Chinese sacbrood virus mediates m6A modification to target and suppress the expression of hemolymph maintenance gene AF9, exacerbating bee infections

ABSTRACT The Chinese sacbrood virus (CSBV) severely threatens the beekeeping industry, wherein 3- to 5-day-old larvae in the critical differentiation stage are highly susceptible to low levels of CSBV exposure. Once infected, larvae cannot undergo normal pupation, but the pathogenic mechanism remains unclear. Previous studies have shown that m6A modification plays an important regulatory role in larval development during the critical differentiation stage. However, it is unknown whether CSBV infection affects the pupation of honeybee larvae by altering m6A modification. Here, a novel immunoregulatory factor, AF9, was identified in honeybee larvae through combined methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis following CSBV infection. Enzyme-linked immunosorbent assay (ELISA) quantification of m6A methylation in total RNA and MeRIP-qPCR further revealed that CSBV infection of honeybee larvae inhibits the expression of AF9 via m6A modification, thereby hindering the host innate immune response and promoting CSBV replication. MeRIP-qPCR was then used to demonstrate that AcMETTL3 targets and modifies AF9 mRNA, thereby inhibiting AF9 expression. Homology and functional analysis of human-derived AF9 (MLLT3) suggested that AF9 exerted a similar effect as MLLT3 on honeybee hemolymph functioning. dsRNA was then fed to silence genes, followed by RNA extraction and expression analysis from hemolymph. Downregulation of AF9 expression led to decreased numbers of live cells in the hemolymph of honeybee larvae and a reduction in phenoloxidase activity, thereby inhibiting the host immune response. Finally, an Apis mellifera pupation infection model was constructed to further explore the antiviral activities associated with AmAF9. AmAF9 exerted a similarly significant antiviral effect against deformed wing virus (DWV) and acute bee paralysis virus (ABPV) infections in Apis mellifera pupae. These results indicate that CSBV infection promotes overall m6A modification in the host and inhibits the expression of AF9 through AcMETTL3 targeting, leading to host immunosuppression and exacerbating honeybee infection. Similarly, AF9 is stably expressed in Apis mellifera and exhibits the same antiviral effect, making it a broad-spectrum target in honeybee viruses. IMPORTANCE The Chinese sacbrood virus (CSBV) poses a serious threat to the health of Apis cerana colonies, yet its specific pathogenic mechanism remains unclear. This study shows that infection with CSBV can enhance overall m6A modification levels in Apis cerana larvae and suppress the expression of AF9 by promoting targeting of AcMETTL3, thereby inhibiting the innate immune response and exacerbating CSBV infection. Further analyses indicated that AF9 functions similarly as the mammalian homologous gene MLLT3 by maintaining normal functions of hemolymph. Moreover, AF9 can also significantly inhibit infections by common Apis mellifera viruses. In summary, a new mechanism is detailed here by which CSBV escapes the host’s innate immune response by enhancing m6A modification to target and suppress the immune response gene AF9 . This study also provides new insights into the mechanisms by which bee viruses inhibit host immune responses and suggests that AF9 may serve as a potential new broad-spectrum antiviral target in bees.