Progress 12/01/05 to 09/30/10
Outputs
We have employed the type baculovirus species called Autographa californica multinucleocapsid nucleopolyhedrosis virus (AcMNPV) for investigations on proteins involved in baculovirus DNA replication and structure. This virus encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, the role for DBP in baculovirus replication remains unclear. To characterize DBP, we expressed it as an N-terminal His6-tag fusion using a recombinant baculovirus and purified it to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3,
for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes, and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with
subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates. We also have a project investigating insect retrovirus (errantiviruses) diversity in cells used for baculovirus-expressed vaccine production. Cloning, sequencing, and phylogenetic analysis of over 20 PCR products from two insect cell lines used for baculovirus expression demonstrated the presence of diverse populations of retrovirus sequences comprising at least seven distinct lineages. We are also collaborating with scientists in the College of Veterinary Medicine to investigate virus infections of livestock. This has led to the investigations of two previously uncharacterized viruses; one of alpacas and the other pathogenic for rabbits. We determined that the alpaca virus is a coronavirus that is closely related to a number of bovine coronaviruses. The rabbit virus is a member of the herpesviridae and is a member of a primate lineage of
these viruses.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide. Understanding the diversity of retroviruses in cell lines used for baculovirus expression may lead to an understanding of whether these viruses are present in vaccine preparations and, if they are present, whether they can transpose into or infect human and other types of cells. This information has implications for vaccine safety. The research on veterinary pathogens is important in preventing the losses caused by these viruses in farm animals.
In addition, since these viruses have not been previously characterized, understanding their biology is critical for evaluating their potential for cross infection to humans or other organisms.
Publications
- Okano, K., Vanarsdall, A.L., and Rohrmann, G.F. 2007. A Baculovirus alkaline nuclease knock out construct produces fragmented DNA and aberrant capsids. Virology 359:46-54.
- Jin, L., Cebra, C.K., Baker, R.J., Mattson, D.E., Cohen, S.A., Alvarado, E.D., Rohrmann, G.F. 2007. Analysis of the Genome Sequence of an Alpaca Coronavirus. Virology 365, 198-203.
- Vanarsdall, A.L., Mikhailov, V.S. and Rohrmann, G.F. (2007) Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene. Virology 364, 475-85.
- Vanarsdall, A.L., Pearson, M.N. and Rohrmann, G.F. (2007) Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame. Virology 367, 187-95.
- Vanarsdall, A.L., Mikhailov, V.S. and Rohrmann, G.F. (2007) Baculovirus DNA Replication and Processing Curr Drug Targets 8, 1096-1102.
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Progress 01/01/06 to 12/31/06
Outputs
We have employed the type baculovirus species called Autographa californica multinucleocapsid nucleopolyhedrosis virus (AcMNPV) for investigations on proteins involved in baculovirus DNA replication and structure. This virus encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, the role for DBP in baculovirus replication remains unclear. A DBP knockout virus was generated and found to be unable to produce budded. Real-time PCR showed that the dbp knockout produced levels of DNA less than the control virus. Analysis of the viral DNA replicated by the dbp knockout by using pulsed-field gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly
bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 hours post-infection, DBP co-localized with replicated DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma. To investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection-infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production. To better characterize the role of these genes in the baculovirus replication
cycle, quantitative DNA replication assays were performed and demonstrated that in cells transfected with the Ac 101, 142, or 144 knockouts, DNA replicated with similar kinetics as a control virus. Western blot analyses of budded virus from cells infected with the repair viruses showed that these proteins are associated with the viral nucleocapsid. Furthermore, immunoelectron microscopy of cells transfected with the knockout bacmids revealed defects in nucleocapsid production for all three constructs. From these results we concluded that the gene products encoded from these open reading frames are essential for virus production and may be involved in DNA processing, packaging, or nucleocapsid morphogenesis. We also have a project investigating insect retroviruses (errantiviruses) with particular emphasis on the relationship between their env protein and a baculovirus envelope fusion protein. We are in the process of surveying cells used for baculovirus-expressed vaccine production for
the presence of retroviruses sequences. We have found about 12 lineages of these viruses in Spodoptera frugiperda cells and also have investigated their presence in Trichoplusia ni cells.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide. The studies on the envelope fusion protein provides information on how retroviruses likely evolved from non-infectious transposable elements to infectious viruses. This likely occurred when they integrated into a baculovirus and obtained an envelope fusion protein. We are also investigating the presence of these viruses in cell lines used for human vaccine production. This information may lead to an understanding of whether these viruses are
present in vaccine preparations and, if they are present, whether they can transpose into or infect human and other types of cells. This information has implications for vaccine safety.
