scholarly journals Single-cell analysis of early antiviral gene expression reveals a determinant of stochasticIFNB1expression

2017 ◽  
Author(s):  
Sultan Doğanay ◽  
Maurice Youzong Lee ◽  
Alina Baum ◽  
Jessie Peh ◽  
Sun-Young Hwang ◽  
...  

AbstractRIG-I-like receptors (RLRs) are cytoplasmic sensors of viral RNA that trigger the signaling cascade that leads to type I interferon (IFN) production. Transcriptional induction of RLRs by IFN is believed to play the role of positive feedback to further amplify viral sensing. We found that RLRs and several other IFN-stimulated genes (ISGs) are induced early in viral infection independent of IFN. Expression of these early ISGs requires IRF3/IRF7 and is highly correlated amongst them. Simultaneous detection of mRNA ofIFNB1, viral replicase, and ISGs revealed distinct populations ofIFNB1expressing and non-expressing cells which are highly correlated with the levels of early ISGs but are uncorrelated with IFN-dependent ISGs and viral gene expression. Individual expression of RLRs madeIFNB1expression more robust and earlier, suggesting a causal relation between levels of RLR and induction of IFN.

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Joshua E. Messinger ◽  
Joanne Dai ◽  
Lyla J. Stanland ◽  
Alexander M. Price ◽  
Micah A. Luftig

ABSTRACTDeciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescencein situhybridization (RNA-FISH) targeting EBVEBNA2orLMP1and humanCCR7orMGST1. This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCEEBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.


2000 ◽  
Vol 58 (3) ◽  
pp. 1148-1159 ◽  
Author(s):  
Wataru Kajiyama ◽  
Jeffrey B. Kopp ◽  
Nancy J. Marinos ◽  
Paul E. Klotman ◽  
Peter Dickie

2019 ◽  
Author(s):  
Nir Drayman ◽  
Parthiv Patel ◽  
Luke Vistain ◽  
Savaş Tay

ABSTRACTViral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.


2021 ◽  
Author(s):  
C. A. Aurubin ◽  
D. A. Knaack ◽  
D. Sahoo ◽  
V. L. Tarakanova

Gammaherpesviruses are ubiquitous pathogens that establish life-long infections in >95% of adults worldwide and are associated with several cancers. We showed that endogenous cholesterol synthesis supports gammaherpesvirus replication. However, the role of exogenous cholesterol exchange and signaling during infection remains poorly understood. Extracellular cholesterol is carried in the serum by several lipoproteins, including low-density lipoproteins (LDL). The LDL-receptor (LDL-R) mediates the endocytosis of these cholesterol-rich LDL particles into the cell, thereby supplying the cell with cholesterol. We found that LDL-R expression attenuates gammaherpesvirus replication during the early stages of the replication cycle, as evident by increased viral gene expression in LDL-R -/- primary macrophages. This was not observed in primary fibroblasts, indicating that the antiviral effects of LDL-R are cell type-specific. Increased viral gene expression in LDL-R -/- primary macrophages was due to increased activity of the endogenous cholesterol synthesis pathway. Intriguingly, despite type I interferon-driven increase in LDL-R mRNA levels in infected macrophages, protein levels of LDL-R continually decreased over the single cycle of viral replication. Thus, our study has uncovered an intriguing tug of war between the LDL-R-driven antiviral effect on cholesterol metabolism and the viral targeting of the LDL-R protein. Importance. LDL-R is a cell surface receptor that mediates the endocytosis of cholesterol-rich low density lipoproteins, allowing cells to acquire cholesterol exogenously. Several RNA viruses usurp LDL-R function to facilitate replication; however, the role of LDL-R in DNA virus infection remains unknown. Gammaherpesviruses are double-stranded DNA viruses that are associated with several cancers. Here, we show that LDL-R attenuates gammaherpesvirus replication in primary macrophages by decreasing endogenous cholesterol synthesis activity, a pathway known to support gammaherpesvirus replication. In response, LDL-R protein levels are decreased in infected cells to mitigate the antiviral effects, revealing an intriguing tug-of-war between the virus and the host.


