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. 2019 Mar:529:169-176.
doi: 10.1016/j.virol.2019.01.025. Epub 2019 Jan 30.

Coxsackievirus B infection induces the extracellular release of miR-590-5p, a proviral microRNA

Juliana F Germano  1 , Savannah Sawaged  1 , Hannaneh Saadaeijahromi  1 , Allen M Andres  1 , Ralph Feuer  2 , Roberta A Gottlieb  1 , Jon Sin  3
Affiliations

Coxsackievirus B infection induces the extracellular release of miR-590-5p, a proviral microRNA

Juliana F Germano et al. Virology. 2019 Mar.

Abstract

Coxsackievirus B is a significant human pathogen and is a leading cause of myocarditis. We and others have observed that certain enteroviruses including coxsackievirus B cause infected cells to shed extracellular vesicles containing infectious virus. Recent reports have shown that vesicle-bound virus can infect more efficiently than free virus. Though microRNAs are differentially regulated in cells following infection, few have been associated with the vesicles shed from infected cells. Here we report exclusive trafficking of specific microRNAs into viral vesicles compared to vesicles from non-infected cells. We found that the most highly-expressed unique microRNA in viral vesicles was miR-590-5p, which facilitates prolonged viral replication by blocking apoptotic factors. Cells over-expressing this miR were significantly more susceptible to infection. This may be a mechanism by which coxsackievirus B boosts subsequent rounds of infection by co-packaging virus and a select set of pro-viral microRNAs in extracellular vesicles.

Keywords: Apoptosis; Coxsackievirus; MicroRNA; Vesicles.

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Figures

Figure 1.
Figure 1.. Viral EV-bound miRs promote cell survival.
(A) List of miRs expressed exclusively in EVs shed from CVB infected cells, and absent in EVs shed from mock infected cells. Black box indicates miR-590–5p, which was the most abundant miR in the list. (B) Ingenuity pathways analysis (IPA) of miRs expressed exclusively in EVs shed from CVB-infected HL-1 cardiomyocytes. Gray bars represent percentage of target mRNAs that overlap with IPA Knowledge Base for specific biological attributes. Numbers indicate total amount of molecules in IPA dataset that represent corresponding attribute. Yellow line corresponds to the padj (log(B-H p-value)) of attributes.
Figure 2.
Figure 2.. miR-590–5p plays roles in cell survival and viral infection.
Network analysis of miR-590–5p and cellular targets. Blue lines indicate inhibited interactions while orange lines indicate activated interactions. General cellular pathways predicted to be affected by miR-590–5p are shown at the right of the network.
Figure 3.
Figure 3.. miR-590–5p enhances CVB infection.
HL-1s were transfected with either scrambled miR, miR-590–5p mimic, or miR-590–5p antagomir and subsequently infected with eGFP-CVB at MOI 1. (A) Fluorescence microscopy of infected HL-1s at 8 h and 24 h postinfection (PI). Phase contrast images show cell number at 24 h PI. Scale bars represent 200 μm. (B) Plaque assays on culture media of infected HL-1s 24 h PI (*p<0.05, **p<0.01, ***p<0.001; Student’s t-test; n=3). (C) Western blots detecting VP1 in infected cells at 0 h, 8 h, and 24 h PI. (D) Densitometric quantification of VP1 levels in western blots of cells 24 h PI (**p<0.01, ***p<0.001; Student’s t-test; n=3–4).
Figure 4.
Figure 4.. miR-590–5p suppresses sprouty-1 which is an antiviral factor.
(A) Western blot detecting Spry1 in HL-1s infected with eGFP-CVB at MOI 10. (B) Western blot detecting Spry1 in HL-1s treated with EVs isolated from cells infected with eGFP-CVB. (C) Densitometric quantification of western blot in B (Student’s t-test; n=3). (D) Western blot detecting Spry1 in HL-1s transfected with scrambled miR or miR-590–5p mimic. (E) Densitometric quantification of western blot in D (****p<0.0001; Student’s t-test; n=3). (F) Fluorescence microscopy images on HL-1s transfected with either scrambled RNA (siSCRAMBLE) or siRNA targeting Spry1 (siSPRY1) infected with eGFP-CVB at MOI 1. Images were taken 24 h PI. Scale bars represent 200 μm. (G) Plaque assays on culture media of cells in F (*p<0.05; Student’s t-test; n=3). (H) Western blot detecting VP1 in cells in F. (I) Densitometric quantification of western blot in H (*p<0.05; Student’s t-test; n=3).
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