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. 2021 Apr 19;22(8):4218.
doi: 10.3390/ijms22084218.

Differential miRNA Expression Profiling Reveals Correlation of miR125b-5p with Persistent Infection of Japanese Encephalitis Virus

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Differential miRNA Expression Profiling Reveals Correlation of miR125b-5p with Persistent Infection of Japanese Encephalitis Virus

Chih-Wei Huang et al. Int J Mol Sci. .

Abstract

MicroRNAs (miRNAs) play versatile roles in multiple biological processes. However, little is known about miRNA's involvement in flavivirus persistent infection. Here, we used an miRNA array analysis of Japanese encephalitis virus (JEV)-infected cells to search for persistent infection-associated miRNAs in comparison to acute infection. Among all differentially expressed miRNAs, the miR-125b-5p is the most significantly increased one. The high level of miR-125b-5p in persistently JEV-infected cells was confirmed by Northern analysis and real-time quantitative polymerase chain reaction. As soon as the cells established a persistent infection, a significantly high expression of miR-125b-5p was readily observed. Transfecting excess quantities of a miR-125b-5p mimic into acutely infected cells reduced genome replication and virus titers. Host targets of miR125b-5p were analyzed by target prediction algorithms, and six candidates were confirmed by a dual-luciferase reporter assay. These genes were upregulated in the acutely infected cells and sharply declined in the persistently infected cells. The transfection of the miR125b-5p mimic reduced the expression levels of Stat3, Map2k7, and Triap1. Our studies indicated that miR-125b-5p targets both viral and host sequences, suggesting its role in coordinating viral replication and host antiviral responses. This is the first report to characterize the potential roles of miR-125b-5p in persistent JEV infections.

Keywords: Japanese encephalitis virus; miR-125b-5p; persistent infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MiR-125b-5p is highly expressed in persistently JEV-infected cells. (a) Northern blot analysis of miR-125b-5p expression. Total RNAs were extracted from uninfected BHK-21 cells (lane 1), cells infected with JEV RP-9 at an MOI of 0.1 at the indicated times post-infection (hpi), and persistently infected cells at passage 17 (P17). Northern blot analysis was done using in vitro transcribed radiolabeled riboprobe specific to miR-125b-5p. The positions of precursor miR-125b-5p (pre-miR-125b-5p), mature miR-125b-5p (miR-125b-5p), and U6 snRNA are indicated. (b) The same RNAs were subjected to RT-qPCR analysis to determine the expression of miR-125b-5p. The relative fold change was normalized to endogenous U6 snRNA. Bars represent the mean ± SD (n = 3). *: p-value < 0.05 comparison between acute (24 hpi) and persistent infection (P17) was analyzed by Student’s t-test.
Figure 2
Figure 2
The miR-125b-5p was immediately elevated once established persistent infection. The total RNA was extracted from uninfected, acutely infected (24 and 48 hpi at an MOI of 0.1), and persistently infected cells from passages 1 to 6 (P1–P6). The RNA was subjected to an RT reaction and analyzed by RT-qPCR. The relative fold change was normalized to endogenous U6 snRNA. Bars represent the mean ± SD (n = 3). **: p-value < 0.01, ***: p-value < 0.001 comparison between acute (48 hpi) and persistent infection was analyzed by Student’s t-test.
Figure 3
Figure 3
Transfected miR-125b-5p mimics lasted for 72 h. BHK-21 cells were mock-transfected or transfected with miR-125b-5p at 0.5 nM. Total RNA was extracted at 6, 12, 24, 36, 48, and 72 hpt. The RNA was subjected to an RT reaction and analyzed by RT-PCR. The relative fold change was normalized to endogenous U6 snRNA. Bars represent the mean ± SD (n = 3).
Figure 4
Figure 4
Transfecting excess amounts of miR-125b-5p inhibited JEV replication. BHK-21 cells were transfected with miR-125b-5p (0.5 nM) or a scramble control and then infected with JEV at an MOI of 0.1 at 24 h post-transfection. (a) Cytoplasmic RNA was extracted at the indicated time post-infection. The fold change of viral RNA was analyzed using RT-qPCR. (b) The supernatant was collected, and virus titers were determined by plaque assay. Bars represent the mean ± SD (n = 3). *: p-value < 0.05; **: p-value < 0.01; *** p-value < 0.001 compared with scramble control and analyzed by Student’s t-test.
Figure 5
Figure 5
Downregulation of miR-12b-5p-targeted genes in the persistently JEV-infected cells. (a) Six predicted miR-125b-5p target genes were downregulated in persistently infected cells compared with the acutely infected cells. Total RNAs were extracted from uninfected, acutely infected, and persistently infected cells at 24 hpi (P10), and the amounts of the six genes were analyzed by RT-qPCR. The relative fold change was normalized to mock-infection. Bars represent the mean ± SD (n = 3). *: p-value < 0.05; ***: p-value < 0.001 comparison between acute and persistent infections was analyzed by a Student’s t-test. (b) Decreased expressions of both Stat3 and Map2k7 in persistently JEV-infected cells in comparison with acutely infected cells. Cell lysates from acute or persistent infection, as indicated on the top, were analyzed by 10% SDS-PAGE and Western blotting with antibodies, as indicated on the left.
Figure 6
Figure 6
MiR-125b-5p targeted the six predicted genes, as demonstrated in luciferase reporter assays. Diagrams of the dual-luciferase reporter plasmids (pmirGLO vector; Table S3) and the microRNA (miRNA) are shown on the top. The activity was evaluated by the insertion of miRNA target sites at the 3′-UTR of the firefly luciferase gene (luc) and Renilla luciferase acting as a control reporter for normalization. The deletion of the miRNA target site was used as a negative control. BHK-21 cells were co-transfected with 100 ng of a luciferase reporter construct containing a gene target or the seed sequences deleted in conjunction with 0.5 nM of the miR-125b-5p mimic or the scramble miRNA as a negative control. After 24 h, cells were lysed for a dual-luciferase assay. Data are shown as means ± SD (n = 3). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001 compared with the scramble control and analyzed by Student’s t-test.
Figure 7
Figure 7
The overexpression of miR125b-5p downregulated the target genes. BHK-21 cells were transfected with miR-125b-5p or an miRNA scramble control. At 24 h post-transfection, cells were infected with JEV at an MOI of 0.1. Total RNA was extracted at 48 hpt and analyzed by RT-qPCR. The relative fold change was normalized to endogenous GAPDH RNA. *: p-value <0.05 in the comparison between mock infection and acute JEV infection analyzed by Student’s t-test. **: p-value < 0.01 in comparison between the miRNA scramble control and miR125b-5p transfection in the JEV-infected cells.

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