Peptidoglycan Recognition Protein S5 of Bombyx mori Facilitates the Proliferation of Bombyx mori Cypovirus 1.

Bombyx mori cypovirus 1 (BmCPV1), a primary pathogen of the silkworm, is a typical dsRNA virus belonging to the Reoviridae family. In this study, a total of 2520 differentially expressed genes (DEGs) were identified by RNA-seq analysis of the silkworm midgut after BmCPV1 infection and Gene Ontology (GO) functional annotation showed that the DEGs predominantly functioned in binding (molecular function), cell (cellular component), and cellular processes (biological process). Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation revealed that the DEGs were mainly distributed in global and overview metabolism maps, translation, and signal transduction. Among the identified DEGs, BmPGRP-S5 belongs to the peptidoglycan recognition protein (PGRP) family. Previous studies have revealed that PGRPs were involved in the interactions between silkworm and BmCPV1. Here, we explored the effect of BmPGRP-S5 on BmCPV1 replication and demonstrated that BmPGRP-S5 promotes the proliferation of BmCPV1 in BmN cells through overexpression or knockdown experiments. Knocking down of BmPGRP-S5 in silkworm larvae similarly promoted the proliferation of BmCPV1. Through experimental validation, we therefore determined that BmPGRP-S5 acts as a proviral host factor for BmCPV1 infection. This study clarifies the proliferation mechanism of BmCPV1 and provides new insights into the functional role of BmPGRP-S5.

Characterization of virus-like particles assembled by co-expression of BmCPV capsid shell protein and large protrusion protein.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the Reoviridae family of the Cypovirus genus. Previous studies have shown that the BmCPV major capsid shell protein (CSP) has the ability to self-assemble into virus-like particles (VLPs), and cryo-electron microscopy of the BmCPV virions has revealed a tight mutual binding region between CSP and another capsid protein known as the Large Protrusion Protein (LPP), which further stabilizes the capsid shell. In this study, the multi-gene baculovirus expression system, Ac-MultiBac, was used to produce both solely CSP-based and CSP-LPP co-assembled VLPs. Transmission electron microscopy (TEM) results showed that addition of LPP did not affect the assembly of VLPs resulting in almost identical structure in both cases. However, ex vivo administration of VLPs to silkworm midgut tissue showed that CSP-based VLPs did not induce a significant transcriptional response in the innate immunity and RNAi gene cascades, compared to the co-assembled CSP-LPP based VLPs and the natural BmCPV virions isolated from polyhedra. The experimental results indicate that CSP and LPP attach tightly ("Plug and Display" model with CSP acting as "catcher" and LPP as "tag") to form VLPs that have a structure similar to that of the native CPV virions. Moreover, our results showed that the formation of VLPs with the two BmCPV capsid proteins is feasible, which can form the basis for the production of BmCPV-based VLPs as a new type of biological material to display exogenous proteins.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the genus Cypovirus in the family Reoviridae. The results of cryo-electron microscopy showed that the BmCPV capsid consists of 60 asymmetric units, and each asymmetric unit contains one turret protein (TP), two large protrusion proteins (LPP) and two capsid shell proteins (CSP). CSP has the ability to self-assemble into virus-like particles (VLPs), and the small protrusion domain (SPD) in CSP may play an essential role in the assembly of viral capsids. In this study, three critical amino acid sites, D828, S829 and V945, in the SPD were efficiently mutated (point mutation) based on the principle of PCR circular mutagenesis. Moreover, a multi-gene expression system, Ac-MultiBac baculovirus, was used to produce eight different recombinant VLPs in vitro. Transmission electron microscopy showed that the single site and double site mutations had little effect on the efficiency and morphology of the assembly of VLPs. Still, the simultaneous mutation of the three sites had a significant impact. The experimental results demonstrate that the SPD of CSP plays an essential role in assembly of the viral capsid, which lays the foundation for further analysis of the molecular and structural mechanism of BmCPV capsid assembly.

Alkaline phosphatase can promote the replication of Bombyx mori cypovirus 1 by interaction with its turret protein.

Bombyx mori cypovirus 1 (BmCPV1) is a member of the Reoviridae family which is characterized by its single-layered capsid. Similar with other turreted viruses in the Reoviridae, transcription of BmCPV1 occurs inside the capsid, and the nascent mRNA is released to the turret which consists of five turret proteins (TPs) and located at the 5-fold axis of the outer capsid, then the capping enzyme TP will guanylate and methylate the nascent viral mRNA to produce a matured mRNA. However, during these processes, how the BmCPV1 draws other cellular proteins to facilitate its replication is still lesser-known. Here we used an ELISA to investigate the interaction between ALP and BmCPV1. A co-immunoprecipitation technique was employed to detect the interaction of ALP with the Methylase domain of TP. We further studied whether ALP affects the replication of BmCPV1 inside the cell, results show that reducing the expression of ALP through RNAi reduced the transcription level of the BmCPV1 VP1 gene, which was increased by overexpression of ALP. In summary, our data demonstrate an interaction between ALP and BmCPV1 and that ALP promoted the replication of BmCPV1, and support our hypothesis of the ALP is an RTPase to facilitate the capping process of BmCPV1.