Biology subjects

Stern-Ginossar, N.

Publications and source records attributed to Stern-Ginossar, N..

11 recordsLinked to original sources

bioRxiv2024

Virus Entry is a Major Determinant of HCMV Latency in Monocytes

Human cytomegalovirus (HCMV) infection can result in either productive or latent infection, the latter enabling life-long viral persistence. Monocytes support latent infection but become permissive to productive infection upon differentiation into macrophages. The molecular basis for these differentiation-driven differences has been largely attributed to chromatin-mediated repression of the viral genome. Using metabolic labeling of newly synthesized RNA during the early stages of infection, we observed markedly lower viral transcription in monocytes compared to macrophages. Unbiased comparison of the two cell types revealed that this difference is partly due to reduced viral entry in monocytes: fewer viruses enter, and correspondingly fewer genomes reach the nucleus. Indeed, ectopic expression of known HCMV entry receptors in monocytes enhanced viral entry and consequently facilitated productive infection, demonstrating that these cells can support full replication if entry is efficient. We further identified integrin {beta}3 as a surface protein upregulated upon differentiation that plays an important role in HCMV entry into macrophages, partially accounting for the observed differences in entry efficiency. Finally, we show that cells receiving fewer viral genomes are the ones that establish latent infection and have the capacity to reactivate. Overall, our findings reveal that entry is a previously unrecognized factor contributing to latent infection in monocytes, adding a critical layer to the paradigm of HCMV latency.

microbiology↗

bioRxiv2024

Essentiality and dynamic expression of the human tRNA pool during viral infection

Human viruses depend on the translation resources of the host cell. A significant translation resource is the tRNA pool of the cell, as human viruses do not encode tRNA genes. Through tRNA sequencing, we inspected the human tRNA pool upon infection of human Cytomegalovirus (HCMV) and SARS-CoV-2. HCMV-induced alterations in tRNA expression were predominantly virus-driven, with minimal influence from the cellular immune response. Notably, specific tRNA post-transcriptional modifications appeared to modulate stability and were susceptible to HCMV manipulation. In contrast, SARS-CoV-2 infection did not significantly impact tRNA expression or modifications. We compared the codon usage of viral genes to the proliferation-differentiation codon-usage signatures of human genes. We found a marked difference between the viruses, with HCMV genes aligning with differentiation codon usage and SARS-CoV-2 genes reflecting proliferation codon usage. We further found that codon usage of structural and gene expression-related viral genes displayed high adaptation to host cell tRNA pools. Through a systematic CRISPR screen targeting human tRNA genes and modification enzymes, we identified specific tRNAs and enzymes that improve or reduce HCMV infectivity and cellular growth. These findings highlight the dynamic interplay between the tRNA pool and viral infection dynamics, shedding light on mechanisms governing host-virus interactions.

genomics↗

bioRxiv2023

A virally encoded high resolution screen of cytomegalovirus host dependencies

Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins role along the infection cycle, providing a roadmap for in-depth dissection of host-herpesvirus interactions.

microbiology↗

bioRxiv2022

The molecular principles governing HCMV infection outcome

Infection with Human cytomegalovirus (HCMV) can result in either productive or non-productive infection, the latter potentially leading to establishment of latency, but the molecular factors that dictate these different infection outcomes are elusive. Macrophages are known targets of HCMV and considered to be permissive for productive infection, while monocytes, their precursors, are latently infected. Here we reveal that infection of macrophages is more complex than previously appreciated and can result in either productive or non-productive infection. By analyzing the progression of HCMV infection in monocytes and macrophages using single cell transcriptomics, we uncover that the level of viral gene expression, and specifically the expression of the major immediate early proteins, IE1 and IE2, is the principal barrier for establishing productive infection. On the cellular side, we reveal that the cell intrinsic levels of interferon stimulated genes (ISG), but not their induction, is a main determinant of infection outcome and that intrinsic ISG levels are downregulated with monocyte differentiation, partially explaining why macrophages are more susceptible to productive HCMV infection. We further show that, compared to monocytes, non-productive macrophages maintain higher levels of viral transcripts and are able to reactivate, raising the possibility that they may serve as latency reservoirs. Overall, by harnessing the tractable system of monocyte differentiation we decipher underlying principles that control HCMV infection outcome, and propose macrophages as a potential HCMV reservoir in tissues.