Publications
- Okano, K., Vanarsdall, A.L., Mikhailov, V.S., and Rohrmann, G.F. 2006. Conserved molecular systems of the Baculoviridae. Invited review. Virology,344:77-87.
- Vanarsdall, A.L., Okano, K. and Rohrmann, G.F. 2006. Characterization of the role of baculovirus very late factor-1 in capsid structure and DNA processing. J. Virol. 80: 1724-1733.
- Mikhailov, V.S., Okano, K. and Rohrmann, G.F. 2006. Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3. Virology 346: 469-478
- Pearson, M.N. and Rohrmann, G.F. 2006. Envelope gene capture and insect retrovirus evolution: the relationship between errantivirus and baculovirus envelope proteins. Virus Res., 118, 7-15.
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Progress 01/01/05 to 12/31/05
Outputs
We have employed the type baculovirus species called Autographa californica multinucleocapsid nucleopolyhedrosis virus (AcMNPV) for investigations on proteins involved in baculovirus DNA replication. One of these, very late expression factor-1 (VLF-1) is a putative tyrosine recombinase and is required for both very late gene expression and budded virus production in cell culture. We have demonstrated that a vlf-1 knockout bacmid is able to synthesize viral DNA similar to wt levels similar and is not required for synthesizing complete genomes. However without VLF-1, defective capsids are produced. Immunoelectron microscopy demonstrated that VLF-1 localized to the end region of the nucleocapsid. We have characterized the DNA replication intermediates using by field inversion gel electrophoresis (FIGE). These investigations indicated that the VLF-1 knockout produced patterns of DNA was similar to wt. In contrast, DNA generated by the alkaline nuclease knockout bacmid had
much higher concentrations of sub-genome size DNA. In addition, FIGE analysis of partially digested DNA from wt virus infected cells failed to detect viral DNA in the form of multiples of unit length suggesting that long concatemers are not synthesized. The single-stranded DNA-binding protein LEF-3 of AcMNPV consists of 385 amino acids, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not required for interaction with DNA and are readily accessible to proteolytic digestion. LEF-3 promotes both Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted
unwinding at oversaturation of DNA substrates. In addition, the balance between annealing and unwinding activities of LEF-3 is determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. We also have a project investigating insect retroviruses (errantiviruses) with particular emphasis on the relationship between their env protein and a baculovirus envelope fusion protein. We have characterized selected features of the env protein from the errantivirus Dme17.6V. These include characterization of the 21 amino acid predicted fusion peptide sequence that is highly homologous to a region of baculovirus envelope fusion proteins. We found that, although this sequence could not substitute for the homologous sequence in the baculovirus LD130 envelope fusion protein, by changing four amino acids, the hybrid construct became active for low-pH induced cell fusion. In addition, a Dme17.6V env-egfp construct was found to localize to
cell membranes.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The studies on the envelope fusion protein provide information on how retroviruses likely evolved from non-infectious transposable elements to infectious viruses. This likely occurred when they integrated into a baculovirus and obtained an envelope fusion protein. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide.
Publications
- Mikhailov, V.S., Okano, K. and Rohrmann, G.F. 2005. The Redox State of the Baculovirus Single-stranded DNA-binding Protein LEF-3 Regulates Its DNA Binding, Unwinding, and Annealing Activities. J Biol Chem 280, 29444-53.
