Abstract 15678: Dicer-1 Prevents Coxsackievirus B3 (CVB3) Infection of the Heart by Inhibiting the Viral Replication in a RNaseIII Domain Dependent Manner
While Dicer-2 plays an important role in innate antiviral mechanisms in Drosophila, it remains unclear why mammalian cells have preserved only Dicer-1 during evolution. In addition, a paralog of mammalian Dicer-1, melanoma differentiation-associated protein 5, has recently been identified as a sensor for incoming CVB3 infection. We assessed the hypothesis that Dicer-1 prevents CVB3 infection of the heart and subsequent cardiomyopathy by inhibiting the viral replication in the cardiomyocyte.
Methods: 1) The effect of cardiac Dicer-1 on the susceptibility to CVB3 infection was examined using tamoxifen-inducible, cardiac-specific Dicer-1 KO mice (Dicer-1f/f, α-MHC/mer-Cre-mer). The viral infection of the heart was evaluated at 4 days post infection by %Evans blue dye (EBD) uptake area, a marker for the viral infected area, and the virus titer with standard plaque forming assay. 2) The effect of Dicer-1 on CVB3 replication was assessed by examining one-step growth curve of CVB3 in Dicer-1 KO (Dicer-1f/f, adenovirus-Cre) and WT (Dicer-1+/+, adenovirus-Cre) embryonic cardiomyocytes. 3) Interaction between Dicer-1 and CVB3 RNA during the replication was examined by immunoprecipitation of Dicer-1 complex followed by strand-specific RT-PCR. 4) The requirement of RNaseIII activity for the antiviral effect of Dicer-1 was evaluated by overexpressing WT Dicer-1 (wtD) or mutant Dicer-1 (muD) lacking RNaseIII activity in HeLa cells.
Results: 1) %EBD area and virus titer were significantly increased in Dicer-1 KO heart compared to WT heart (EBD: mean±SD; 29.5±11.6 vs 1.78±1.45, n=3, p<0.05, Virus titer: 37263±3642 vs 3679±561 pfu/mg, n=5, p=0.006). 2) Dicer-1 ablation increased virus replication in isolated cardiomyocytes. 3) Interaction between Dicer-1 and CVB3 RNA was detectable from 3 hours post CVB3 infection. 4) The viral VP1 protein expression was significantly decreased in cells with wtD compared to those with muD at 8 hours post infection (0.862±0.080 vs 1.10±0.172, p<0.05, n=3). No such differences were observed between muD and the empty vector expressed cells.
Conclusion: These results demonstrated that Dicer-1 interacts with CVB3 RNA and inhibits the viral replication in a RNaseIII dependent manner, thus preventing CVB3 infection of the heart.
- © 2012 by American Heart Association, Inc.