Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.
The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera.


OBJECTIVE
This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.
Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens, Greece azogra@bio.demokritos.gr.
Aris
A
Zografidis
Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Science, Ghent University, Ghent, Belgium.
Filip
F
Van Nieuwerburgh
Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens, Greece.
Anna
A
Kolliopoulou
Theoretical Biology and Computational Genomics Laboratory, Institute of Biosciences and Applications, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens, Greece.
Konstantinos
K
Apostolou-Karampelis
Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, California, USA.
Steven R
SR
Head
Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Science, Ghent University, Ghent, Belgium.
Dieter
D
Deforce
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Guy
G
Smagghe
Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens, Greece.
Luc
L
Swevers
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000276:immunology* / Q000821:virology*; D041981:Gastrointestinal Tract / Q000821:virology; D016679:Genome, Viral / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions / Q000235:genetics; D007113:Immunity, Innate / Q000235:genetics* / Q000276:immunology; D007814:Larva / Q000821:virology; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000378:metabolism; D034741:RNA, Small Interfering; D012367:RNA, Viral / Q000235:genetics; D012087:Reoviridae / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D043244:Ribonuclease III / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D015678:Viral Structural Proteins / Q000235:genetics
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012367:RNA, Viral; D015678:Viral Structural Proteins; D043244:Ribonuclease III