Functional Regulation of an Autographa californica Nucleopolyhedrovirus-Encoded MicroRNA, AcMNPV-miR-1, in Baculovirus Replication

Abstract

An Autographa californica nucleopolyhedrovirus-encoded microRNA (miRNA), AcMNPV-miR-1, downregulates the ac94 gene, reducing the production of infectious budded virions and accelerating the formation of occlusion-derived virions. In the current study, four viruses that constitutively overexpress AcMNPV-miR-1 were constructed to further explore the function of the miRNA. In addition to the ac94 gene, two new viral gene targets (ac18 and ac95) of AcMNPV-miR-1 were identified, and the possible interacting proteins were verified and tested. In the context of AcMNPV-miR-1 overexpression, ac18 was slightly upregulated, and ac95 was downregulated. Several interacting proteins were identified, and a functional pathway for AcMNPV-miR-1 was deduced. AcMNPV-miR-1 overexpression decreased budded virus infectivity, reduced viral DNA replication, accelerated polyhedron formation, and promoted viral infection efficiency in Trichoplusia ni larvae, suggesting that AcMNPV-miR-1 restrains virus infection of cells but facilitates virus infection of larvae. IMPORTANCERecently, microRNAs (miRNAs) have been widely reported as moderators or regulators of mammalian cellular processes, especially disease-related pathways in humans. However, the roles played by miRNAs encoded by baculoviruses, which infect numerous beneficial insects and agricultural pests, have rarely been described. To explore the actions of virus-encoded miRNAs, we investigated an miRNA encoded by Autographa californica nucleopolyhedrovirus (AcMNPV-miR-1). We previously identified this miRNA through the exogenous addition of AcMNPV-miR-1 mimics. In the current study, we constitutively overexpressed AcMNPV-miR-1 and analyzed the resultant effects to more comprehensively assess what is indeed the function of this miRNA during viral infection. In addition, we widely explored the target genes for the miRNA in the viral and host genomes and proposed a possible functional network for AcMNPV-miR-1, which provides a better general understanding of virus-encoded miRNAs. In brief, our study implied that AcMNPV-miR-1 constrains viral replication and cellular infection but enhances larval infection. MicroRNAs (miRNAs) are short RNAs ϳ22 nucleotides (nt) in length that are derived from transcripts that fold back on themselves to form distinctive hairpin structures (1). miRNAs modulate the stability and/or translational potential of their mRNA targets (2, 3) to achieve regulatory control over virtually every biological process. In many cases, a single miRNA can regulate abundant targets (4, 5), and one mRNA can interact with several miRNAs (6). Therefore, the network formed between miRNAs and their target mRNAs is comprehensive and complex.Over the last decade, there have been many developments in the understanding of virus-encoded miRNAs. In 2005, miRNAs encoded by simian vacuolating virus 40 (SV40) were reported to reduce the expression of viral T antigens (7). Since then, several virus-encoded miRNAs have been reported. For example, miR-BART, which is encoded by Epstein-B...

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“…miRNAs also play important roles in baculovirus infection and virus–host interaction. Virally encoded miRNAs have been found in baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) ( Zhu et al, 2013 ; Zhu M. et al, 2016 ), Bombyx mori nucleopolyhedrovirus ( Singh et al, 2010 , 2012 , 2014 ), and Spodopera litura nucleopolyhedrovirus ( Kharbanda et al, 2015 ), etc., and shown to regulate virus gene expression and replication. Meanwhile, baculovirus infection also alters the expression profiles of host miRNA, suggesting their function in virus–host interactions ( Mehrabadi et al, 2013 ; Shi et al, 2016 ).…”

Characterization of miRNAs encoded by Autographa californica nucleopolyhedrovirus

“…To verify a candidate target as a true target of one of the miRNAs, the recombinant plasmid psiCHECK-target was constructed by inserting the predicted miRNA binding site region into the psiCHECK-2 vector downstream of the Renilla luciferase gene. We also constructed a psiCHECK-target-mut plasmid with mutated binding sites and a psiCHECK-A3 plasmid with the B. mori actin 3 CDS inserted into the psiCHECK-2 vector as a control ( Fig 5) [8,13].…”

“…The predicted candidate targets have been verified by dual-luciferase reporter assay (details can refer to the section of Materials and Methods). * Targets for AcMNPV-miR-1 and -miR-3 have been reported previously [8,12,13]. a referenced from [19].…”

“…These AcMNPV encoded miRNAs predominantly down-regulate target genes expression. However, up-regulation also exists, such as AcMNPV-miR-1 up-regulating ac18 [13], though it is not common. Nevertheless, down-or up-regulation of viral miRNA targets depends on the function of the target gene and serves for the ultimate aim to achieve infection.…”

“…AcMNPV-miR-1 can down-regulate viral gene ac94, reducing the production of infectious budded virions (BVs) and accelerating the formation of occlusion-derived virions (ODVs) [8]. In 2016, we further reported two more target genes (ac95 and ac18) of AcMNPV-miR-1, and depicted the function pathway of this miRNA [13]. As for AcMNPV-miR-3, it mainly down-regulates viral gene ac101, meanwhile, it also regulates other five viral genes [12].…”