A Comprehensive Study of MicroRNA in Baculoviruses

Abstract

Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological characteristics and the biotechnological applications derived from these entities, the Baculoviridae family is an important subject of study and manipulation in the natural sciences. With the advent of RNA interference mechanisms, the presence of baculoviral genes that do not code for proteins but instead generate transcripts similar to microRNAs (miRNAs) has been described. These miRNAs are functionally associated with the regulation of gene expression, both in viral and host sequences. This article provides a comprehensive review of miRNA biogenesis, function, and characterization in general, with a specific focus on those identified in baculoviruses. Furthermore, it delves into the specific roles of baculoviral miRNAs in regulating viral and host genes and presents structural and thermodynamic stability studies that are useful for detecting shared characteristics with predictive utility. This review aims to expand our understanding of the baculoviral miRNAome, contributing to improvements in the production of baculovirus-based biopesticides, management of resistance phenomena in pests, enhancement of recombinant protein production systems, and development of diverse and improved BacMam vectors to meet biomedical demands.

Keywords: RNA folding; baculovirus; gene regulation; microRNA.

Figure 1

Scientific publications and miRBase database content related to miRNAs. Panel (A) depicts the number of publications (original articles) per year on miRNAs from different entities (except for 2023, where the results are partial). The data were obtained using the PubMed search (https://pubmed.ncbi.nlm.nih.gov/; last accessed on 3 April 2023) with the keyword “microRNA” and corresponding combinations for the entities considered. Panel (B) presents a bar graph illustrating the reported miRNA quantities in different updates of the miRBase database (https://www.mirbase.org/; last accessed on 3 April 2023) for various entities (when this information was available). This figure is original and has not been previously published.

Figure 2

miRNA-mediated silencing mechanism. The illustration depicts the canonical pathway of endogenous miRNA-mediated silencing. 1. Transcription from the cellular genome (gDNA) or a viral nucleic acid (vDNA) to generate the pri-miRNA (usually carried out by RNA polymerase II). 2, 3. Association with Drosha and Pasha proteins (DGCR8 in humans) to generate the pre-miRNA. 4. Export to the cytoplasm facilitated by Exportin5 and RanGTP. 5. Association with DICER and TRBP, which, along with the presence of Argonaut 2 (AGO2), complete miRNA maturation and form the RNA-induced silencing complex (RISC). 6. Binding of the miRISC to the target transcript (typically in the 3′ UTR of an mRNA). 7. Degradation of the target transcript or inhibition of its translation; reduction in target RNA molecules and/or a decrease in the production of the encoded protein (in the case of mRNA), leading to subsequent effects on biological functions. As the protein machinery involved in RNA interference in the “baculovirus/insect” system is derived from host genes, GenBank accessions of the orthologs of the main proteins are indicated in some of the lepidopterans that host baculoviruses where bac-miRs were reported: NP_001095931.1 (Ago1), NP_001036995.2 (Ago2), NP_001180543.1 (DICER-2), XP_012547603.2 (Drosha), XP_037872173.1 (Pasha), of Bombyx mori; XM_022976077.1 (AGO), AHC98010.1 (AGO2), XM_022972435.1 (DICER), XP_022826597.1 (Drosha), XP_022831748.1 (Pasha), of Spodoptera Litura; AVK59452.1 (AGO1), XP_035453543.1 (AGO2), XP_035450082.2 (DICER), XP_050556783.1 (Drosha), XP_050553060.1 (Pasha), of Spodoptera frugiperda. This figure is original and has not been previously published.

Figure 3

Bac-miR common sequential features. Box plot showing the GC percentage (A) and length (B) of the baculoviral pre-miRNAs and mature miRNAs, generated using an ad hoc script implemented with Biopython [53]. For these analyses, the sequences reported in publications where the 13 bac-miRs are described were considered. This figure is original and has not been previously published.

Figure 4

Secondary structures of baculoviral pre-miRNAs. The secondary structures of reported experimental pre-miRNAs from AcMNPV, BmNPV, AgMNPV, and SpltNPV are depicted along with their corresponding minimum free energy (MFE) values. Mature miRNA sequences are highlighted in orange. The RNAStructure server (https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html; last accessed on 20 September 2023) [54], using a temperature of 299.15 degrees Kelvin (26 °C, a probable temperature for baculovirus-infected larvae), and default parameters, was employed for generating the structures. The base pair probabilities are indicated by the colors of the bases. This figure is original and has not been previously published.

Figure 5

Conservation of secondary structures in baculoviral pre-miRNAs. The LocARNA-P server (http://rna.informatik.uni-freiburg.de/LocARNA/Input.jsp; last accessed on 20 September 2023) [55], with a temperature setting of 26 °C, was employed in this work to perform a comparative analysis of the secondary structures of miRNAs against experimentally determined structures. The generated guide tree encompasses all the sequences (A) and showcases hierarchical clustering to depict similar relationships, guiding the structural multiple alignments. This tree reveals the emergence of two distinct groups of structures that exhibit varying degrees of conservation (Group 1 and Group 2). With the identification of these two groups, the analysis was reiterated, utilizing only sequences belonging to each one (B,C), leading to the identification of structural conservation conforming to the typical pattern. Compatible base pairs are color coded to indicate sequence conservation by hue (C-G, G-C, A-U, U-A, G-U, or U-G) and structural conservation by saturation. This figure is original and has not been previously published.