Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery

Bullard, Whitney; Kara, Mehmet; Sethuraman, Sunantha; Wang, Yiping; Nirmalan, Shreya; Esemenli, Alim; Feswick, April; Hoffman, Brett; Renne, Rolf; Tibbetts, Scott A.

doi:10.1371/journal.ppat.1007843

Cited by 27 publications

(23 citation statements)

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“…To determine whether enhanced representation of viral miRNAs in RISC is a conserved feature of γHVs, we supplemented data from our previously published KSHV[ 42 ] and MHV68[ 66 ] qCLASH studies with small RNA sequencing to similarly compare KSHV and MHV68 hybrid levels to their respective miRNA expression. Similar to our findings with in vitro EBV miRNA expression, KSHV miRNAs were found to be expressed at low levels in KSHV infected LTC-TIVE cells ( S8A Fig ).…”

Section: Resultsmentioning

confidence: 99%

“… Raw CLASH sequencing reads were obtained for KSHV infected TIVE-EX-LTC[ 42 ] (SRA accessions: SRR5876950-SRR5876952) and MHV68 infected HE2.1[ 66 ] (SRA accessions: SRR8395245-SRR8395247) cell lines, with small fraction sequencing performed on the same cell lines. (A-B; left) Yellow triangles display total viral microRNA expression, ; green triangles represent the percent of all microRNA-mRNA hybrids containing a viral microRNA, .…”

Section: Supporting Informationmentioning

confidence: 99%

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EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

et al. 2021

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The Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt’s Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in BL and GC datasets. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing host immune response.

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Section: Resultsmentioning

confidence: 99%

Section: Supporting Informationmentioning

confidence: 99%

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

et al. 2021

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“…To determine whether enhanced representation of viral miRNAs in RISC is a conserved feature of γ HVs, we supplemented data from our previously published KSHV(42) and MHV68(65) qCLASH studies with small RNA sequencing to similarly compare KSHV and MHV68 hybrid levels to their respective miRNA expression. Similar to our findings with in vitro EBV miRNA expression, KSHV miRNAs were found to be expressed at low levels in KSHV infected LTC-TIVE cells ( Figure S7A ).…”

Section: Resultsmentioning

confidence: 99%

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

Ungerleider

Bullard²,

Kara³

et al. 2020

Preprint

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Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt’s Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and a BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in TCGA GC dataset. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing the host immune response.AUTHOR SUMMARYBurkitt’s Lymphoma and gastric cancer are both associated with EBV, a prolific DNA tumor virus that latently resides in nearly all human beings. Despite mostly restricting viral gene expression to noncoding RNAs, EBV has important influences on the fitness of infected tumor cells. Here, we show that the miRNA class of viral noncoding RNAs are a major viral contributor to remodeling the tumor cell regulatory machinery in patient tumor samples. First, an assessment of miRNA expression in clinical tumor samples showed that EBV miRNAs are expressed at unexpectedly high levels relative to cell miRNAs. Using a highly specific miRNA target identification approach, CLASH, we comprehensively identified both viral and cellular microRNA targets and the relative abundance of each microRNA-mRNA interaction. We also show that viral microRNAs bind to and alter the expression of their mRNA targets more effectively than their cellular microRNA counterparts. Pathway analysis of the most effectively targeted mRNAs revealed enrichment of immune signaling pathways and we show a corresponding inverse correlation between EBV miRNA expression and infiltrating immune cells in EBV positive primary tumors. Altogether, this study shows that EBV miRNAs are key regulators of the tumor cell phenotype and the immune cell microenvironment.

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“…The TMERs are dispensable for lytic replication and establishment of latency; however, these transcripts are required for pathogenesis during acute infection of an immunocompromised host (7,24,25). The TMERs contain bi-functional elements with a tRNA-like structure at the 5' end and hairpins that are processed into biologically-active miRNAs (7), capable of targeting a number of RNAs for post-transcriptional regulation (26). Our lab has previously shown that the tRNA-like on May 10, 2021 by guest http://jvi.asm.org/ Downloaded from γHV68 infection activates pol III promoters 5 structure is sufficient to rescue pathogenesis of a TMER deficient viral recombinant, suggesting that like the EBERs, the TMERs may contribute to pathogenesis through their interactions with host proteins (25).…”

Section: Introductionmentioning

confidence: 99%

Lytic Infection with Murine Gammaherpesvirus 68 Activates Host and Viral RNA Polymerase III Promoters and Enhances Noncoding RNA Expression

Knox

Mueller

Medina

et al. 2021

J Virol

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RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCE Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

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Identification of murine gammaherpesvirus 68 miRNA-mRNA hybrids reveals miRNA target conservation among gammaherpesviruses including host translation and protein modification machinery

Cited by 27 publications

References 76 publications

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape

Lytic Infection with Murine Gammaherpesvirus 68 Activates Host and Viral RNA Polymerase III Promoters and Enhances Noncoding RNA Expression

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