- Vanarsdall, A.L., Okano, K. and Rohrmann, G.F. 2005. Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene. Virology, 331:175-180.
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Progress 01/01/04 to 12/31/04
Outputs
We have demonstrated the baculovirus DNA polymerase is essential for viral DNA replication and that it is unlikely that host DNA polymerases play a role in this process. We made major progress in both the biochemical and biological characterization of gene products involved in DNA replication and processing including a gene with homology to lambda integrase called very late factor 1 (VLF-1) and another with homology to exonucleases from lambda phage and herpes viruses. We characterized the role of these proteins in baculovirus biology by deleting their genes from the viral genome and then characterizing the effects of the deletions on virus replication. We were able to accomplish this by the application of bacterial artificial chromosome (BAC) technology to the study of baculoviruses. This technology allowed us to make lethal mutations to the viral genome in the context of a BAC and then investigate the effects of these mutations by transfecting the bacmid DNA into
insect cells. In addition, construction of the deletion mutants has provided us with useful tools for further investigation of the roles that the deleted genes play in DNA replication. The investigations of baculovirus replication intermediates of wt virus along with bacmids deleted for specific genes is providing information on the roles the genes play in the production of complete genomes. We have also investigated a gene encoding an envelope protein of an insect retrovirus. The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV
Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR from the Lymantria dispar nucleopolyhedrovirus fusion peptide (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env- green fluorescent protein construct localized to cell membranes, no cell fusion was observed.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The studies on the envelope fusion protein provide information on how retroviruses likely evolved from non infectious transposable elements to infectious viruses. This likely occurred when they integrated into a baculovirus and obtained an envelope fusion protein. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide.
Publications
- Pearson, M.N. and Rohrmann, G.F. 2004. Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) env protein and a baculovirus envelope fusion protein. Virology 322. 61-68
- Mikhailov, V., Okano, K. and Rohrmann, G. 2004. Specificity of the Endonuclease Activity of the Baculovirus Alkaline Nuclease for Single-stranded DNA. J. Biol. Chem., 279, 14734-14745.
- Okano, K., Vanarsdall, A.L. and Rohrmann, G.F. 2004. Characterization of a baculovirus lacking the Alkaline Nuclease Gene. J. Virol. 78. 10650-10656.
- Vanarsdall, A.L., Okano, K. and Rohrmann, G.F. 2004. Characterization of a baculovirus with a deletion of vlf-1. Virology. 326(1): 191-201.
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Progress 01/01/03 to 12/31/03
Outputs
We have been doing research on baculovirus DNA replication and homologous recombination. All baculoviruses encode and alkaline nuclease (AN) which likely participates in the maturation of virus genomes and in DNA recombination. We expressed one such AN in a recombinant baculovirus as a His6-tagged fusion and obtained in pure form (*AN), or as a complex with the baculoviral ssDNA-binding protein LEF-3 (*AN/L3). Both AN preparations possessed a potent 5 to 3 prime exonuclease and an endonuclease activities. Mutant *AN(S146A)/L3 with a change from serine to alanine at position 146 in a conservative motif was impaired in both activities. This proved that the endonuclease is an intrinsic activity of baculovirus AN. The AN endonuclease showed specificity for ssDNA and converted supercoiled plasmid DNA (Replicative Form I, RFI) into the open circular form (RFII) by a single strand break. Plasmid DNA relaxed with topoisomerase I was resistant to *AN/L3 indicating that the
partially single-stranded regions in negatively supercoiled molecules served as targets for the endonuclease. Unwinding the supercoiled DNA with ethidium bromide also made DNA resistant to AN/L3. In reaction with nicked circular DNA (RFII), AN and AN/L3 hydrolyzed exonucleolytically the broken strand or cut endonucleolytically the intact strand at position opposite the nick (gap). When LEF-3 was added to the assay, the balance between the exonucleolytic and endonucleolytic modes of hydrolysis shifted in favor of the exonuclease. The data suggest that the AN endonuclease may digest the intermediates in replication and recombination at positions of structural irregularities in DNA duplexes, whereas LEF-3 may further regulate processing of the intermediates by AN via the endonuclease and exonuclease pathways. In order to investigate the utility of using recombinant baculoviruses as fish vaccines, five fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a
