The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells.

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

NISES-AnPe-428 cell line derived from the Chinese oak silkworm Antheraea pernyi is permissive for multiple nucleopolyhedrovirus species from insects of four different families.

The cell line NISES-AnPe-428 (AnPe), derived from the Chinese oak silkworm Antheraea pernyi, was characterized for its permissiveness and productivity for six different nucleopolyhedrovirus (NPV) species. These NPVs included homologous Antheraea pernyi NPV (AnpeNPV) and heterologous Autographa californica multiple NPV (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea MNPV (HycuMNPV), Spodoptera exigua MNPV (SeMNPV), and Lymantria dispar MNPV (LdMNPV), representing viruses that had been isolated from insect species belonging to five different families (Saturniidae, Noctuidae, Bombycidae, Arctiidae, and Lymantriidae). We found that AnPe cells supported productive replication of AnpeNPV, AcMNPV, BmNPV, HycuMNPV, and SeMNPV to varying degrees. Upon infection with SeMNPV, a subset of AnPe cell population in the culture underwent apoptosis, while remaining cells produced limited amounts of progeny virions and polyhedra. AnPe cells were refractory to LdMNPV infection and failed to support replication of viral DNA, indicating that viral replication was restricted at or prior to the step of viral DNA replication. These results indicated that AnPe cells have the potential to provide excellent systems for studying the molecular mechanisms underlying cellular permissiveness for NPV replication and host-range determination of NPVs.

Identification of the minimal AcMNPV P143 protein region responsible for triggering apoptosis and rRNA degradation of Bombyx mori cells.

Bombyx mori cells induce antiviral responses including global protein synthesis shutdown, rRNA degradation, and apoptosis upon infection with Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We previously demonstrated that five and six amino acid residues located at positions between 514 and 599 of AcMNPV P143 (Ac-P143) protein are important for induction of apoptosis and rRNA degradation, respectively. However, it remains unexplored whether other residues of Ac-P143 protein also participate in antiviral immune responses. Here, we conducted transient expression analysis using a number of Ac-P143 protein deletion and truncation mutants and found that some of the N-terminal 413 residues (amino acids 1-413), besides previously identified residues between amino acids 514 and 599, are indispensable, whereas C-terminal 622 residues (amino acids 600-1221) are dispensable, for Ac-P143 protein to induce apoptosis or rRNA degradation. In addition, we found that the N-terminal 413 sequence (amino acids 1-413) of Ac-P143 protein can be substituted with corresponding BmNPV P143 (Bm-P143) protein sequence. Further analysis demonstrated that mutant Ac-P143 protein consisting of 275 residues (amino acids 325-599), but not 274 residues (amino acids 326-599) lacking glutamine residue at position 325 (Q325), is sufficient for triggering apoptosis and rRNA degradation of B. mori cells. These 275 residues are located outside the region of DNA helicase motifs of Ac-P143 protein, indicating that induction of apoptosis or rRNA degradation occurs independently of viral DNA replication-related function of the Ac-P143 protein. Moreover, Ac-P143(325-599/Q325A) and Ac-P143(1-599/Q325A) proteins harboring Q325A substitution retain the ability to induce apoptosis and rRNA degradation in B. mori cells. These findings suggest that the Ac-P143 protein needs minimal sequence length starting from the Q325 residue that contains a specific effector domain to induce apoptosis and rRNA degradation.

Novel genes participating in the formation of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution and RNA interference knockdown.

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.