Functional Analysis of a Cellular MicroRNA in Insect Host-Ascovirus Interaction

Abstract

MicroRNAs (miRNAs) have emerged as key regulators in many biological processes, from development to defense, at almost all organismal levels. Recently, their role has been highlighted in pathogen-host interactions. Emerging evidence from a variety of virus-host systems indicates that cellular as well as virally encoded miRNAs influence viral replication. Here, we report changes in expression levels of host miRNAs upon ascovirus infection in an insect cell line and investigated the role of a host miRNA, Hz-miR24, in the host-virus system. We found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Experimental evidence demonstrated that Hz-miR24 downregulates ascoviral DNA-dependent RNA polymerase and its ␤ subunit transcript levels late in infection. The specific miRNA-target interactions were investigated and confirmed using the ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Further, the expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. To our knowledge, this is the first cellular miRNA reported to interact with an insect virus.

An Ascovirus-Encoded RNase III Autoregulates Its Expression and Suppresses RNA Interference-Mediated Gene Silencing

“…Nevertheless, other possible role(s) for the RNase III protein cannot be ruled out (e.g., interfering with microRNA biogenesis). Recently, we showed that expression of a subset of host cell (HzFB) microRNAs (miRNA) was downregulated after HvAV-3e infection at 24 and 48 hpi when RNase III expression is at its peak and later upregulated (18). If the protein is involved in the degradation of miRNA duplexes, then its function has to be specific against certain subset of miRNAs rather than blocking the process in general.…”

miR-281, an abundant midgut-specific miRNA of the vector mosquito Aedes albopictus enhances dengue virus replication

“…Based on the mfe scores, one potential miR-281 target was found: nt37-nt55 (mfe = −21.4 kcal/mol), which is within the 5′-UTR SLA structure of the DENV-2 genome ( Figure 10A). To investigate the interaction between the seed region of miR-281 and the predicted biding site in the 5′-UTR, we used an EGFP plasmid-based reporter system in the C6/36 cell line [22,30,31]. The DENV 5′-UTR and the mutant 5′-UTR sequence (see Methods) were cloned upstream of the EGFP gene in the pIB expression vector, resulting in the constructs pIB/5UTR-EGFP (target-5UTR) and pIB/mut-5UTR-EGFP (mut-5UTR), The RT-qPCR data were analyzed using the 2 -Δct method.…”

Multiple miRNAs jointly regulate the biosynthesis of ecdysteroid in the holometabolous insects, Chilo suppressalis

“…It should be noticed that 1351 miRNAs were from 12 Drosophila species, which accounted for about half of known insect miRNAs (Kozomara and Griffiths-Jones 2014). Many studies to elucidate the functions of miRNAs in regulating a variety of insect physiological processes have been carried out, such as in metamorphosis development (Sokol et al 2008;Gomez-Orte and Belles 2009;Ling et al 2014;Belles 2017;Wu et al 2017), cell growth (Wang et al 2010), behavior (Cohen et al 2017), sex determination (McJunkin and Ambros 2017), oogenesis , embryogenesis (Jannot et al 2016), immunity (Zhu et al 2013), insect-pathogen interactions (Hussain and Asgari 2010;Asgari 2013;Wu et al 2016), etc. Several miRNAs have been reported to regulate insect metamorphosis by targeting genes either in the ecdysone cascade or the JH cascade (Zhang et al 2009;Belles 2017).…”