Titer determination is a prerequisite for the study of viruses. However, the current available methods are tedious and time-consuming. To improve the efficiency of titer determination, we have developed a rapid and simple method for the routine detection of baculovirus titers using a quantitative real-time PCR. This method is based on the amplification of approximately 150-bp fragments located in the coding regions of selected genes. The PCR was found to be quantitative in a range of 10(3) to 10(9) virus particles per 200 microL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID(50)) of baculovirus. This quantitative real-time PCR requires only 30 min to perform, and the entire titer determination can be accomplished within 1 h without the need for cell seeding or further virus dilution and infection. Because this technology is easy to operate, generates data with high precision, and most importantly is very quick, it will certainly be broadly applied for titer determination of baculoviruses in the future.

In recent years, baculovirus has emerged as a tool for high-efficiency gene transfer into mammalian cells. However, the level of gene expression is often limited by the strength of the mammalian promoter used. Here, we show that the baculovirus RING protein IE2 is a strong, promiscuous trans-activator in mammalian cells, dramatically upregulating the cytomegalovirus (CMV) promoter in both Vero E6 and U-2OS cells. Further study of the cellular mechanism for the activation led to the discovery of a novel IE2 nuclear body structure which contains a high concentration of G-actin and closely associates with RNA polymerase II, PML, and SUMO1. IE2 mutagenesis studies indicated that the RING and coiled-coil domains of IE2 were necessary for nuclear body formation, as well as for strong activation of the CMV promoter in mammalian cells. Overall, this study shows that the IE2 trans-activator could significantly advance the use of baculovirus in mammalian gene transfer and protein production.

A DNA sequence upstream from the polyhedrin gene of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) was found to activate strongly the expression of full or minimal promoters derived from AcMNPV and other sources. Promoters tested included the minimal CMV (CMVm) promoter from human cytomegalovirus, the full heat shock 70 promoter from Drosophila, and the minimal p35 promoter from baculovirus. Deletion and mutagenesis analyses showed that this functional polyhedrin upstream (pu) activator sequence contains three open reading frames (ORFs), ORF4, ORF5, and lef2. In plasmid transfection assays, the pu sequence was able to confer high level luciferase expression driven by all of these full or minimal promoters in insect Sf21 cells. A known baculovirus enhancer, the homologous region (hr) of AcMNPV, further enhanced the expression of these promoters. Experiments showed that although multiple hr sequences function in an additive manner, pu and hr together function synergistically, resulting in as much as 18,000-fold promoter activation. Furthermore, a modified CMVm promoter containing pu and/or hr was inserted into the baculovirus genome to drive the luciferase coding region. The CMVm promoter expressed luciferase much earlier, and although it expressed a bit less than did the p10 promoter, the CMVm promoter gave rise to greater luciferase activity. Therefore, we have uncovered a cryptic viral sequence capable of activating a diverse group of promoters. Finally, these experiments demonstrate that synthetic sequences containing pu, hr, and different full or minimal promoters can generate a set of essentially unlimited novel promoters for weak to very strong expression of foreign proteins using baculovirus.

The baculovirus-insect cell system has long been deployed for a variety of applications including for use as biopesticides, for recombinant protein production, transient transgene expression, tissue therapy, and for vaccine production. Apart from the advantage of large-scale heterologous protein production with appropriate eukaryotic post-translational modification, foreign proteins can also be displayed on the viral envelope. This surface-display technology preserves the native multimeric structure of the protein, thereby expanding the clinical and pharmaceutical utility of the baculovirus system. Recombinant baculoviruses displaying major antigens for human or animal viruses can serve as appropriate vaccines. They can also serve as effective diagnostic platforms and various cell-based assay systems. In this review, we discuss progress in applying baculovirus surfacedisplay, including protein display on the envelope, capsid, and occlusion bodies of baculoviruses, as well as on cells. We will also describe strategies for improvement of this biotechnological approach.

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7 % cell lysis, whereas L-BEVS showed 60 % lysis of cells. The quality of protein expressed in both N-and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRETbased in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.