Efficient Production of Enterovirus 71 (EV71) Virus-like Particles by Controlling Promoter Strength in Insect Cells.

This study was conducted to efficiently produce virus-like particles (VLPs) of enterovirus 71 (EV71), a causative virus of hand, foot, and mouth disease (HFMD). The expression level of the P1 precursor, a structural protein of EV71, was modified to increase VLP production, and the optimal expression level and duration of the 3CD protein for P1 cleavage were determined. The expression level and duration of 3CD were controlled by the p10 promoter, which was weakened by repeated burst sequence (BS) applications, as well as the OpIE2 promoter, which was weakened by the insertion of random untranslated region sequences of various lengths. The cleavage and production efficiency of the P1 precursor were compared based on the expression time and level of 3CD, revealing that the p10-BS5 promoter with four repeated BSs was the most effective. When P1 and 3CD were expressed using the hyperexpression vector and the p10-BS5 promoter, high levels of structural protein production and normal HFMD-VLP formation were observed, respectively. This study suggests that the production efficiency of HFMD-VLPs can be significantly enhanced by increasing the expression of the P1 precursor and controlling the amount and duration of 3CD expression.

Baculovirus entire ORF1629 is not essential for viral replication.

It is generally accepted that ORF1629 is essential for baculovirus replication, which has enabled isolation of recombinant viruses in a baculovirus expression system using linearized viral DNA. ORF1629-defective viruses cannot replicate in insect cells; only recombinant virus with complete ORF1629 restoration by recombination can propagate, allowing for pure isolation and the development of bacmids for easy selection of recombinant viruses. We inadvertently found proliferation in insect cells of a bacmid lacking a complete ORF1629. PCR indicated no other viruses but a lack of complete ORF1629 in the proliferated bacmid, suggesting that the baculovirus propagated without a complete ORF1629. Lack of ORF1629 decreased the virus growth rate and yield; it also increased the occlusion body (OB) size but decreased its yield. These results were confirmed for Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV). Thus, entire ORF1629 is not essential for viral replication, though it does affect the virus growth rate, yield, and size and OB production.

Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System.

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins.

Complete genomic sequences and comparative analysis of Mamestra brassicae nucleopolyhedrovirus isolated in Korea.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected M. brassicae (Lepidoptera: Noctuidae) larvae in Korea. The full genome sequences of MabrNPV-K1 were determined, analysed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,710 bp and had an overall G + C content of 39.9%. Computer-assisted analysis predicted 158 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Two inhibitor of apoptosis (iap) and six baculovirus repeated ORFs were interspersed in the MabrNPV-K1 genome. The unique MabrNPV-K1 ORF133 was identified in the MabrNPV-K1 genome that was not previously reported in baculoviruses. The gene content and arrangement in MabrNPV-K1 had the highest similarity with those of Helicoverpa armigera MNPV (HearMNPV) and Mamestra configurata NPV-B (MacoNPV-B), and their shared homologous genes were 99% collinear. The MabrNPV-K1 genome contained four homologous repeat regions (hr1, hr2, hr3 and hr4) that accounted for 3.3% of the genome. The genomic positions of the four MabrNPV-K1 hr regions were conserved among those of HearMNPV and MacoNPV-B. The gene parity plot, percent identity of the gene homologues and a phylogenetic analysis suggested that these three viruses are closely related not only to each other but also to the same virus strains rather than different virus species.

Hyper-enhanced production of foreign recombinant protein by fusion with the partial polyhedrin of nucleopolyhedrovirus.

To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.

Characterization of Beauveria bassiana MsW1 isolated from pine sawyers, Monochamus saltuarius.

Monochamus saltuarius is a vector for pine wilt disease that causes enormous damage to native pine trees in Korea. To develop a biological control method for this pine wilt disease vector, an entomopathogenic fungus was isolated from the cadaver of an adult M. saltuarius supporting fungal conidiation. This fungus was named MsW1 and identified as Beauveria bassiana by microscopic examination, PCR amplification using B. bassiana -specific primers and genetic sequencing of the ITS and EF1-α regions. Virulence tests against M. saltuarius were conducted with conidial suspensions (1 × 10(8) conidia/ml) of B. bassiana MsW1 in laboratory conditions. The median lethal times (LT(50)) of adults and larvae were 7.2 and 7 days, and 100% mortality was observed at 11 and 13 days after inoculation, respectively. This is the first characterization of B. bassiana from M. saltuarius.