METABOLISM OF INTRAVENOUS METHYLNALTREXONE IN MICE, RATS, DOGS AND HUMANS

were observed in rats. Dogs produced only one metabolite, MNTX-3-glucuronide (M9). In conclusion, MNTX was not extensively metabolized in humans. Conversion to methyl-6-naltrexol isomers (M4 and M5) and MNTX-3-sulfate (M2) were the primary pathways of metabolism in humans. MNTX was metabolized to a higher extent in mice than in rats, dogs, and humans. Glucuronidation was a major metabolic pathway in mice, rats and dogs, but not in humans. Overall, the data suggested species differences in the metabolism of MNTX

A novel chimeric RNA originating from BmCPV S4 and Bombyx mori HDAC11 transcripts regulates virus proliferation.

Polymerases encoded by segmented negative-strand RNA viruses cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching") to generate chimeric RNA, and trans-splicing occurs between viral and cellular transcripts. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an RNA virus belonging to Reoviridae, is a major pathogen of silkworm (B. mori). The genome of BmCPV consists of 10 segmented double-stranded RNAs (S1-S10) from which viral RNAs encoding a protein are transcribed. In this study, chimeric silkworm-BmCPV RNAs, in which the sequence derived from the silkworm transcript could fuse with both the 5' end and the 3' end of viral RNA, were identified in the midgut of BmCPV-infected silkworms by RNA_seq and further confirmed by RT-PCR and Sanger sequencing. A novel chimeric RNA, HDAC11-S4 RNA 4, derived from silkworm histone deacetylase 11 (HDAC11) and the BmCPV S4 transcript encoding viral structural protein 4 (VP4), was selected for validation by in situ hybridization and Northern blotting. Interestingly, our results indicated that HDAC11-S4 RNA 4 was generated in a BmCPV RNA-dependent RNA polymerase (RdRp)-independent manner and could be translated into a truncated BmCPV VP4 with a silkworm HDAC11-derived N-terminal extension. Moreover, it was confirmed that HDAC11-S4 RNA 4 inhibited BmCPV proliferation, decreased the level of H3K9me3 and increased the level of H3K9ac. These results indicated that during infection with BmCPV, a novel mechanism, different from that described in previous reports, allows the genesis of chimeric silkworm-BmCPV RNAs with biological functions

Interaction between Bombyx mori Cytoplasmic Polyhedrosis Virus NSP8 and BmAgo2 Inhibits RNA Interference and Enhances Virus Proliferation

ABSTRACT Some insect viruses encode suppressors of RNA interference (RNAi) to counteract the antiviral RNAi pathway. However, it is unknown whether Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) encodes an RNAi suppressor. In this study, the presence of viral small interfering RNA (vsiRNA) in BmN cells infected with BmCPV was confirmed by small RNA sequencing. The Dual-Luciferase reporter test demonstrated that BmCPV infection may prevent firefly luciferase (Luc) gene silencing caused by particular short RNA. It was also established that the inhibition relied on the nonstructural protein NSP8, which suggests that NSP8 was a possible RNAi suppressor. In cultured BmN cells, the expressions of viral structural protein 1 (vp1) and NSP9 were triggered by overexpression of nsp8, suggesting that BmCPV proliferation was enhanced by NSP8. A pulldown assay was conducted with BmCPV genomic double-stranded RNA (dsRNA) labeled with biotin. The mass spectral detection of NSP8 in the pulldown complex suggests that NSP8 is capable of direct binding to BmCPV genomic dsRNA. The colocalization of NSP8 and B. mori Argonaute 2 (BmAgo2) was detected by an immunofluorescence assay, leading to the hypothesis that NSP8 interacts with BmAgo2. Coimmunoprecipitation further supported the present investigation. Moreover, vasa intronic protein, a component of RNA-induced silencing complex (RISC), could be detected in the coprecipitation complex of NSP8 by mass spectrum analysis. NSP8 and the mRNA decapping protein (Dcp2) were also discovered to colocalize to processing bodies (P bodies) for RNAi-mediated gene silencing in Saccharomyces cerevisiae. These findings revealed that by interacting with BmAgo2 and suppressing RNAi, NSP8 promoted BmCPV growth. IMPORTANCE It has been reported that the RNAi pathway is inhibited by binding RNAi suppressors encoded by some insect-specific viruses belonging to Dicistroviridae, Nodaviridae, or Birnaviridae to dsRNAs to protect dsRNAs from being cut by Dicer-2. However, it is unknown whether BmCPV, belonging to Spinareoviridae, encodes an RNAi suppressor. In this study, we found that nonstructural protein NSP8 encoded by BmCPV inhibits small interfering RNA (siRNA)-induced RNAi and that NSP8, as an RNAi suppressor, can bind to viral dsRNAs and interact with BmAgo2. Moreover, vasa intronic protein, a component of RISC, was found to interact with NSP8. Heterologously expressed NSP8 and Dcp2 were colocalized to P bodies in yeast. These results indicated that NSP8 promoted BmCPV proliferation by binding itself to BmCPV genomic dsRNAs and interacting with BmAgo2 through suppression of siRNA-induced RNAi. Our findings deepen our understanding of the game between BmCPV and silkworm in regulating viral infection