2017 ◽  
pp. JVI.01451-17 ◽  
Author(s):  
Nora Freudenberger ◽  
Tina Meyer ◽  
Peter Groitl ◽  
Thomas Dobner ◽  
Sabrina Schreiner

Human Adenoviruses (HAdV) are non-enveloped containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear-pore-complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood.The core protein V is speculated to bridge core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultanously, these altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than observed during wildtype infection.Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies.ImportanceMany decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion, as the functions are more diverse. We showed that capsid protein protein VI is regulating antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, being beneficial for efficient viral gene expression. Here, we were able to show that also core protein V represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. Conclusively, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency.


Open Biology ◽  
2017 ◽  
Vol 7 (10) ◽  
pp. 170115 ◽  
Author(s):  
Stephan Hofmann ◽  
Sandra Dehn ◽  
Ramona Businger ◽  
Sebastian Bolduan ◽  
Martha Schneider ◽  
...  

Viruses interact with multiple host cell factors. Some of these are required to promote viral propagation, others have roles in inhibiting infection. Here, we delineate the function of the cellular factor PHF13 (or SPOC1), a putative HIV-1 restriction factor. Early in the HIV-1 replication cycle PHF13 increased the number of integrated proviral copies and the number of infected cells. However, after HIV-1 integration, high levels of PHF13 suppressed viral gene expression. The antiviral activity of PHF13 is counteracted by the viral accessory protein Vpr, which mediates PHF13 degradation. Altogether, the transcriptional master regulator and chromatin binding protein PHF13 does not have purely repressive effects on HIV-1 replication, but also promotes viral integration. By the functional characterization of the dual role of PHF13 during the HIV-1 replication cycle, we reveal a surprising and intricate mechanism through which HIV-1 might regulate the switch from integration to viral gene expression. Furthermore, we identify PHF13 as a cellular target specifically degraded by HIV-1 Vpr.


2008 ◽  
Vol 82 (24) ◽  
pp. 12543-12554 ◽  
Author(s):  
Vera Lukashchuk ◽  
Steven McFarlane ◽  
Roger D. Everett ◽  
Chris M. Preston

ABSTRACT The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression.


2005 ◽  
Vol 79 (22) ◽  
pp. 14371-14382 ◽  
Author(s):  
Heesoon Chang ◽  
Dirk P. Dittmer ◽  
Shin-Young Chul ◽  
Youngkwon Hong ◽  
Jae U. Jung

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) RTA transcription factor is recruited to its responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jκ, indicating that RTA mimics cellular Notch signal transduction to activate viral lytic gene expression. To test whether cellular Notch signal transduction and RTA are functionally exchangeable for viral gene expression, human Notch intracellular (hNIC) domain that constitutively activates RBP-Jκ transcription factor activity was expressed in KSHV-infected primary effusion lymphoma BCBL1 cells (TRExBCBL1-hNIC) in a tetracycline-inducible manner. Gene expression profiling showed that like RTA, hNIC robustly induced expression of a number of viral genes, including viral interleukin 6 (vIL-6), K3, and K5. Unlike RTA, however, hNIC was not capable of evoking the full repertoire of lytic viral gene expression and thereby lytic replication. To further understand the role of Notch signal transduction in KSHV gene expression, vIL-6 growth factor and K5 immune modulator genes were selected for detailed analysis. Despite the presence of multiple RBP-Jκ binding sites, hNIC targeted the specific RBP-Jκ binding sites of vIL-6 and K5 promoter regions to regulate their gene expression. These results indicate that cellular Notch signal transduction not only is partially exchangeable with RTA in regard to activation of viral lytic gene expression but also provides a novel expression profile of KSHV growth and immune deregulatory genes that is likely different from that of RTA-independent standard latency program as well as RTA-dependent lytic reproduction program.


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