RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.     

Contribution of the caspase gene sequence diversification to the specifically antiviral defense in invertebrate

Vertebrates achieve adaptive immunity of all sorts against pathogens through the diversification of antibodies. However the mechanism of invertebrates' innate immune defense against various pathogens remains largely unknown. Our study used shrimp and white spot syndrome virus (WSSV) to show that PjCaspase, a caspase gene of shrimp that is crucial in apoptosis, possessed gene sequence diversity. At present, the role of gene sequence diversity in immunity has not been characterized. To address this issue, we compared the PjCaspase gene sequence diversities from WSSV-free and WSSV-resistant shrimp. The sequence analysis indicated that the PjCaspase gene from the WSSV-resistant shrimp contained a special fragment, designated as fragment 3 (221-229 aa). Down-regulation or overexpression of the PjCaspase gene containing fragment 3 led to significant inhibition or enhancement of virus-induced apoptosis, but had no effect on bacterium challenge. We found evidence that the silencing or overexpression of this gene led to a 7-fold increase or 11-fold decrease of WSSV copies, respectively. Our results suggested that the PjCaspase gene containing fragment 3 provided the molecular basis for the antiviral defense of shrimp. This study represented the first report of the role of gene sequence diversity in the immunity of an invertebrate against virus infection. Invertebrates may employ this gene sequence diversity as a system to avoid pathogen interference with their immune response.     

TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp.     

Functional Analysis of a Crustacean MicroRNA in Host-Virus Interactions

Growing evidence from mammals suggests that host microRNAs (miRNAs) play important roles in the antiviral immune response. However, the roles of invertebrate miRNAs in response to virus infection remain to be investigated. Based on our previous studies, the shrimp miR-7 was found to be upregulated in response to white spot syndrome virus (WSSV) infection. In this study, the results showed that shrimp miR-7 could target the 3′-untranslated region (3′UTR) of the WSSV early gene wsv477, implying that miR-7 was involved in viral DNA replication. In insect High Five cells, the synthesized miR-7 significantly decreased the expression level of the fluorescent construct bearing the 3′UTR of wsv477 compared with the expression of the control constructs. When the activity of transfected miR-7 was blocked by locked-nucleic-acid (LNA)-modified anti-miR-7 oligonucleotide (AMO-miR-7), the repression of luciferase gene expression by miR-7 was relieved. In vivo, when the synthesized miR-7 was injected into shrimp, the numbers of WSSV genome copies/mg gills were 1,000-fold lower than those of WSSV only at 72 and 96 h postinfection. The results indicated that the blocking of endogenous miR-7 by AMO-miR-7 led to about a 10-fold increase of WSSV genome copies/mg gills in WSSV-infected shrimp compared with the control WSSV only. Further, it was revealed that the host Dicer1 was an important component for the biogenesis of miR-7, which had a large effect on virus infection. Therefore, our study revealed a novel regulatory function for an invertebrate miRNA in host-virus interactions by targeting the viral early gene.     

Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.     

Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.     

Involvement of Viral MicroRNA in the Regulation of Antiviral Apoptosis in Shrimp

Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific in vivo. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes in vivo. As a result, the number of WSSV copies in shrimp in vivo was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the in vivo molecular events of viral miRNA in antiviral apoptosis.     

The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.     

对虾抗病毒免疫研究现状

白斑综合征病毒（white spot syndrome virus,wssv）,是对虾养殖中主要的致病病毒,在全球范围内引起急性感染和致死反应,带来巨大的经济损失。之前的研究主要集中在对虾的先天免疫反应上,在抗病毒反应方面的尚所知有限。总结了近年来对虾抗病毒免疫研究取得的主要成果,概括了病毒侵染对虾后分子水平上的改变,旨在为更加有效的预防和治疗白斑综合症提供参考。     

Comparison of antiviral efficiency of immune responses in shrimp

The antiviral effectiveness of three major immune responses including phagocytosis, apoptosis and proPO system in the shrimp Marsupenaeus japonicus was characterized. It was found that the suppression of apoptosis and phagocytosis in vivo by their corresponding inhibitors resulted in the increase of white spot syndrome virus (WSSV) copies and shrimp mortality, whereas the inhibition of phenol oxidase generated the least influence on WSSV infection and shrimp mortality. These results suggest that apoptosis and phagocytosis were the essential immune responses to protect shrimp from virus infection, while the phenol-oxidase-dependent proPO system plays a comparatively minor role in antiviral defense of shrimp.     

