Introduction

Atlastins belong to the dynamin superfamily, which is involved in several physiological functions of endomembrane in eukaryotic cells1, including formation of transport vesicles2, mitochondrial fusion and fission3, membrane fusion4 and pathogen resistance5. Nearly a decade ago, genetic mutation of Atlastin was identified as a cause of a form of autosomal dominant hereditary spastic paraplegia (HSP), which is characterized by progressive lower-extremity weakness and spasticity6. Atlastin has an N-terminal conserved GTPase domain, a three-helix-bundle (3HB) middle domain, two tandem transmembrane (TM) segments and a C-terminal cytoplasmic tail (CT)7. Current studies have reported that Atlastin is required for the formation and maintenance of ER networks8 and mediates the homotypic ER fusion and vesicle trafficking in the ER/Golgi interface9. In Drosophila melanogaster, overexpression of Atlastin causes expansion of the ER membranes and inhibition of Atlastin results in ER fragmentation10. The depletion of Atlastin2/3 can cause unbranched ER tubules in Hela cells8; in Xenopus egg extracts, Atlastin antibodies inhibit formation of the ER network11. Atlastin-1 is highly expressed in the brain and is enriched in growth cones, also benefits axon elongation during neuronal development12,13. Danio rerio Atlastin also controls larval mobility and spinal motor axon growth by inhibiting the bone morphogenetic protein (BMP) pathway14.

Atlastin has been found widely in many different species. Some of them, such as Homo sapiens, Xenopus laevis and Danio rerio, have three Atlastin isoforms. In others, such as Drosophila melanogaster and Caenorhabditis elegans, only one is present15. Bombyx mori, the silkworm, has four known Atlastin homeotic genes16. Silkworms are an important lepidopteran insect and its silk is an essential biological material in both textiles and medicine17,18,Preparation of BmAtlastin-n antibody (anti-BmAtlastin-n)

The primers (forward 5′ cgggatccAGTCTCGGTGTGAAGCCAAAGG 3′ and reverse 5′ cggaattcCGCCTTGTTGTGGAGTGAATC 3′) were used to amplify fragments from vector pMD19-BmAtlastin-n and then constructed with pET32a-c (+) (Novagen). Vector pET32a-BmAtlastin-n was transformed into BL21 and induced by 0.5 mM IPTG at 37 °C for 5 h. The recombinant protein was purified by Ni affinity chromatography and the products were used for the immunization of New Zealand white rabbits (0.4 mg/each). Immune treatments were performed once per week for a total of four times and then the serum was collected to prepare antibody of BmAtlastin-n.

Western blotting

Cells were lysed with lysis buffer (Beyotime, China) followed by washing twice with PBS and denatured at 100 °C with 5 × SDS-PAGE Loading Buffer (Beyotime). Proteins were subjected to 12% SDS–PAGE and the bands were transferred to PVDF membranes. They were then incubated with primary antibody anti-BmAtlastin-n/anti-VP39/anti-α-Tubulin (Beyotime) (1/5000, 1 h, 25 °C) and secondary antibody HRP conjugated anti-rabbit IgG/HRP conjugated anti-mouse IgG (Beyotime). The final results were analyzed with ECL Western Blotting Detection System (Bio-Rad).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 15 min at 25 °C and permeated in 0.5% Triton X-100 (10 min, 25 °C), then blocked with Immunol Staining Blocking Buffer (Beyotime, 1 h, 37 °C). Cells were incubated with anti-BmAtlastin-n primary antibody (1:200, 1 h, 37 °C). Alexa Fluor 555 conjugated donkey anti-rabbit polyclonal (Beyotime) was used as the secondary antibody. Then the cells were imaged under a laser scanning confocal microscope (Olympus).

Microinjection and screening

The transgenic vector piggyBac [3 × P3-EGFP, IE1P-BmAtlastin-n] and the helper vector pHA3PIG were co-injected into the silkworm eggs within 4 h of spawning using a microinjector (Eppendorf)37,38. Then the G0 silkworms were reared at 25 °C. The G1 silkworms were produced by inbreeding and backcrosses of G0. BmAtlastin-n-OE transgenic silkworms were selected in G1 broods using a fluorescent stereomicroscope (Olympus), which were found to be EGFP positive in their compound eyes.

Analysis of the insertion site

The genomic DNA was extracted from BmAtlastin-n-OE transgenic silkworm and digested with Hae III for 16 h at 37 °C, then self-ligated in solution I (Takara). The products were used for performing the inverse PCR with the primers pBacL (forward 5′ ATCAGTGACACTTACCGCATTGACA 3′ and reverse 5′ TGACGAGCTTGTTGGTGAGGATTCT 3′) and pBacR (forward 5′ TACGCATGATTATCTTTAACGTA 3′ and reverse 5′ GTACTGTCATCTGATGTACCAGG 3′)34 and the fragments were cloned into pMD19-T vector (Takara) and sequenced. The results were analyzed in silkDB (http://www.silkdb.org/silkdb/).

Oral inoculation and injection of BmNPV

Fresh mulberry leaves were cut into circles 1 cm in diameter, then each piece was smeared with 1 × 106 ODVs. First-day fourth-instar larvae of BmAtlastin-n-OE transgenic silkworm and Dazao control silkworm were fed mulberry leaves with ODVs (one piece/larva). The silkworms, which each ate a whole piece of mulberry leaf, were collected for assessment of the mortality rate. Then a capillary tube was used to inject 1 × 106 BV particles/larva in the two lines form valve. Each treatment was performed in triplicate and each replicate included 100 larvae.

Statistical analysis

The student’s t-test was used to assess any statistically significant differences between treatments. P-value < 0.05 was considered significant, here indicated with “*” and P-value < 0.01 was considered very significant, here indicated with “**”. Data from three independent experiments are here presented as means ± SEM.

Additional Information

How to cite this article: Liu, T.- et al. A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in Bombyx mori. Sci. Rep. 6, 28946; doi: 10.1038/srep28946 (2016).