microbiology↗

bioRxiv2021

Temporal Dynamics of HCMV Gene Expression in Lytic and Latent Infections

Primary infection with Human cytomegalovirus (HCMV) results in a persistent lifelong infection due to its ability to establish latent infection. During productive HCMV infection, viral genes are expressed in a coordinated cascade that is characteristic of all herpesviruses and traditionally relies on the dependencies of viral genes on protein synthesis and viral DNA replication. In contrast, the transcriptional landscape associated with HCMV latency is still disputed and poorly understood. Here, we examine viral transcriptomic dynamics during the establishment of both productive and latent HCMV infections. Our temporal measurements reveal that viral gene expression dynamics along productive infection and their dependencies on protein synthesis and viral DNA replication, do not fully align. This illustrates that the regulation of herpesvirus genes does not represent a simple sequential transcriptional cascade, and surprisingly, many viral genes are regulated by multiple independent modules. Using our improved classification of viral gene expression kinetics in conjunction with transcriptome-wide measurements of the effects of a wide array of chromatin modifiers, we unbiasedly show that a defining characteristic of latent cells is the unique repression of immediate early (IE) genes. Altogether, our findings provide an elaborate definition of HCMV gene expression patterns and reveal novel principles that govern viral gene expression in lytic and latent infection states.

microbiology↗

bioRxiv2021

CRISPR screens for host factors critical for infection by SARS-CoV-2 variants of concern identify GATA6 as a central modulator of ACE2.

The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

microbiology↗

bioRxiv2020

SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 (COVID-19) pandemic. Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis and its ability to antagonize innate immune responses. Here, we use RNA-sequencing and ribosome profiling along SARS-CoV-2 infection and comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show SARS-CoV-2 infection leads to a global reduction in translation but that viral transcripts are not preferentially translated. Instead, we reveal that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we show that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired, implying infection prevents newly transcribed cellular mRNAs from accessing the ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses.

microbiology↗

bioRxiv2020

Context-dependent functional compensation between Ythdf m6A readers

The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay, translation and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is dynamically regulated by a set of writer, eraser and reader proteins. The YTH-domain family of proteins: Ythdf1, Ythdf2, and Ythdf3, are three homologous m6A binding proteins, which 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 for each of the Ythdf readers and for the three readers together (triple-KO). We then estimated the effect in-vivo, in mouse gametogenesis and viability, and in-vitro, in mouse embryonic stem cells (mESCs). We show that in gametogenesis, Mettl3-KO 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, possibly due to differences in readers expression, both in quantity and in spatial location. By knocking out the three readers together and systematically testing offspring genotypes, we have revealed a redundancy in the readers role during early development, a redundancy which is dosage-dependent. Additionally, we show that in mESCs there is compensation between the three readers, since the inability 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 novel model for the Ythdf readers function. There is a dosage-dependent redundancy when all three readers are co-expressed in the same location in the cells.

developmental biology↗

bioRxiv2020

The coding capacity of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic 1,2. In order to understand SARS-CoV-2 pathogenicity and antigenic potential, and to develop diagnostic and therapeutic tools, it is essential to portray the full repertoire of its expressed proteins. The SARS-CoV-2 coding capacity map is currently based on computational predictions and relies on homology to other coronaviruses. Since coronaviruses differ in their protein array, especially in the variety of accessory proteins, it is crucial to characterize the specific collection of SARS-CoV-2 proteins in an unbiased and open-ended manner. Utilizing a suite of ribosome profiling techniques 3-8, we present a high-resolution map of the SARS-CoV-2 coding regions, allowing us to accurately quantify the expression of canonical viral open reading frames (ORF)s and to identify 23 novel unannotated viral translated ORFs. These ORFs include upstream ORFs (uORFs) that are likely playing a regulatory role, several in-frame internal ORFs lying 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; rather, virus translation dominates host translation due to high levels of viral transcripts. Overall, our work reveals the full coding capacity of SARS-CoV-2 genome, providing a rich resource, which will form the basis of future functional studies and diagnostic efforts.

microbiology↗

bioRxiv2019

Comprehensive Annotations of Human Herpesvirus 6A and 6B Genomes Reveal Novel and Conserved Genomic Features

Human herpesvirus 6 (HHV-6) A and B are highly ubiquitous betaherpesviruses, infecting the majority of the human population. Like other herpesviruses, 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 the HHV-6 translation products and to follow their temporal expression. We identify hundreds of new open reading frames (ORFs), including many upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. Furthermore, by integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs that are conserved across betaherpesviruses and we show that uORFs are specifically enriched in late viral genes. Using our transcriptome measurements, we identified three highly abundant HHV-6 encoded long non-coding RNAs (lncRNAs), one of which generates a non-polyadenylated stable intron that appears to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features that are conserved between betaherpesviruses, providing a rich resource for future functional studies.

microbiology↗

bioRxiv2017

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

Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, 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 RNA-seq atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence in-vivo is prevalent in diverse tissues. Unexpectedly, we find only viral transcripts that resemble gene expression during 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 current views on latency, we also find no evidence for a specific 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.

microbiology↗