Involvement of interaction between viral VP466 and host tropomyosin proteins in virus infection in shrimp

The accumulating evidence indicates that the viral structural proteins play critical roles in virus infection. However, the interaction between the viral structural protein and host cytoskeleton protein in virus infection remains to be addressed. In this study, the viral VP466 protein, one of the major structural proteins of shrimp white spot syndrome virus (WSSV), was characterized. The results showed that the suppression of VP466 gene expression led to the inhibition of WSSV infection in shrimp, indicating that the VP466 protein was required in virus invasion. It was found that the VP466 protein was interacted with the host cytoskeleton protein tropomyosin. As documented, the VP466–tropomyosin interaction facilitated the WSSV infection. Therefore our findings revealed a novel molecular mechanism in the virus invasion to its host, which would be helpful to better understand the molecular events in virus infection in invertebrate.     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (ie1), WSSV303 and WSSV371

The Toll-like receptor (TLR)-mediated NF-κB pathway is essential for defending against viruses in insects and mammals. Viruses also develop strategies to utilize this pathway to benefit their infection and replication in mammal hosts. In invertebrates, the TLR-mediated NF-κB pathway has only been well-studied in insects and has been demonstrated to be important in antiviral responses. However, there are few reports of interactions between viruses and the TLR-mediated NF-κB pathway in invertebrate hosts. Here, we studied Litopenaeus vannamei Pelle, which is the central regulator of the Toll pathway, and proposed that a similar TLR/MyD88/Tube/Pelle/TRAF6/NF-κB cascade may exist in shrimp for immune gene regulation. After performing genome-wild analysis of white spot syndrome virus (WSSV) encoded proteins, we found that WSSV449 shows 15.7-19.4% identity to Tube, which is an important component of the insect Toll pathway. We further found that WSSV449 activated promoters of Toll pathway-controlled antimicrobial peptide genes, indicating WSSV449 has a similar function to host Tube in activating the NF-κB pathway. We suspected that WSSV449 activated the Toll-mediated NF-κB pathway for regulating viral gene expression. To test this hypothesis, we analyzed the promoters of viral genes and found 40 promoters that possess NF-κB binding sites. A promoter screen showed that the promoter activities of WSSV069 (ie1), WSSV303 and WSSV371 can be highly induced by the shrimp NF-κB family protein LvDorsal. WSSV449 also induced these three viral promoter activities by activating the NF-κB pathway. To our knowledge, this is the first report of a virus that encodes a protein similar to the Toll pathway component Tube to upregulate gene expression in the invertebrate host.     

Viral MicroRNAs Targeting Virus Genes Promote Virus Infection in Shrimp In Vivo

Viral microRNAs (miRNAs), most of which are characterized in cell lines, have been found to play important roles in the virus life cycle to avoid attack by the host immune system or to keep virus in the latency state. Viral miRNAs targeting virus genes can inhibit virus infection. In this study, in vivo findings in Marsupenaeus japonicus shrimp revealed that the viral miRNAs could target virus genes and further promote the virus infection. The results showed that white spot syndrome virus (WSSV)-encoded miRNAs WSSV-miR-66 and WSSV-miR-68 were transcribed at the early stage of WSSV infection. When the expression of WSSV-miR-66 and WSSV-miR-68 was silenced with sequence-specific anti-miRNA oligonucleotides (AMOs), the number of copies of WSSV and the WSSV-infected shrimp mortality were significantly decreased, indicating that the two viral miRNAs had a great effect on virus infection. It was revealed that the WSSV wsv094 and wsv177 genes were the targets of WSSV-miR-66 and that the wsv248 and wsv309 genes were the targets of WSSV-miR-68. The data demonstrate that the four target genes play negative roles in the WSSV infection. The targeting of the four virus genes by WSSV-miR-66 and WSSV-miR-68 led to the promotion of virus infection. Therefore, our in vivo findings show a novel aspect of viral miRNAs in virus-host interactions.     

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.     

Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp’s immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections.