Hemagglutinin (HA) is the major immunogen on the envelope of avian influenza virus (AIV). Therefore we constructed two recombinant baculoviruses: Bac-HA, expressing histidine-tagged HA with the cytoplasmic domain (CTD) derived from HA, and Bac-HA64, expressing histidine-tagged HA with the CTD derived from baculovirus envelope protein gp64. After infection, HA with either CTD was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that both Bac-HA and Bac-HA64 displayed HA on the viral surface. However, analyses of purified viruses revealed that significantly more HA was incorporated into Bac-HA64 than into Bac-HA. In comparison with Bac-HA, Bac-HA64 significantly improved the gene delivery and transgene expression in mammalian cells, as determined by quantitative real-time polymerase chain reaction and flow cytometry. Bac-HA64 elicited significantly higher hemagglutination inhibition titers in mouse models than Bac-HA and the negative controls. These data collectively confirmed that the gp64 CTD, in comparison with HA CTD, resulted in more efficient HA incorporation into baculovirus, more efficient transgene delivery and expression, and elevated immunogenicity. This is the first report demonstrating the potential of HA-pseudotyped baculovirus as an avian influenza vaccine and that the choice of CTD tremendously affects baculovirus properties and vaccine efficacy.
To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.
It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.