Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

Abstract

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. Central to the defense of insects against RNA viruses is the triggering of the so-called exogenous RNA interference pathway (exo-RNAi), by which viral double-stranded RNA (dsRNA) is cleaved intracellularly into viral small interfering RNAs (siRNAs) that in turn silence homologous transcripts (1). It is established that Dicer-2, a cytoplasmic RNase III class enzyme, recognizes and cuts successively the dsRNA template to produce small interfering RNA duplexes of ϳ21 nucleotides (nt) characterized by a signature of 2-nt 3=-hydroxyl overhangs (2). These duplexes are subsequently incorporated into the effector complex RISC (RNA-induced silencing complex) via interactions with Argonaute-2, which, upon discarding one of the two strands (passenger strand), is followed by selection and cleavage of viral sequences bearing perfect complementarity to the remaining strand (guide strand) (1, 3). Dicer-2 may act upon the genome of the virus itself-as in the case of dsRNA viruses-and/or on viral replication and transcription intermediates, although structured, single-stranded RNA (ssRNA) may also be utilized (4). Interestingly, several deepsequencing data have revealed that approximately 18-to 30-nt viral small RNAs (vsRNAs) of infected insects are not evenly distributed along the genomes but map preferentially in distinct genomic areas (hot spots) versus genomic stretches with unmapped or less-mapped vsRNAs (cold spots) (4-13). The origin of these profiles, as well as the hypothetical functional distinction between vsRNAs deriving from hot or cold spots, remains substantially elusive, although hot spots near the end of the genome have been attributed to structured regulatory viral regions (14) and an RNAi decoy-like mechanism driven by abundant vsRNAs ...

PIWI pathway against viruses in insects

“…When Aag2 cells were infected with the dsRNA virus BTV‐1, piRNA‐like molecules of viral origin were detected which however did not possess the specific sequence bias being usually observed in piRNAs (Schnettler, Ratinier, et al, ). A similar finding has also been commented on in a study of dsRNA virus (BmCPV) infection in the silkworm B. mori (Zografidis et al, ). According to Morazzani et al (), dsRNA molecules could activate the production of vpiRNA‐like molecules, possibly because specific regions are recognized as pathogen‐associated molecular patterns (PAMPs).…”

“…Finally, although the number of vpiRNA‐related studies in the order of Lepidoptera is limited, it is apparent that BmN4 cells produce BmMLV‐specific vpiRNAs with a Ping‐Pong pattern where Siwi is mainly found to bind 1U‐sense and BmAgo3 to bind preferentially 10A‐antisense vpiRNAs (Katsuma et al, ). However, neither in Bm5 cells nor in B. mori larvae was any evidence for an active Ping‐Pong mechanism detected (L. Wang et al, ; Zografidis et al, ).…”

“…In another B. mori ovary‐derived cell line, Bm5, no piRNA‐sized peak appeared among viral small RNAs corresponding to the persistently infecting BmMLV and the acutely infecting CrPV viral genomes (L. Wang et al, ). Finally, data from a previous work of our team (Zografidis et al, ) related to acute B. mori cytoplasmic polyhedrosis virus (BmCPV; Reoviridae) infection of the silkworm midgut revealed the production of vpiRNA‐sized small RNAs, which derived mainly from segment 10 of the viral genome and had a clear positive strand bias but did not possess the characteristic sequence signatures (1U, 10A) connected to the Ping‐Pong pathway.…”

Antimicrobial Peptides as Potential Antiviral Factors in Insect Antiviral Immune Response

“…Although there exist specific strains of silkworm that are resistant to some viruses, the specific mechanism is unclear ( 88 – 90 ). Like other insects, RNAi was considered as the major defense strategy against viral infections in B. mori ( 91 ). However, the antiviral innate immune response of silkworm has not been systematically studied although specific antiviral molecules such as PP2A ( 92 ), BmSTING ( 93 ), BmAtlastin-n ( 94 ), BmNOX ( 95 ), Bmlipase-1 ( 96 ), were identified.…”

The evolving world of small RNAs from RNA viruses

“…64 Deep sequencing of viral small RNAs elucidated the Lepidopteran antiviral response against infection by CrPV (Reoviridae), a segmented dsRNA virus. 65 The analysis showed that the silkworm Bombyx mori generates virus-derived siRNAs when infected by Bombyx cytoplasmic polyhedrosis virus (BmCPV). Moreover, in addition to cleavage of viral dsRNAs by Dicer, an uncharacterized RNase cleaves the viral RNA to generate vsRNAs.…”