Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

Jn, Berger; Sanford, Bridget; Kimball, Abigail K.; Lm, Oko; Kaspar, Rachael E.; Niemeyer, Brian F.; Kl, Jones; Et, Clambey; Dyk, Linda F. van

doi:10.1101/2020.08.11.203117

Cited by 5 publications

(5 citation statements)

References 66 publications

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“…S6b). This agrees with two recent studies that showed the single-cell heterogeneity in herpes simplex virus type 1 (HSV-1) infection 22 , and low correlation between viral gene expression in a murine gammaherpesvirus infection model 23 . One partial explanation is that the K8.1+ cells were at different stages of reactivation.…”

Section: Fd-seq Reveals Heterogeneity In Kshv Reactivated Single Tumosupporting

confidence: 92%

Fixed single-cell RNA sequencing for understanding virus infection and host response

Phan

Gent

Drayman

et al. 2020

Preprint

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Single-cell RNA sequencing studies requiring intracellular protein staining, rare-cell sorting, or pathogen inactivation are severely limited because current high-throughput methods are incompatible with paraformaldehyde treatment, a very common and simple tissue/cell fixation and preservation technique. Here we present FD-seq, a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, stained and sorted single-cells. We used FD-seq to address two important questions in virology. First, by analyzing a rare population of cells supporting lytic reactivation of the human tumor virus KSHV, we identified TMEM119 as a host factor that mediates reactivation. Second, we studied the transcriptome of lung cells infected with the coronavirus OC43, which causes the common cold and also serves as a safer model pathogen for SARS-CoV-2. We found that pro-inflammatory pathways are primarily upregulated in abortively-infected or uninfected bystander cells, which are exposed to the virus but fail to express high level of viral genes. FD-seq is suitable for characterizing rare cell populations of interest, for studying high-containment biological samples after inactivation, and for integrating intracellular phenotypic with transcriptomic information.

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Section: Fd-seq Reveals Heterogeneity In Kshv Reactivated Single Tumosupporting

confidence: 92%

Fixed single-cell RNA sequencing for understanding virus infection and host response

Phan

Gent

Drayman

et al. 2020

Preprint

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“…6a). This agrees with two recent studies that showed heterogeneity in host cell factor abundance at the single-cell level in herpes simplex virus type 1 (HSV-1) infection 24 , and low correlation in expression of viral genes in a murine gammaherpesvirus infection model 25 . This may partially be caused by the possibility that the sorted K8.1+ cells were at different stages of reactivation.…”

Section: Resultssupporting

confidence: 92%

High-throughput RNA sequencing of paraformaldehyde-fixed single cells

et al. 2021

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Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

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“…It is notable that while the LANA protein is expressed throughout infection, it is generally expressed at a low level, and thus, combination with the enzymatic amplification of beta-lactamase is critical. Further development of this beta-lactamase tagged virus system allowed tracking of cells infected with panels of mutant viruses both in vitro and in vivo, as well as live cell sorting of rare infected cells for downstream analyses of function and multiparametric mass cytometry and transcriptomics (25, 26).…”

Section: Introductionmentioning

confidence: 99%

Optimized detection of acute MHV68 infection with a reporter system identifies large peritoneal macrophages as a dominant target of primary infection

Riggs

Medina

Perrenoud

et al. 2020

Preprint

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Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally-infected cells. Here, we combine detection of a beta-lactamase activated fluorescent substrate with full spectrum flow cytometry to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages by 8 days post-infection, and a concordant increase in small peritoneal macrophages and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh large peritoneal macrophages emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 to 3 days post-infection, MHV68 infects ~5-10% of peritoneal cells, with >75% being large peritoneal macrophages. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in small peritoneal macrophages, with few infected dendritic cells and B cells. MHV68 infection at 3 days post-infection contains both lytic and latent infection, consistent with the identification of cells with active reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.ImportanceIdentifying virally-infected cells in vivo is key to tracking viral infection and understanding host-pathogen interactions. The ability to further characterize and phenotype virally-infected cells is technically challenging. We use a mouse gammaherpesvirus, MHV68, expressing a reporter gene to identify infected cells during primary infection via flow cytometry. Optimization using this reporter system allowed us to further characterize infected cells via multiparameter full spectrum flow cytometry. Our study provides a technical model for high throughput single-cell immunophenotyping methods in the context of gammaherpesvirus infection. Furthermore, we show that acute MHV68 infection in the peritoneal cavity dramatically changes the immune landscape of this tissue, results in a high number of infected macrophages at early times, and is characterized by both lytic and latent infection within immune cells.

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Redefining De Novo Gammaherpesvirus Infection Through High-Dimensional, Single-Cell Analysis of Virus and Host

Cited by 5 publications

References 66 publications

Fixed single-cell RNA sequencing for understanding virus infection and host response

Fixed single-cell RNA sequencing for understanding virus infection and host response

High-throughput RNA sequencing of paraformaldehyde-fixed single cells

Optimized detection of acute MHV68 infection with a reporter system identifies large peritoneal macrophages as a dominant target of primary infection

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