Studies in our laboratory are focused on understanding the biology and pathology of viral infections. We study a group of viruses called "baculoviruses" which are large DNA viruses that have been used for biological control of insect pest species. In addition, baculoviruses have been developed as powerful gene expression systems for producing eukaryotic proteins in both research and industrial applications. More recently, baculoviruses have been examined as potential vectors for human gene therapy. A major thrust of our work is focused on basic studies of the glycoproteins found in the envelopes of baculoviruses, and the roles of specific envelope proteins in viral attachment, entry, and exit. We also have interests in the evolutionary origins of viral envelope proteins and the acquisition of envelope protein genes by viruses. Another major research focus in our lab is the study of viral gene expression. We are using model baculovirus promoters, a recently developed genetic knockout system, and a viral microarray to examine the regulation of early and late phases of viral gene expression.
Baculoviruses are large DNA viruses that infect and are highly pathogenic to a number of very important insect pest species.
Because they represent a highly host-specific and thus environmentally friendly alternative to chemical methods of insect control, baculoviruses will likely comprise an important component of future strategies for biological control in both agriculture and forestry. Baculoviruses have also been developed and used extensively in research, serving as invaluable expression vectors for high level production of recombinant proteins. In addition, baculoviruses serve as convenient model systems for studies of virus entry and exit, transcriptional regulation, DNA replication, and other host-pathogen interactions.
Viral entry and exit. To enter a host cell, the envelope of the baculovirus must fuse with the host plasma membrane, releasing the nucleocapsid (which carries the viral genome) into the cell. Membrane fusion occurs at a low pH, during a process known as receptor-mediated endocytosis. In early studies, we found that a viral envelope glycoprotein known as GP64, was necessary and sufficient for the low pH-activated membrane fusion activity that occurs during endocytosis. We also demonstrated that the GP64 protein serves as a viral attachment protein at the cell surface (at the first step of cellular entry). Our studies also showed that the GP64 protein is involved in virus assembly during the final phase of the infection cycle, when progeny virus particles bud from the cell surface. Current studies are focused on understanding how the structure and organization of this important protein facilitate these diverse and essential functions. For these studies, we are using a variety of physical, biochemical, genetic, and molecular techniques to identify and define specific functional domains of the GP64 protein. Of particular importance, we recently developed a GP64 knockout virus, and a system for replacing the essential wild type gp64 gene with mutant forms of gp64. These powerful molecular tools are allowing us to perform detailed studies of the roles of this envelope protein, and the functions of specific protein domains - all in the context of the viral infection, as well as with infected host animals (insects).
Viral gene expression. We are also examining the regulation of baculovirus gene expression. The large genomes of baculoviruses may encode over 150 genes and these genes are expressed in two major phases, early and late. Early phase genes are transcribed by the host cell’s RNA polymerase II, whereas late phase genes are transcribed by a viral encoded have focused on the identification of sequences regulating early and late viral transcription, and on host and viral regulatory proteins that interact with viral regulatory sequences. We are currently using a genetic knockout system to examine the roles of specific late expression factor (LEF) genes in regulating late gene transcription. A major goal of these studies is to more broadly understand the regulation of viral gene expression and to develop regulatory paradigms within this virus family. By studying DNA sequence motifs and the host and viral proteins required for the activation and modulation of baculovirus gene expression, we are developing a better understanding of the intricate mechanisms regulating gene expression from these large viral genomes.
Viruses make great villains. They infect cells with ruthless efficiency, commandeering and sometimes destroying their hosts to reproduce themselves before moving on to the next victim.
But can these highly-evolved killing machines be made to work to our advantage? That's the question Gary Blissard is helping to answer. He works with baculoviruses, which can liquefy their insect hosts in a matter of days, but don't induce so much as a sneeze in mammals. “They're like the Ebola virus of the insect world,” Blissard explains. Once a baculovirus gains entry to an insect cell, it hijacks its host's genetic machinery and produces massive quantities of proteins to build more viruses. Baculoviruses can easily enter mammal cells, too, but once inside the virus is stymied by the differences in the gene expression system. Researchers hope that with some tweaking, baculoviruses could be used in gene therapy to induce the production of needed proteins in patients with a defective gene. Specialized baculoviruses could also replace chemicals in controlling some crop pests and disease-carrying mosquitoes, thus reducing the impact on farm workers and the environment.
First, though, scientists must learn more about what makes baculoviruses tick. To that end, Blissard studies what enables them to get into and out of cells. His lab is now characterizing a crucial protein, GP64, which the virus uses both to fuse with and enter the host cell, and to assemble new viruses at the end of its infection cycle. Using microarrays, Blissard's group is also learning what proteins are needed at which stages of the infection cycle, and how the virus makes them at the right time. Ultimately, what they learn may be used to recruit baculoviruses—at least in modified form—to our side.
Certain viruses are our allies in the fight against insect pests. Research that leads to a better understanding of the viral infection process could in turn lead to more environmentally friendly, natural insect control.
Among other research projects,Gary Blissard is studying how certain viruses, called baculoviruses, infect insects. He and his colleagues have focused on how a particular baculovirus envelope protein, called GP64, enables the virus to invade an insect cell, insert its DNA into the cell, and then multiply and exit in massive numbers.
Blissard’s group has found that GP64 has three major functions in the viral infection cycle. First, they showed that GP64 is an attachment protein – a protein that enables the virus to bind to receptors on the surface of the host insect cell,which is the first step in the process of infection. In 2007, Blissard’s lab identified the particular portion of GP64 that is responsible for this binding activity.
After the virus binds to the host cell, it enters the cell where it is surrounded by the cell’s membrane. To cause infection, the virus must fuse with that membrane and deliver its DNA into the cell nucleus. Having proved that GP64 is independently able to fuse membranes, Blissard’s team is now investigating how this process occurs.
The third step in the infection cycle calls for new virus particles to emerge, or “bud,”from the cell surface. To determine whether GP64 played a role in virus budding, Blissard’s lab “knocked out” the gene for the GP64 protein, which severely limited virus budding, and the remaining new virus particles were not infectious. These studies show that GP64 plays a critical role in the assembly of the new virus particles. Current studies aim to understand these three major functions of GP64 in much greater detail.
Knowing how baculoviruses infect insect cells may enable scientists to improve the virus’ insect control capabilities, which could reduce the use of chemical pesticides. But this work also has other exciting applications, such as in gene therapy. Because baculoviruses cause disease only in insects and because they are highly effective at entering cells and depositing DNA in the cell nucleus, they may be excellent vehicles for inserting beneficial new genes into mammalian cells – an advance that could improve our ability to safely correct genetic disorders in humans.
