The Roles Of Shrimp MiR-71-mediated Autophagy And WSSV-encoded MiRNAs In Virus Infection

Since the first outbreak in1993, white spot syndrome virus (WSSV) has caused shrimp farming industry huge economic losses worldwide. Although a lot of scientific work has been done on the pathogenesis of WSSV, no efficient strategy is available to control the virus. Studies have shown that the regulation of gene expression plays a key role in the virus-host interaction. As reported, miRNAs take great effects on the regulation of gene expression.MicroRNA (miRNA) is a class of about22nt non-coding RNA. MiRNA is involved in regulation of almost all life processes. It was reported that at least50percent of mammal genes are regulated by miRNAs. For miRNAs, pri-miRNA is formed under the action of RNA polymerase Ⅱ. Pri-miRNA is cut into about70nt pre-miRNA by Drosha and DGCR8in the nucleus, and then pre-miRNA is transported to the cytoplasm by Ran and exportin5. In the cytoplasm, the Dicer-TRBP complex cut the pre-miRNA into20-22nt mature miRNAs duplexes. Most miRNAs target mRNA by recognition and binding of the target mRNA3’UTR by the seed sequences (2-8bases). Mature miRNA under the help of Dicer goes into the silencing complex (RISC) in which the single-stranded miRNA binds with the target mRNA through complementary base pairing. In recent years, many studies report that DNA virus could encode its own miRNA during infection. However, almost all these studies come from vertebrate viruses. Investigations on miRNA encoded by invertebrate DNA virus are very limited.Guided by miRNAs, RNAi plays an important role in virus-host interactions by fine-tuning gene expression. Many viral and cellular miRNAs are involved in virus infection, though no comprehensive general model for miRNAs derived from invertebrate DNA viruses exists for their function in eukaryotic systems, despite extensive research on miRNAs. To address this issue, the miRNAs from shrimp white spot syndrome virus (WSSV), a DNA virus with a305kb double-stranded circular DNA genome, were characterized. Based on WSSV miRNA microarray and northern blot analyses, WSSV was shown to possess the capacity to encode40distinct viral miRNAs, a miRNA content roughly360times greater than that of humans. These findings suggested that the high content of viral miRNAs might greatly contribute to viral variability in response selective pressures in the host environment. Transcription analysis revealed that80%of WSSV miRNAs were expressed during early stages of viral infection, indicating their importance in initial infective processes. The results showed that the biogenesis of viral miRNAs was dependent on host Drosha and Dicer1and mediated by Ago1. The viral miRNAs including WSSV-miR211and WSSV-miR212were required for successful infection of WSSV in shrimp. In this study, the findings presented that viral miRNAs WSSV-miR66and WSSV-miR68could promote the replication of the virus by the inhibition of viral mRNA. In WSSV infection, many WSSV genes are regulated by viral miRNAs. Based on miRNA target prediction by bioinformatics softwares, it was found that many viral miRNA target genes were involved in the innate immune response of the host, and some of them were critical viral genes. During WSSV infection, numerous viral genes were targeted by WSSV miRNAs. This study presented the first comprehensive view of viral miRNAs encoded by an invertebrate DNA virus, providing insight into the molecular events of virus-host interactions. In the process of viral infection, the host miRNA expression profile will change significantly. After WSSV infection, Marsupenaeus japonicas miRNA expression profile changed. Among them, a conservative miRNA miR-71was significantly upregulated, suggesting that miR-71might be involved in the process of virus infection. The results showed that the WSSV infection led to the increase of shrimp autophagy. Thus, miR-71might play an important role in the virus infection by the regulation of autophagy.Autophagy (autophgy) is a process that cells engulf their own proteins or organelles, and fusion them with lysosomes to degrade damaged proteins or organelles. Autophagy is a mechanism that all eukaryotic cells have to maintain homeostasis. As reported, autophagy plays a vital role in development and differentiation processes of cells. Autophagy also plays a role in innate immunity and adaptive immunity, such as resistance to the invasion of bacteria. It is found that autophagy could be induced by various stimuli (e.g., growth factors and nutrients consumption, hypoxia, drugs and radiation therapy). Many signal transduction pathways and second messengers involved in the formation have been shown to regulate the autophagy activity. Many of these pathways converge in evolutionarily conserved kinase TOR (target of rapamycin). However, there are other independent autophagy signaling pathways, especially in mammalian cells (e.g., phosphoinositide pathway). Moreover, in mammalian cells, the mechanism of autophagy regulation is not very clear.Previous studies showed that specific miRNAs could regulate autophagy. In this study, the miRNA expression profiling of hemolymph from normal shrimp and autophagy-induced shrimp were analyzed using miRNA chips. The results indicated that15shrimp miRNAs were significantly upregulated and17miRNAs significantly downregulated. It was found that the expression level of miR-71increased significantly when autophagy was enhanced. The data presented that the WSSV infection could enhance the autophagy of shrimp hemolymph, suggesting that autophagy might play an important role in WSSV infection. The results showed that miR-71could significantly enhance the level of autophagy in shrimp. Autophagy took great effects on virus infection. The findings revealed that miR-71-mediated autophagy facilitated the WSSV infection. MiR-71could regulate the immune-related genes of shrimp, and then induce the shrimp autophagy to facilitate the virus infection.