Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

Borah, Sumit; Nichols, Lisa A.; Hassman, Lynn M.; Kedes, Dean H.; Steitz, Joan A.

doi:10.1261/rna.033126.112

Cited by 24 publications

(19 citation statements)

References 35 publications

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“…MM cells, however, reportedly undergo transformation following KSHV infection and efficiently retain multiple copies of the episome even in the absence of positive selection by drug treatment [11] . As a result, we predicted that we would be able to evaluate, at the level of individual cells, the extent of infection in rKSHV.219-infected MM cultures on the basis of characteristic punctate nuclear LANA dots using multispectral imaging flow cytometry (MIFC) [20] - [22] . LANA dots correspond to the presence of the KSHV episome [23] , [24] , and we have shown previously in HeLa cells that the number of dots per cell correlates with intracellular viral load [20] .…”

Section: Resultsmentioning

confidence: 99%

Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

Ellison

Kedes

2014

PLoS ONE

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The availability of reliable recombinant reporter virus systems has been a great boon to the study of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). Unexpectedly, we found that expression of the ostensibly constitutive green fluorescent protein (GFP) marker was progressively lost during unselected passage in primary rat mesenchymal precursor cells (MM), despite efficient maintenance of latent viral gene expression and episomal partitioning. This repression of EF1-α promoter-driven GFP expression appeared to be passage-dependent, however, since functionally immortalized MM cells derived from long serial passage retained stable expression of GFP following rKSHV.219 infection. Chromatin analysis of cultures that we had infected in parallel demonstrated an increase in repressive H3K27 tri-methylation across the viral episome with the exception of the LANA control region in MM cells infected at early rather than late passage post-isolation. The silencing of GFP expression in the MM cells was reversible in a dose-dependent fashion by the histone deacetylase inhibitor valproic acid, further implicating cellular silencing on incoming viral genomes, and underscoring potential differences in viral gene regulation between primary and functionally immortalized cells. Furthermore, using multispectral imaging flow cytometry, we also determined that the extent of GFP expression per cell among those that were positive did not correlate with the number of LANA dots per nucleus nor the extent of overall LANA expression per cell. This suggests a more complex mode of local gene regulation, rather than one that simply reflects the relative intracellular viral copy number. In sum, we have demonstrated the significant potential for false-negative data when using a constitutive marker gene as a sole means of evaluating herpesviral infection, especially in primary cells.

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Section: Resultsmentioning

confidence: 99%

Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

Ellison

Kedes

2014

PLoS ONE

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“…This supports the hypothesis that this class of RBPs would be significantly impacted by the mRNA abundance and availability. PABPC nuclear translocation in particular has been well documented in the context of infection with viruses that drive mRNA decay (Bablanian et al, 1991; Borah et al, 2012; Harb et al, 2008; Lee, 2009; Park et al, 2014; Piron et al, 1998; Salaun et al, 2010), and our unbiased proteomics approach establishes it as one of the most robustly relocalized RBPs under these conditions. Several features of PABPC render it an ideal indicator of mRNA abundance.…”

Section: Discussionmentioning

confidence: 82%

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

Gilbertson

Federspiel

Hartenian

et al. 2018

Preprint

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“…To extend these findings, we analyzed viral gene expression in the KSHV infected B cell tumor line, BCBL-1, focused on detection of an abundant viral ncRNA, the KSHV polyadenylated nuclear RNA (PAN, nut1, or T1.1) [20]. PAN RNA is known to be highly inducible upon induction of reactivation in KSHV latently infected B cell lymphoma cell lines [10, 20]. The frequency of PAN RNA+ cells was low in untreated BCBL-1 cells, with ~1% of cells spontaneously expressing this ncNRA (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

Oko

Kimball

Kaspar

et al. 2018

Preprint

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24Virus-host interactions are frequently studied in bulk cell populations, obscuring 25 cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at 26 the single-cell level, combining a sensitive and robust fluorescent in situ hybridization 27 platform with multiparameter flow cytometry, to study the expression of 28 gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection 29 and reactivation in vitro. This method allowed robust detection of viral ncRNAs of 30 murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and 31 Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying 32 the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA 33 regulation during γHV68 infection, we find heterogeneous and asynchronous gene 34 expression during latency and reactivation, with reactivation from latency identified by a 35 distinct gene expression profile within rare cells. Further, during lytic replication with 36 γHV68, we find many cells have limited viral gene expression, with only a fraction of 37 cells showing robust gene expression, dynamic RNA localization, and progressive 38 infection. Lytic viral gene expression was enhanced in primary fibroblasts and by 39 conditions associated with enhanced viral replication, with multiple subpopulations of 40 cells present in even highly permissive infection conditions. These findings, powered by 41 single-cell analysis integrated with automated clustering algorithms, suggest inefficient 42 or abortive γHV infection in many cells, and identify substantial heterogeneity in viral 43 gene expression at the single-cell level. 44 45 3 AUTHOR SUMMARY 46 The gammaherpesviruses are a group of DNA tumor viruses that establish 47 The Herpesviridae are a family of large dsDNA viruses that include multiple 63 prominent human and animal pathogens [1]. Although these viruses infect different cell 64 types, and are associated with diverse pathologies, they share conserved genes and 65 two fundamental phases of infection: lytic replication and latent infection [1]. Lytic 66 replication is characterized by a cascade of viral gene expression, active viral DNA 67 replication and the production of infectious virions. Conversely, latency is characterized 68 4 by limited viral gene expression and the absence of de novo viral replication. While 69 latent infection is a relatively quiescent form of infection, the herpesviruses can 70 reactivate from latency, to reinitiate lytic replication. 71 Among the herpesviruses, the gammaherpesviruses (γHV) are lymphotropic 72 viruses that include the human pathogens Epstein-Barr virus (EBV) [2] and Kaposi's 73 sarcoma associated herpesvirus (KSHV) [3]. Murine gammaherpesvirus 68 (γHV68, or 74 MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4), is a well-described small 75 animal model for the γHVs [4]. While these viruses establish a lifelong infection that is 76 often clinically inapparent, immune-suppressed individuals are part...

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Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

Cited by 24 publications

References 35 publications

Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

Variable Episomal Silencing of a Recombinant Herpesvirus Renders Its Encoded GFP an Unreliable Marker of Infection in Primary Cells

Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression

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