(2):173-182. Epub 2018 Dec 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

N-methyladenosine (mA) is the most common mRNA modification. Recent studies have revealed that depletion of mA machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the mA 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was mA modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that mA-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role mA serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
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http://dx.doi.org/10.1038/s41590-018-0275-zDOI Listing
February 2019

Virus-Induced Changes in mRNA Secondary Structure Uncover cis-Regulatory Elements that Directly Control Gene Expression.

Mol Cell 2018 12 11;72(5):862-874.e5. Epub 2018 Oct 11.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

mRNAs carry two layers of information, the genetic code and the information that dictates their post-transcriptional fate. The latter function relies on a complex interplay between cis-elements and trans-regulators, and unbiased identification of these elements is still challenging. To identify cis-elements that control gene expression, we use dimethyl sulfate (DMS) mutational profiling with sequencing and map changes in mRNA secondary structure following viral infection. Our dynamic structural data reveal a major role for ribosomes in unwinding secondary structures, which is further supported by the relationship we uncover between structure and translation efficiency. Moreover, our analysis revealed dozens of regions in viral and cellular mRNAs that exhibit changes in secondary structure. In-depth analysis of these regions reveals cis-elements in 3' UTRs that regulate mRNA stability and elements within coding sequences that control translation. Overall, our study demonstrates how mapping dynamic changes in mRNA structure allows unbiased identification of functional regulatory elements.
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http://dx.doi.org/10.1016/j.molcel.2018.09.003DOI Listing
December 2018

Defining the Transcriptional Landscape during Cytomegalovirus Latency with Single-Cell RNA Sequencing.

mBio 2018 03 13;9(2). Epub 2018 Mar 13.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression. Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression.
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http://dx.doi.org/10.1128/mBio.00013-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850328PMC
March 2018

The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution.

Nature 2017 11 25;551(7679):251-255. Epub 2017 Oct 25.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N-methyladenosine (mA), which disrupts Watson-Crick base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of mA at single-nucleotide resolution. Within the cytosol, mA is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single mA site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. mA leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that mA on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over mA levels was adopted as a potential means of post-transcriptional regulation.
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http://dx.doi.org/10.1038/nature24456DOI Listing
November 2017

Dissecting the Effect of Genetic Variation on the Hepatic Expression of Drug Disposition Genes across the Collaborative Cross Mouse Strains.

Front Genet 2016 5;7:172. Epub 2016 Oct 5.

Department of Cell Research and Immunology, Faculty of Life Sciences, Tel-Aviv University Tel-Aviv, Israel.

A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC) mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs). By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences.
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http://dx.doi.org/10.3389/fgene.2016.00172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050206PMC
October 2016

POEM: Identifying Joint Additive Effects on Regulatory Circuits.

Front Genet 2016 19;7:48. Epub 2016 Apr 19.

Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University Tel Aviv, Israel.

Motivation: Expression Quantitative Trait Locus (eQTL) mapping tackles the problem of identifying variation in DNA sequence that have an effect on the transcriptional regulatory network. Major computational efforts are aimed at characterizing the joint effects of several eQTLs acting in concert to govern the expression of the same genes. Yet, progress toward a comprehensive prediction of such joint effects is limited. For example, existing eQTL methods commonly discover interacting loci affecting the expression levels of a module of co-regulated genes. Such "modularization" approaches, however, are focused on epistatic relations and thus have limited utility for the case of additive (non-epistatic) effects.

Results: Here we present POEM (Pairwise effect On Expression Modules), a methodology for identifying pairwise eQTL effects on gene modules. POEM is specifically designed to achieve high performance in the case of additive joint effects. We applied POEM to transcription profiles measured in bone marrow-derived dendritic cells across a population of genotyped mice. Our study reveals widespread additive, trans-acting pairwise effects on gene modules, characterizes their organizational principles, and highlights high-order interconnections between modules within the immune signaling network. These analyses elucidate the central role of additive pairwise effect in regulatory circuits, and provide computational tools for future investigations into the interplay between eQTLs.

Availability: The software described in this article is available at csgi.tau.ac.il/POEM/.
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http://dx.doi.org/10.3389/fgene.2016.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4835676PMC
May 2016

Linking traits based on their shared molecular mechanisms.

Elife 2015 Mar 17;4. Epub 2015 Mar 17.

Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

There is growing recognition that co-morbidity and co-occurrence of disease traits are often determined by shared genetic and molecular mechanisms. In most cases, however, the specific mechanisms that lead to such trait-trait relationships are yet unknown. Here we present an analysis of a broad spectrum of behavioral and physiological traits together with gene-expression measurements across genetically diverse mouse strains. We develop an unbiased methodology that constructs potentially overlapping groups of traits and resolves their underlying combination of genetic loci and molecular mechanisms. For example, our method predicts that genetic variation in the Klf7 gene may influence gene transcripts in bone marrow-derived myeloid cells, which in turn affect 17 behavioral traits following morphine injection; this predicted effect of Klf7 is consistent with an in vitro perturbation of Klf7 in bone marrow cells. Our analysis demonstrates the utility of studying hidden causative mechanisms that lead to relationships between complex traits.
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http://dx.doi.org/10.7554/eLife.04346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362207PMC
March 2015