Bombyx Mori Cypovirus Research Articles

Identification and characterization of two putative microRNAs encoded by Bombyx mori cypovirus

Viral microRNAs (miRNAs) have been demonstrated to play important roles in virus-host interactions. Some RNA virus-encoded miRNAs have been reported to promote viral replication and may be used as potential drug targets. Bombyx mori cypovirus (BmCPV), an important pathogen of silkworm, is a double-stranded RNA virus frequently causing serious damages in sericulture. Research on miRNA encoded by BmCPV may be useful to elucidate the BmCPV-host interaction and to develop new anti-viral methods. In our previous study, small RNA libraries of the midgut of BmCPV-infected silkworm have been generated by deep sequencing and several BmCPV-encoded putative miRNAs were predicted. In this study, two putative miRNAs encoded by BmCPV were identified and then validated by stem-loop qRT-PCR and northern blot. They are BmCPV-miR-3 encoded by the third genomic RNA segment of BmCPV (478–497bp) and BmCPV-miR-5 encoded by the fifth genomic RNA segment (2481–2500bp), both are 20bp and encoded by ORF regions. miRNA expression could be detected as early as 5h after BmCPV infection, and the expression level of BmCPV-miR-3 is much higher than that of BmCPV-miR-5 in the course of infection. Three potential target genes were predicted in the host genome, two for BmCPV-miR-3 and one for BmCPV-miR-5, but just one in the virus genome for BmCPV-miR-3 only, with the binding sites all in coding regions. Dual luciferase assay and qRT-PCR indicated that BmCPV-miR-3 could down-regulate the expression of both its two target genes, but no regulatory effect by BmCPV-miR-5 on its target gene was detected. In contrast, BmCPV-miR-3 could up-regulate the viral target. This is the first report that an insect double stranded RNA virus may generate miRNAs and the results obtained will benefit the future study of the functions of BmCPV-encoded miRNAs on viral replication and virus-host interaction.

Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

Receptor-mediated endocytosis using a β1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

Nonvirus encoded proteins could be embedded into Bombyx mori cypovirus polyhedra

To explore whether the nonvirus encoded protein could be embedded into Bombyx mori cypovirus (BmCPV) polyhedra. The stable transformants of BmN cells expressing a polyhedrin (Polh) gene of BmCPV were constructed by transfection with a non-transposon derived vector containing a polh gene. The polyhedra were purified from the midguts of BmCPV-infected silkworms and the transformed BmN cells, respectively. The proteins embedded into polyhedra were determined by mass spectrometry analysis. Host derived proteins were detected in the purified polyhedra. Analysis of structure and hydrophilicity of embedded proteins indicated that the hydrophilic proteins, in structure, were similar to the left-handed structure of polyhedrin or the N-terminal domain of BmCPV structural protein VP3, which were easily embedded into the BmCPV polyhedra. The lysate of polyhedra purified from the infected transformation of BmN cells with modified B. mori baculovirus BmPAK6 could infect BmN cells, indicating that B. mori baculovirus could be embedded into BmCPV polyhedra. Both the purified polyhedra and its lysate could be coloured by X-gal, indicating that the β-galactosidase expressed by BmPAK6 could be incorporated into BmCPV polyhedra. These results suggested that some heterologous proteins and baculovirus could be embedded into polyhedra in an unknown manner.

Bone regeneration by polyhedral microcrystals from silkworm virus

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

Characterization of the complete genome segments from BmCPV-SZ, a novelBombyx moricypovirus 1 isolate

A novel Bombyx mori cypovirus 1 isolated from infected silkworm larvae and tentatively assigned as Bombyx mori cypovirus 1 isolate Suzhou (BmCPV-SZ). The complete nucleotide sequences of genomic segments S1-S10 from BmCPV-SZ were determined. All segments possessed a single open reading frame; however, bioinformatic evidence suggested a short overlapping coding sequence in S1. Each BmCPV-SZ segment possessed the conserved terminal sequences AGUAA and GUUAGCC at the 5' and 3' ends, respectively. The conserved A/G at the -3 position in relation to the AUG codon could be found in the BmCPV-SZ genome, and it was postulated that this conserved A/G may be the most important nucleotide for efficient translation initiation in cypoviruses (CPVs). Examination of the putative amino acid sequences encoded by BmCPV-SZ revealed some characteristic motifs. Homology searches showed that viral structural proteins VP1, VP3, and VP4 had localized homologies with proteins of Rice ragged stunt virus , a member of the genus Oryzavirus within the family Reoviridae. A phylogenetic tree based on RNA-dependent RNA polymerase sequences demonstrated that CPV is more closely related to Rice ragged stunt virus and Aedes pseudoscutellaris reovirus than to other members of Reoviridae, suggesting that they may have originated from common ancestors.

