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 10³ to 10⁹ virus particles per 200 μL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID₅₀) 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.

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 3₁₀ helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found thatAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vectorin vitroandin vivothat will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.

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 FRET-based 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.

Heliothis zea nudivirus 1 ( Hz NV-1 or Hz -1 virus), previously regarded as a nonoccluded baculovirus, recently has been placed in the Nudivirus genus. This virus generates Hz NV-1 HindIII-I 1 ( hhi1 ) and many other transcripts during productive viral infection; during latent viral infection, however, persistency-associated gene 1 ( pag1 ) is the only gene expressed. In this report, we used transient expression assays to show that hhi1 can trigger strong apoptosis in transfected cells, which can be blocked, at least partially, by the inhibitor of apoptosis genes Autographa californica iap2 (Ac-iap2 ) and H. zea iap2 ( Hz-iap2) . In addition to these two genes, unexpectedly, pag1 , which encodes a noncoding RNA with no detectable protein product, was found to efficiently suppress hhi1 -induced apoptosis. The assay of pro- Sf -caspase-1 processing by hhi1 transfection did not detect the small P12 subunit at any of the time intervals tested, suggesting that hhi1 of Hz NV-1 induces apoptosis through alternative caspase pathways.

The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus ( Ac MNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin ( ori ) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143 -3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143 -3 fragment showed that subfragment p143 -3.2a contained the essential sequence of this putative ori . Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143 -3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori . Further study showed that the p143 -3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions ( hr s), the major baculovirus ori , or non- hr ori s during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral ori s, viral proteins are generally required for their proper replication in host cells. However, the p143 -3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCE Baculovirus DNA replication relies on both hr and non- hr ori s; however, so far very little is known about the latter ori s. Here we have identified a new non- hr ori , the p143 ori , which resides in the coding region of p143 . By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori . This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.