Our laboratory focuses on the studies of gene expression and protein engineering using insect and mammalian systems. One of the major molecular biological tools used is baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). This is a rod-shaped insect-specific virus with a dsDNA genome of 130 kb. It is a non-human infectious virus and thus safe and easy for manipulation. Baculovirus has long been used for the expression of engineered proteins in insect cells for animal vaccine, medical studies and biotechnological applications. More and more evidences in recent years showed baculovirus can produce engineered proteins, especially vaccines, with very good quality. It can also be used as a safe and efficient tool for gene delivery in mammalian cells and organisms. In our laboratory, we are not only manipulating baculovirus for better expression of engineered proteins, but also applying it as a versatile tool for molecular biological studies. We have found that a baculovirus gene product, IE2, which can recruit G-actin and RNA polymerase II to form a novel micro-machine, for high level transcription of target genes in mammalian cells. We have also found that human miRNA can interact with viral dsDNA as a novel triplex structure to control viral DNA replication. Recently, we have further used baculovirus as pseudotyped viruses for the safe study of detrimental human viruses, and cooperated with vaccine companies for practical applications.
Our research work and various findings are described as follows:
Influenza virus is a family of RNA viruses, individual virus contains eight RNAs which encodes for 11 proteins including hemagglutinin (HA), neuraminidase (NA). HA is known to mediate virus entry into the cells and is the major antigen to stimulate antibodies against it. HA is produced by the cell as a monomer; however it must be assembled as a trimmer on the surface of the virus to be functional. Antisera which can recognize the steric trimeric structure are thus crucial for serotype detection. Previously, we have successfully expressed HA protein from flu (USP 7,527,967 patent, 2009) and spike protein from coronavirus (Chang et al., 2004) onto the surface of baculovirus as functional trimeric HA and as a pseudotyped virus. The HA-displaying baculovirus (HA-Bac) can serve as a pseudotyped flu virus for antiserum production, the sera neutralization tests and vaccine potency assay. Purified HA1 was known to be useful for human vaccination, however, the purified HA lacks proper steric conformation, and cannot properly recognize its receptor, also, lost the recognition of neutralizing antibodies. HA-Bac, which displays trimeric HAs, can be purified easier together with the virus particle as convenient antigen for animal vaccination. These baculovirus-based pseudotyped viruses can also be used as a useful tool for the safe study of influenza viruses. Similar strategy can be extended to Ebola, SARS, MERS, and many other highly detrimental human and animal viruses as safe pseudoviruses for academic studies or industrial applications with great future.
We have identified baculovirus IE2 as a strong activator to stimulate CMV promoter expression in mammalian cells (Fig. 2, 3). The mechanism by which IE2 activates CMV promoter expression in mammalian cells was further studied. We found that IE2 can form a unique clathrate cage-like structure, which encloses G-actin and further recruits activated RNA polymerase II. This clathrate cage-like structure (CCLS) can serve as a very strong micro-machine to generate large amounts of mRNA for gene expression (Fig. 2). Since the CCLS is a unique machinery for gene expression visible at the light microscopic level, the mechanism by which IE2 and G-actin work in concert with RNA polymerase II could be a notable emerging subject in the molecular biological studies. Also, since IE2 can strongly enhance baculovirus-mediated gene expression in the mammalian cells (Fig. 3), these studies also make baculovirus as a useful tool for gene delivery in the mammalian cells.
MicroRNAs are a class of small RNAs so far known to function as post-transcriptional regulators in cellular processes such as mRNA degradation, translational repression, and deadenylation. Recently, we have identified a group of miRNAs that can recognize and bind directly to the replication origin of DNA and repress DNA replication (Fig. 4), thus uncovering a new function for miRNA. Since miRNA is very likely to regulate DNA replications in many viral and cellular DNA replication origins, this novel discovery is very important for the future study of DNA replication as controlled or mediated by miRNA. Now, the important question is to identify what is the mechanism mediate miRNA to recognize the origin of DNA replication, what are proteins involve in this novel mechanism. The results will be very fruitful, and extend to many replication origins of cells and viruses in the future.
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