Shaofang Yu

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. yipinghong

Summary.Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yipinghong with yellowing at the leaf edge in China. Purified viral particles of 28–30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5′ untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3′ UTR, with an identical terminal motif (5′-AAAAUAAAACC-3′). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.

Autographa californica multiple nucleopolyhedrovirus odv-e66 is an essential gene required for oral infectivity.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e66 is a core gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E66. The N-terminal 23 amino acid of the envelope protein ODV-E66 are sufficient to direct native and fusion proteins to induced membrane microvesicles and the viral envelope during infection with AcMNPV. In this study, an odv-e66-knockout bacmid can not express N-terminal hydrophobic domains was constructed via homologous recombination in Escherichia coli. The odv-e66 deletion had no effect on budded virus (BV) production and viral DNA replication in infected Sf9 cells. Larval bioassays demonstrated that injection of odv-e66 deletion BV into the hemocoel could kill P. xylostella larvae as efficiently as repaired and control viruses; however, odv-e66 deletion mutant resulted in a 50% lethal dose that was 10(3) higher than that of the repaired and control viruses when inoculated per os. These results indicated that ODV-E66 envelope protein most likely played an important role in the oral infectivity of AcMNPV, but is not essential for virus replication.

The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication.

Construction of a BmNPV polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, Bombyx mori

The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.

Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV)

In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection, a large amount of polyhedra appear in the infected cell nucleolus, these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment. To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV, two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh+) Bac-to-Bac system, designated as vBmBac(polh+)-enhanced green fluorescent protein (EGFP) and vBmBac(polh+)-LacZ, which can express the polyhedrin and foreign protein simultaneously. Light microscopy analysis showed that all viruses produced polyhedra of normal appearance. Green fluorescence can be apparently detected on the surface of the vBmBac(polh+)-EGFP polyhedra, but not the BmNPV polyhedra. Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra. As expected, the vBmBac(polh+)-LacZ polyhedra contained an amount of LacZ and had a higher β-galactosidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra. This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.

The formation of occlusion-derived virus is affected by the expression level of ODV-E25.

Odv-e25 is a core gene of baculoviruses and encodes a 25.5 kDa protein located on both budded virus (BV) and occlusion-derived virus (ODV). Our previous study demonstrated that ODV-E25 was required for the formation of intranuclear microvesicles and ODV, and an odv-e25 deletion mutant could be rescued by re-expression of odv-e25 under its native promoter. To investigate the functions of ODV-E25 expression level on ODV formation, the promoter of ie-1 (pIE1), the odv-e25 native promoter, and the polyhedrin promoter (pPH) were used to direct odv-e25 expression. Our results showed that the production of ODV-E25 under its native promoter was higher than that under pIE1 but lower than that under pPH. Viral DNA replication and budded viruses (BVs) production showed that expression of odv-e25 under pIE1 and pPH could not completely repair the defects caused by the deletion of ODV-E25, while expression under its native promoter did. Electron microscopy showed that intranuclear microvesicles were found in all the constructs transfected cells except the odv-e25-null virus. However, mature ODVs only were detected in cells transfected with virus in which odv-e25 was expressed under its native or polyhedrin promoter. These results indicated that the formation occlusion-derived virus was affected by the expression level of ODV-E25.