Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication

Abstract

Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV) infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5' untranslated region (UTR) of at least three viral genes (vp26, vp28, and wssv102), and plasmids that were controlled by the 5' UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host-virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

Keywords: Penaeus vannamei; miR-10a; microRNA; virus–host interaction; white spot syndrome virus.

Figure 1

Expression level of miR-10a after white spot syndrome virus (WSSV) infection. (A) Northern blot of shrimp miR-10a. Total RNA extracted from shrimp stomach before WSSV infection (0 dpi) and after WSSV infection (48 dpi) was blotted with γ[³²P]-labeled miR-10a probe (upper panel). tRNA was used as a loading control (lower panel). (B) Real-time PCR analysis of the expression of miR-10a at 0, 12, 24, and 48 hpi in shrimp stomach. 2^−ΔΔCt was used to analyze the expression of miR-10a relative to the expression of shrimp U6 (internal control). Data are presented as mean ± SD from three shrimp at each time point (*p < 0.05 and **p < 0.01 by Student’s t-test).

Figure 2

The role of host miR-10a in white spot syndrome virus (WSSV)-infected shrimp. Shrimp (L. vannamei; mean weight 7 g) were injected simultaneously with WSSV and control anti-miRNA inhibitor (1 nmol/shrimp); WSSV and anti-miR-10a inhibitor (1 nmol/shrimp); WSSV and phosphate-buffered saline (PBS). (A) Real-time PCR analysis of miR-10a expression 1 day after the injection of anti-miR-10a inhibitor. miR-10a expression levels were determined in cDNA derived from shrimp pleopods. 2^−ΔΔCt was used to analyze the expression of miR-10a, and results are presented relative to the expression of shrimp U6. Values shown are the mean ± SD from four shrimp at each time point (*p < 0.05 by Student’s t-test). (B) An IQ REAL™ WSSV Quantitative System kit was used to determine the WSSV copy number in shrimp pleopods. Values shown are the mean ± SD from four shrimp at each time point (*p < 0.05 by Student’s t-test.) (C) Specific anti-IE1, anti-ICP11 and anti-VP28 antibodies were used to detect WSSV protein levels in shrimp stomach of anti-miR-10a inhibitor treated shrimp, non-specific microRNA inhibitor treated shrimp, and PBS-injected shrimp. Shrimp actin was detected with anti-actin antibody as internal control.

Figure 3

Suppression of miR-10a significantly reduces the expression of at least three WSSV genes. (A–D) Luciferase reporter assay for wssv234, vp26, vp28, and wssv102, respectively. Sf9 cells were transfected as indicated with the respective ie1/pGL3/5′-UTR firefly luciferase expression construct (500 ng) plus miR-10a mimic (32 pmol), along with either anti-miR-10a (32 pmol) or the anti-miRNA control (32 pmol). At 48 h post-transfection, firefly luciferase signals were normalized relative to those of the Renilla luciferase internal control, and data are shown relative to the anti-miRNA control (set to 100%). Data represent mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.005 by Student’s t-test.

Figure 4

Mutation of the target gene’s 5′ UTR prevents the downregulation caused by miR-10a suppression. Sf9 cells were transfected with the miR-10a minic (23 pmole) plus 500 ng of the non-mutant or mutant ie1/pGL3/5′-UTR firefly luciferase expression construct for (A) VP26, (B) VP28, and (C) WSSV102, along with either anti-miR-10a (32 pmol) or the anti-miRNA control (32 pmol) as indicated. At 48 h post-transfection, the firefly luciferase signals were normalized relative to those of the Renilla luciferase internal control, and data are shown relative to the anti-miRNA control (set to 100%). Data represent mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.005 by Student’s t-test. The mutated nucleotides are shown in bold.

Figure 4

Mutation of the target gene’s 5′ UTR prevents the downregulation caused by miR-10a suppression. Sf9 cells were transfected with the miR-10a minic (23 pmole) plus 500 ng of the non-mutant or mutant ie1/pGL3/5′-UTR firefly luciferase expression construct for (A) VP26, (B) VP28, and (C) WSSV102, along with either anti-miR-10a (32 pmol) or the anti-miRNA control (32 pmol) as indicated. At 48 h post-transfection, the firefly luciferase signals were normalized relative to those of the Renilla luciferase internal control, and data are shown relative to the anti-miRNA control (set to 100%). Data represent mean ± SD from three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.005 by Student’s t-test. The mutated nucleotides are shown in bold.

Figure 5

Schematic representation of how shrimp miR-10a is co-opted by white spot syndrome virus (WSSV) to enhance viral replication. Pv-miR-10a upregulates the expression of at least three WSSV genes (vp26, vp28, and wssv102) by targeting their 5′ untranslated region. Alignment followed by RNAhybrid prediction suggests that several other important WSSV genes are also likely to be upregulated by host miR-10a.