Rethinking human cytomegalovirus latency reservoir Schwartz, Michal; Stern‐Ginossar, Noam
Annals of the New York Academy of Sciences,
June 2023, 2023-Jun, 2023-06-00, 20230601, Letnik:
1524, Številka:
1
Journal Article
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Human cytomegalovirus (HCMV) is a prevalent herpesvirus, infecting the majority of the human population. Like other herpesviruses, it causes lifelong infection through the establishment of latency. ...Although reactivation from latency can cause significant morbidity and mortality in immunocompromised hosts, our understanding of HCMV latency and how it is maintained remains limited. Here, we discuss the characterized latency reservoir in hematopoietic cells in the bone marrow and the gaps in our knowledge of mechanisms that facilitate HCMV genome maintenance in dividing cells. We further review clinical evidence that strongly suggests the tissue origin of HCMV reactivation, and we outline similarities to murine cytomegalovirus where latency in tissue‐resident cells has been demonstrated. Overall, we think these observations call for a rethinking of HCMV latency reservoirs and point to potential sources of HCMV latency that reside in tissues.
In this perspective, we discuss the characterized latency reservoir in hematopoietic cells in the bone marrow and the gaps in our knowledge with regard to mechanisms that facilitate HCMV genome maintenance in dividing cells. We further review clinical evidence that strongly suggests the tissue origin of HCMV reactivation, and we outline similarities to murine cytomegalovirus where latency in tissue resident cells was demonstrated.
Definition of functional genomic elements is one of the greater challenges of the genomic era. Traditionally, putative short open reading frames (sORFs) coding for less than 100 amino acids were ...disregarded due to computational and experimental limitations; however, it has become clear over the past several years that translation of sORFs is pervasive and serves diverse functions. The development of ribosome profiling, allowing identification of translated sequences genome wide, revealed wide spread, previously unidentified translation events. New computational methodologies as well as improved mass spectrometry approaches also contributed to the task of annotating translated sORFs in different organisms. Viruses are of special interest due to the selective pressure on their genome size, their rapid and confining evolution, and the potential contribution of novel peptides to the host immune response. Indeed, many functional viral sORFs were characterized to date, and ribosome profiling analyses suggest that this may be the tip of the iceberg. Our computational analyses of sORFs identified by ribosome profiling in DNA viruses demonstrate that they may be enriched in specific features implying that at least some of them are functional. Combination of systematic genome editing strategies with synthetic tagging will take us into the next step—elucidation of the biological relevance and function of this intriguing class of molecules.
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.
Protection from harmful pathogens depends on activation of the immune system, which relies on tight regulation of gene expression. Recently, the RNA modification N
-methyladenosine (m
A) has been ...found to play an essential role in such regulation. Here, we summarize newly discovered functions of m
A in controlling various aspects of immunity, including immune recognition, activation of innate and adaptive immune responses, and cell fate decisions. We then discuss some of the current challenges in the field and describe future directions for uncovering the immunological functions of m
A and its mechanisms of action.
The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the ...virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show 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 shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent 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.
Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome ...profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.
Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N
-methyladenosine (m
A), 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 m
A at single-nucleotide resolution. Within the cytosol, m
A 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 m
A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m
A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m
A 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 m
A levels was adopted as a potential means of post-transcriptional regulation.
Translational Control in Virus-Infected Cells Stern-Ginossar, Noam; Thompson, Sunnie R; Mathews, Michael B ...
Cold Spring Harbor perspectives in biology,
03/2019, Letnik:
11, Številka:
3
Journal Article
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As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded ...functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.
How bacteria switch between tracks
Bacterial riboswitches prevent the formation of full-length messenger RNA, and hence proteins, via transcriptional termination in response to metabolites. However, ...identifying riboswitches within the genome has previously required comparative analysis, which may miss species- and environmentally specific responses. Dar
et al.
developed a method called term-seq to document all riboswitches in a bacterial genome, as well as their metabolite counterparts (see the Perspective by Sommer and Suess). The method revealed a role for pathogenic bacterial riboswitches in antibiotic resistance. Thus, transcription may be one way pathogens fend off antibiotic attack.
