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.
Selected PublicationsThe Transcriptome of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus in Trichoplusia ni Cells. J Virol 87: (11) 6391-405 doi: 10.1128/JVI.00194-13. Epub 2013 Mar 27
Dong, S. and Blissard, G.W. 2012. Functional analysis of the Autographa californica multiple nucleopolyhedrovirus GP64 terminal fusion loops and interactions with membranes.. J Virol 86: 9617-9628
Hashimoto, Y., Zhang, S., Zhang, S., Chen, Y.R. and Blissard, G.W. 2012. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins. BMC Biotechnology 12: 12
Li, Z. and Blissard, G.W. 2012. Cellular VPS4 is required for efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus. J Virol 86: 459-472
Su, J., Lung, O. and Blissard, G.W. 2011. The Autographa californica multiple nucleopolyhedrovirus lef-5 gene is required for productive infection. Virology 416: 54-64
Li, Z. and Blissard, G.W. 2011. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Roles of histidine residues in triggering membrane fusion and fusion pore expansion. J Virol 85: 12492-12504
Li, Z. and Blissard, G.W. 2010. Baculovirus GP64 disulfide bonds: the intermolecular disulfide bond of Autographa californica multicapsid nucleopolyhedrovirus GP64 is not essential for membrane fusion and virion budding. J Virol 84: 8584-8595
Hashimoto, Y., Zhang, S. and Blissard, G.W. 2010. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins. BMC Biotechnol 10: 50
Li, Z. and Blissard, G.W. 2009. The pre-transmembrane domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein is critical for membrane fusion and virus infectivity. J Virol 83: 10993-11004.
Li, Z. and Blissard, G.W. 2009. The Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: analysis of transmembrane domain length and sequence requirements. J Virol 83: 4447-4461
Theilmann, D.A. and Blissard, G.W. 2008. Baculoviruses: Molecular Biology of Nucleopolyhedroviruses. In Encylopedia of Virology (Mahy, B.W.J. and van Regenmortel, M. eds). Oxford 0: Elsevier
Li, Z. and Blissard, G.W. 2008. Functional analysis of the transmembrane (TM) domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: Substitution of heterologous TM domains. Journal of Virology 82: 3329-3341
Zhou, J. and Blissard, G.W. 2008. Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct. Journal of Virology 82: 1368-1377
Zhou, J. and Blissard, G.W. 2008. Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus. Journal of Virology 82: 4449-4460
Yamagishi, J., E. D. Burnett , S. H. Harwood, G. W. Blissard. 2007. The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts. Virology 365: 34-47
Granados, R.R., G. Li, G.W. Blissard. 2007. Insect cell culture and biotechnology. Virol. Sinicia 365: 34-47
Zhou, J., G. W. Blissard. 2006. Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein. 352: 427-437
Jehle, J.A., G.W. Blissard, B.C. Bonning, J.S. Cory, E.A. Herniou, G.F. Rohrmann, D.A. Theilmann, S.M. Thiem, J.M.Vlak. 2006. On the classification and nomenclature of baculoviruses: A proposal for revision. Arch. Viology 151: 1257-1266
Sinn, P. L., E. R. Burnight, M. A. Hickey, G. W. Blissard, P. B. McCray. 2005. Persistent Gene Expression in Mouse Nasal Epithelia Following Feline Immunodeficiency Virus-based Vector Gene Transfer. Journal of Virology 79: 12818-12827
Lung, O., G. W. Blissard. 2005. A Cellular Drosophila melanogaster Protein with Similarity to Baculovirus F Envelope Fusion Proteins. Journal of Virology 79: 7979-7989
Theilmann, D.A., G.W. Blissard, B. Bonning, J.Jehle, D.R. O'Reilly, G.F. Rohrmann, S. Thiem, J.M. Vlak. 2005. Baculoviridae. In: H. V. Van Regenmortel, D.H.L. Bishop, M. H. Van Regenmortal and C.M. Fauquet (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, Elsevier, NY. 0: 177-185
Zhang, S. X. X., Y. Han, G. W. Blissard. 2003. Palmitoylation of the Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64: mapping, functional studies, and lipid rafts. Journal of Virology 77: 6265-6273
Lung, O. Y., M. Cruz Alvarez, G. W. Blissard. 2003. Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, is a viral pathogenicity factor. Journal of Virology 77: 328-339
Lin, G., G. W. Blissard. 2002. Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication. Journal of Virology 76: 2770-2779
Lin, G., G. W. Blissard. 2002. Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6-Null Virus: LEF-6 is not essential for viral replication but appears to accelerate late gene transcription. Journal of Virology 76: 5503-5514
Lung, O., M. Westenberg, J. M. Vlak, D. Zuidema, G. W. Blissard. 2002. Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64. Journal of Virology 76: 5729-5736
Lin, G., G. Li, R. R. Granados, G. W. Blissard. 2001. Stable cell lines expressing baculovirus P35: Resistance to apoptosis and nutrient stress, and increased glycoprotein secretion. In Vitro Cellular & Developmental Biology - Animal 37: 293-302
Lin, G., J. M. Slack, G. W. Blissard. 2001. Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus infected Sf9 cells. Journal of General Virology 82: 2289-2294
Mangor, J. T., S. A. Monsma, M. C. Johnson, G. W. Blissard. 2001. A GP64null baculovirus pseudotyped with Vesicular Stomatitis Virus G protein. Journal of Virology 75: 2544-2556
Slack, J. M., G. W. Blissard. 2001. Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells. Journal of General Virology 82: 2519-2529
IJkel, W. F. J., M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, D. Zuidema. 2000. A Novel Baculovirus Envelope Fusion Protein with a Proprotein Convertase Cleavage Site. Virology 275: 30-41
The Good Virus
feature released -2009
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.
How do viruses infect insect pests?
feature released -2008
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.
The Enemy Within.
feature released -2006
Viruses need to evolve fast to stay one step ahead of their hosts’ defenses. One shortcut they use is to steal genes from their hosts, then modify them for their own purposes.
Virologists know this because they’ve found many analogs of viral genes in the genomes of their hosts. Baculoviruses multiply in the nuclei of host cells. While replicating, they sometimes mix their DNA with that of their insect host cell, giving them a chance to copy or steal a host gene before departing for fresh prey.
Many mysteries remain surrounding viral evolution, however, including the genesis of the baculovirus fusion proteins that Gary Blissard’s lab studies. Called F and GP64, these proteins are the virus’ Swiss Army Knives. They allow the virus to fuse with and gain entry to hosts by binding to proteins on the cell surface, and later in the infection cycle they help assemble new viral particles and get them out of the cell.
When the fruit fly genome was sequenced, it revealed several genes that resembled the gene for the viral F protein. While most of those genes were left behind by viruses called retroviruses, one appeared to be a genuine host gene. If this turned out to be a real, functioning host gene, it would suggest that the f gene may have originally moved from an insect to a baculovirus.
To see if this was the case, Blissard’s lab investigated whether the fruit fly f gene was actually being expressed, and found that it was. The lab also observed that unlike viral F proteins, which are found on the surfaces of infected cells and later on the virus surface, host F proteins were found in small structures inside the cell.
Blissard speculates that picking up the f gene could have enabled baculoviruses to go from causing mild symptoms to aggressively liquefying their hosts within days. So it might have been a host’s own gene that turned baculoviruses into the malevolent killing machines they are today.