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

Shnayder, Miri; Nachshon, Aharon; Krishna, Benjamin A.; Poole, Emma; Boshkov, Alina; Binyamin, Amit; Maza, Itay; Schwartz, Michal; Stern-Ginossar, Noam

doi:10.1101/208603

Cited by 16 publications

(20 citation statements)

References 66 publications

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“…1) and the noisy expression of transcripts from the iP2 element (Figs. 2C and 4B) is consistent with recent work in the field suggesting that viral transcription is not as silent during latency as previously presumed (37,38). The initial burst of IE gene expression that we observed during the establishment of latency is likewise consistent with reports from other groups (39,40).…”

Section: Discussionsupporting

confidence: 92%

Alternative promoters drive human cytomegalovirus reactivation from latency

Collins-McMillen

Rak

Buehler

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34 + HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.human cytomegalovirus | latency | reactivation R eactivation of latent human cytomegalovirus (HCMV) infection poses a life-threatening risk to immunocompromised individuals, such as stem cell or organ transplant recipients (1). While the HCMV replicative cycle has been studied extensively, our understanding of the mechanisms controlling the entry into and exit from latency is far from complete.During productive infection, the HCMV genome is transcribed in a temporal cascade composed of three kinetic classes of gene expression designated as immediate early (IE), early, and late (2). The IE proteins, in particular IE1-72 kilodalton (kDa) and IE2-86 kDa proteins, referred to as IE1 and IE2, play critical roles in initiating the HCMV lytic cycle by transactivating the expression of cellular and viral genes and suppressing the innate immune response (3). During latency, IE gene expression is repressed, resulting in diminished viral gene expression and the absence of productive replication (4). Signals that stimulate reactivation induce IE gene expression to allow reentry into the viral replicative cycle (5), and this de-repression of IE genes is considered a pivotal event controlling the switch between latent and reactivated states.The major immediate early promoter (MIEP) is a powerful promoter in cells permissive for lytic HCMV replication and drives high level expression of mRNAs encoding IE1 (UL123) and IE2 (UL122) (6-9). In cell types that support HCMV latency, however, such as CD34 + human progenitor cells (HPCs) and CD14 + monocytes, MIEP activity is diminished (10-12). Because re-expression of UL122 and UL123 is required for reactivation (13-15), it has been presumed that de-repression of the MIEP is critical to reinitiate the viral lytic cycle. However, the origin of UL123 and UL122 transcripts during HCMV reactivation has not been formally defined. ResultsRe-Expression of UL123 ...

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Section: Discussionsupporting

confidence: 92%

Alternative promoters drive human cytomegalovirus reactivation from latency

Collins-McMillen

Rak

Buehler

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Contemporary advances in transcriptome analysis have exposed a surprising complexity of CMV gene expression over time ( Marcinowski et al., 2012 ; Stern-Ginossar et al., 2012 ; Weekes et al., 2014 ; Shnayder et al., 2018 ; Erhard et al., 2019 ). The pattern of the viral gene expression varies not only between different cell lines ( Towler et al., 2012 ), but also at the single cell level ( Erhard et al., 2019 ; Shnayder et al., 2020 ).…”

Section: Discussionmentioning

confidence: 99%

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms

Rand

Kubsch

Kasmapour

et al. 2021

Front. Cell. Infect. Microbiol.

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Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.

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“…For example, among the inferred infected granulocytes we correctly predicted 99% by using a strict viral-load cutoff of 1%, and 88% by using a more permissive (0.01%) cutoff. Utilizing a reporter model of in vitro infection (Shnayder et al, 2018), we were able to assess how Immune and non-immune single cells were isolated from the whole lung of control and influenza-treated mice, 48 hr post infection, for massively parallel single-cell RNA-seq (MARS-seq). In each single cell, the host and the vmRNA were simultaneously measured, allowing identification of infected as opposed to bystander cells as well as retrospective annotation of cell types based on transcriptional identities.…”

Section: Dissecting In Vivo Influenza Infection Using Combined Singlementioning

confidence: 99%

“…mRNA-Seq Pre-processing The analysis starts with a joint alignment of MARS-seq reads for both the host and viral genomes, followed by the joint quantification of host and viral transcripts. We performed this joint analysis as previously described (Shnayder et al, 2018), with a few modifications. We used HISAT (Kim et al, 2015) to map the single-cell reads to a joint reference of both the mouse genome build mm9 and the influenza genome (NCBI records: NC_002016, NC_002017, NC_002018, NC_002019, NC_002020, NC_002021, NC_002022, NC_002023).…”

Section: Quantification and Statistical Analysismentioning

confidence: 99%

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing

et al. 2018

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The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells.

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

Cited by 16 publications

References 66 publications

Alternative promoters drive human cytomegalovirus reactivation from latency

Alternative promoters drive human cytomegalovirus reactivation from latency

A Novel Triple-Fluorescent HCMV Strain Reveals Gene Expression Dynamics and Anti-Herpesviral Drug Mechanisms

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing

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