Science
, this issue p.
10.1126/science.aad9822
; see also p.
144
A powerful technique is described for discovering riboswitches and attenuators that respond to previously unknown ligands.
Also see Perspective by
Sommer and Suess
INTRODUCTION
Riboswitches and attenuators are cis-regulatory RNA elements (ribo-regulators), which in most cases control bacterial gene expression via ligand-mediated, premature transcription termination. Depending on the presence or absence of the specific ligand, the formation of a transcription terminator upstream of the gene causes transcription to abort prematurely, generating short unproductive transcripts. In response to changes in the metabolite concentrations, the structure of the ribo-regulator is altered, destabilizing the terminator and allowing read-through into the gene, thus resulting in expression of the full-length mRNA. These ribo-regulators play central roles in bacterial physiology and virulence and have been used for synthetic biology applications as well as recognized as therapeutic targets for antibiotics.
RATIONALE
Despite the importance of riboswitches and attenuators, there is currently no experimental high-throughput method for the discovery of such ribo-regulators across bacterial genomes. Furthermore, given a metabolite or ligand of interest, until now there was no efficient experimental approach to identify natural riboswitches or attenuators that sense and respond to it.
RESULTS
We developed term-seq, a method that enables quantitative mapping of all exposed RNA 3′ ends in bacteria and allows unbiased, genome-wide identification of genes that are regulated by premature transcription termination. This method quantitatively measures the in vivo activities of all expressed ribo-regulators in a given genome simultaneously and under physiological conditions, thus enabling high-throughput discovery of ribo-regulators that respond to a metabolite of interest. Application of term-seq to the model bacteria
Bacillus subtilis
,
Listeria monocytogenes
, and
Enterococcus faecalis
detected the vast majority of known riboswitches as well as multiple previously unidentified regulators that function via conditional termination.
We demonstrate the utility of our approach by screening for ribo-regulators that specifically respond to small antibiotic molecules. We found that numerous antibiotics resistance genes, in both pathogenic bacteria and in the human microbiome, are regulated via termination-based ribo-regulators that allow read-through when the antibiotic is present in the cell.
Focusing on
lmo0919
, one of the antibiotic-regulated genes we detected in
Listeria monocytogenes
, revealed that this locus confers specific resistance to the translation-inhibiting antibiotic lincomycin. In the absence of the antibiotic, transcription is terminated prematurely by the ribo-regulator. However, upon exposure to lincomycin, drug-inhibited ribosomes stall over a conserved three-amino-acid upstream open reading frame found within the ribo-regulator, thus triggering a conformational change in the transcriptional terminator and inducing the expression of the full-length mRNA that encodes the resistance gene.
CONCLUSION
These results describe a high-throughput method for ribo-regulator discovery in either bacterial monocultures or complex bacterial communities such as the human microbiome. Furthermore, they reveal a broad role for conditional termination in regulating multiple classes of antibiotic resistance genes in the human microbiome and provide a general tool for discovering riboswitches and attenuators that respond to specific metabolites of choice.
Genome-wide discovery of antibiotic responsive ribo-regulation in bacteria.
(
A
) Direct mapping of RNA 3′ ends reveals gene regulation by conditional termination in a genome-wide manner. Differential sequencing of monoculture or complex bacterial communities under metabolite-rich and -poor conditions (metabolite in this case being an antibiotic) detects ribo-regulators that specifically respond to the metabolite. (
B
) Mechanism of regulation of the
Listeria monocytogenes
lmo0919
antibiotic responsive ribo-regulator.
Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in vivo activity of all such regulators in parallel and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach, we detected numerous antibiotic-responsive ribo-regulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in
Listeria monocytogenes
revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance and provide a tool for discovering riboswitches and attenuators that respond to previously unknown ligands.
NKG2D is one of the best characterized activating receptors and is expressed on natural killer cells and on various T-cell subsets. This receptor recognizes several different ligands that are induced ...by cellular stresses. In this review, we described the mechanisms controlling the expression of NKG2D ligands, with the emphasis on post-transcriptional and post-translational regulation.
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