Transcriptome profiling reveals infection strategy of an insect maculavirus

Abstract

Bombyx mori macula-like virus (BmMLV) is a positive, single-stranded insect RNA virus that is closely related to plant maculaviruses. BmMLV is currently characterized as an unclassified maculavirus. BmMLV accumulates at extremely high levels in cell lines derived from the silkworm, Bombyx mori, but it does not lead to lethality and establishes persistent infections. It is unknown how this insect maculavirus replicates and establishes persistent infections in insect cells. Here, we showed that BmMLV p15, which is located on a subgenomic fragment and is not found in plant maculaviruses, is highly expressed in BmMLV-infected silkworm cells and that p15 protein is required to establish BmMLV infections in silkworm cells. We also showed that two distinct small RNA-mediated pathways maintain BmMLV levels in BmMLV-infected silkworm cells, thereby allowing the virus to establish persistent infection. Virus-derived siRNAs and piRNAs were both produced as the infection progressed. Knockdown experiments demonstrated that the exogenous RNAi pathway alone or RNAi and piRNA pathways function cooperatively to silence BmMLV RNA and that both pathways are important for normal growth of BmMLV-infected silkworm cells. On the basis of our study, we propose a mechanism of how a plant virus-like insect virus can establish persistent infections in insect cells.

PIWI pathway against viruses in insects

“…On the other hand, studies in several mosquito species have provided redundant proofs that the piRNA pathway is a key mechanism in their antiviral strategy to survive otherwise lethal infections (Aguiar et al, ; Brackney et al, ; Dietrich, Jansen, et al, ; Dietrich, Shi, et al, ; Joosten et al, ; Leger et al, ; Miesen et al, ; Morazzani et al, ; Schnettler, Donald, et al, ; Schnettler, Ratinier, et al, ; Schnettler et al, ; Scott et al, ; Varjak, Maringer, et al, ; Vodovar et al, ; Y. Wang et al, ). Recently, data on the antiviral properties of piRNAs in the silkworm B. mori have also started to accumulate, showing the possibility that piRNAs could act as antivirals also in insect groups other than mosquitoes (Katsuma et al, ). However, examples of no involvement of piRNAs in the control of viral infections also accumulate, since observations in an infected C. sonorensis (midge) cell line showed that, even if PIWI components are expressed, the piRNA pathway is probably not active against the infecting viruses (Dietrich, Shi, et al, ; Schnettler, Ratinier, et al, ).…”

“…These data could indicate that flavivirus infections are relatively refractory to piRNA pathway activation. 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, ).…”

“…Siwi‐and BmAgo3‐bound virus‐specific piRNAs showed a clear sense strand bias as well as a sharp peak at the expected piRNA size (27–29 nt). For BmMLV‐derived vpiRNAs, the strong sense bias is correlated with a strong 10A antisense signature of both the Siwi‐ and BmAgo3‐bound BmMLV‐vpiRNAs; however, enrichment for 1U is reported only for the sense Siwi‐bound vpiRNAs (Katsuma et al, ). The observed differences in the frequencies of specific sequence logos between Siwi‐ and BmAgo3‐bound vpiRNAs may reflect the existence of two different types of Ping‐Pong cycle partners, that is, homotypic (Siwi:Siwi) and heterotypic (Siwi:BmAgo3) (Katsuma et al, ).…”

“…For BmMLV‐derived vpiRNAs, the strong sense bias is correlated with a strong 10A antisense signature of both the Siwi‐ and BmAgo3‐bound BmMLV‐vpiRNAs; however, enrichment for 1U is reported only for the sense Siwi‐bound vpiRNAs (Katsuma et al, ). The observed differences in the frequencies of specific sequence logos between Siwi‐ and BmAgo3‐bound vpiRNAs may reflect the existence of two different types of Ping‐Pong cycle partners, that is, homotypic (Siwi:Siwi) and heterotypic (Siwi:BmAgo3) (Katsuma et al, ). Although the production of vpiRNA‐like molecules was assumed to depend on the infecting virus but not on the infected cell line (Schnettler, Ratinier, et al, ), there are now indications that the production of viral small RNAs in lepidopteran cells can be cell line‐specific and not species‐specific.…”

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line

“…Fractionation replicates correlate very well (average Pearson correlation coe cient >0.97) ( Fig 3A) and cluster together in the rst two PCA dimensions ( Fig 3B). Within the gradient, fraction series [1][2][3], [4][5][6][7] and [8][9] are similar to each other, while fraction 10 is most different from all others. This is consistent with fraction 10 constituting an acetone precipitation of all proteins that were not separated in the previous fractionation steps.…”