Infection of Bombyx mori Cypovirus 1 in the BmN Cell Line.

Infection of Bombyx mori Cypovirus 1 in the BmN Cell Line.

Assembly into single-shelled virus-like particles by major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1.

The major capsid protein VP1 encoded by genome segment S1 of Bombyx mori cypovirus 1 was expressed in a baculovirus system. In the absence of any other capsid proteins, VP1 was found to assemble into single-shelled virus-like particles. The VP1 particles were more sensitive to acidic conditions than were intact particles.

Molecular characterization of segments 7–10 of Dendrolimus punctatus cytoplasmic polyhedrosis virus provides the complete genome

The nucleotide sequences of genomic segments S7–S10 from Dendrolimus punctatus cypovirus strain Hunan (DpCPV-Hn) have been determined. This provides the complete genome sequences of DpCPV-Hn. Each segment of S7–S10 possess a single segment each. Homology searches showed that the nucleotide sequences and the deduced amino acid sequences of DpCPV S7–10 had high level of identities with those of Bombyx mori cypovirus (BmCPV) S7–10, respectively. While the amino acid sequences of the proteins encoded by DpCPV S7 and S8 have low identities with those of the proteins encoded by type 14 Lymantria dispar cypovirus S7 and S8, respectively. DpCPV S7 encodes viral structural protein VP5, S8 and S9 encode viral non-structural proteins, and S10 encodes polyhedrin gene, according to the function of the genome segments of BmCPV. There are glutamic-acid-rich and proline-rich domains in the central region of DpCPV S8 encoded protein. A nuclear localization signal was found in the protein encoded by DpCPV S9. Phylogenetic analysis of RNA-dependent RNA polymerases from nine viruses of the family Reoviridae and polyhedrin from eight viruses of the genus Cypovirus indicate that DpCPV is a type 1 cypovirus, more closely related to BmCPV than to other cypovirus species. These results also support the classification of CPV groups based on the electrophoretic migration of genomic dsRNA.

Comparison of the amino acid sequences of RNA-dependent RNA polymerases of cypoviruses in the family Reoviridae.

The nucleotide sequences of the genome segment S2 of Bombyx mori cypovirus 1, S2 of Lymantria dispar cypovirus 1, S1 of Lymantria dispar cypovirus 14 and S1 of a proposed new electropherotype of Trichoplusia ni cypovirus 15 were determined. These segments encoded putative RNA-dependent RNA polymerases (RDRPs). The deduced amino acid sequences of RDRPs within the genus Cypovirus showed 32% to 94% identities, while extent of homology between RDRPs in the genera Cypovirus and Oryzavirus, a genus most closely related, was approximately 26% identity. Both the genera Cypovirus and Oryzavirus might have originated from a common insect virus ancestor.

Nucleotide sequence of genome segment 5 from Bombyx mori cypovirus 1.

The complete nucleotide sequences of the double-stranded RNA genome segments 5 (S5) from Bombyx mori cypovirus 1 (BmCPV-1) strains I and H were determined. The segments consisted of 2,852 nucleotides encoding putative proteins of 881 amino acids with molecular masses of approximately 101 kDa (p101). A homology search showed that p101 has high similarity (93%) to foot-and-mouth disease virus (FMDV) 2A protease (2Apro) at amino acid position 219 to 235. These findings suggest the possibility that p101 encoded by BmCPV-1 S5 might be cleaved into two non-structural proteins by post-translational autocleavage involving a 2Apro-like protease.

Nucleotide sequences of genome segments 6 and 7 of Bombyx mori cypovirus 1, encoding the viral structural proteins V4 and V5, respectively.

Nucleotide sequence analyses of cDNAs derived from the double-stranded RNA genome segments 6 and 7 (S6 and S7) of Bombyx mori cypovirus 1 (BmCPV-1) have revealed that they consist of 1796 and 1501 nucleotides encoding putative proteins of 561 and 448 amino acids with molecular masses of 63604 and 49875 (p64 and p50), respectively. The amino acid sequence of p64, which has a high leucine residue content (10%), contains a leucine zipper motif. Antiserum raised against p64 specifically bound to a viral structural protein of ca. 68 kDa (V4), while antiserum against p50, which specifically bound to a protein of ca. 56 kDa in BmN4 cells infected with BmCPV-1, reacted with a cluster of four viral structural proteins ranging from ca. 34 to 40 kDa (V5). These observations indicate that p50 might be cleaved to V5 during the formation of virus particles.