recombinant baculovirus that is capable of expressing a beta-galactosidase reporter gene from the CAG promoter (consisting of a CMV enhancer element, a chicken actin promoter and rabbitbeta-globin termination sequences). TO (Tilapia ovary ), EPC (carp), CHH-1 (Chum salmon heart fibroblasts), and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ beta-galactosidase assay, whereas rainbow trout gonad (RTG-2) cells were not. We have also done research on glycoproteins that are involved in virus entry into cells. We have demonstrated that a baculovirus envelope fusion protein is related to a envelope fusion protein from a group of insect retroviruses. We have recently demonstrated that the protease cleavage site has been conserved in sequence location between the envelope proteins of these two types of viruses.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The studies on the envelope fusion protein provide information on how retroviruses likely evolved from non infectious transposable elements to infectious viruses. This likely occurred when they integrated into a baculovirus and obtained an envelope fusion protein. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide. Our research on the ability of baculoviruses to enter and express genes in fish cell lines is providing information on the applicability of using recombinant baculoviruses for fish
vaccines.
Publications
- Mikhailov, V., Okano, K. and Rohrmann, G. (2003) Baculovirus Alkaline Nuclease Possesses a 5 prime to 3 prime Exonuclease Activity and Associates with the DNA-Binding Protein LEF-3. J. Virol. 77(4), 2436-2444.
- Leisy, D., Lewis, T., Leong, J. and Rohrmann, G. (2003) Transduction of Cultured Fish Cells with Recombinant Baculoviruses. J. Gen. Virol. 84, 1173-1178.
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Progress 01/01/02 to 12/31/02
Outputs
We have been doing research on baculovirus DNA replication and homologous recombination. We have also done research on glycoproteins that are involved in virus entry into cells. We have characterized a protein called very late expression factor -1 (vlf-1) and demonstrated that it is capable of binding to cruciform structures suggesting that it may be involved in the resolution of structures generated during DNA replication. We have also characterized a baculovirus encoded nuclease and a single strand DNA binding protein that may be involved in homologous recombination. We have demonstrated that the two proteins form a complex and digest DNA in a 5'->3' direction. This activity likely generates single stranded ends of DNA fragments that then initiate recombination by strand invasion. We have also characterized a baculovirus envelope fusion protein called ld130 that is involved in allowing the virus to enter and infect cells. We have demonstrated that it is related to a
envelope fusion protein from a group of insect retroviruses.
Impacts
Baculoviruses are widely used as expression vectors for the production of proteins for biomedical research. They also show promise as viral insecticides and as vectors for gene therapy. Our research provides fundamental data on how the virus is capable of replicating its DNA and how its DNA is able to recombine during the infection cycle. The studies on the envelope fusion protein provide information on how retroviruses likely evolved from non infectious transposable elements to infectious viruses. This likely occurred when they integrated into a baculovirus and obtained an envelope fusion protein. The data that we have generated is critical for understanding how the virus is able to function as an expression vector and also for assessing its safety if it is to be used as an insecticide.
Publications
- Mikhailov, V. S., and G. F. Rohrmann. 2002. The Baculovirus Replication Factor LEF-1 is a DNA Primase. J. Virol. 76:2287-2297.
- Pearson, M. N., R. L. Q. Russell, and G. F. Rohrmann. 2002. Transcriptional mapping of two genes encoding baculovirus envelope-associated proteins. J. Gen. Virol. 83:937-943.
- Pearson, M. N., and G. F. Rohrmann. 2002. Transfer, incorporation, and substitution of envelope fusion proteins among members of the Baculoviridae, Orthomyxoviridae, and Metaviridae (insect retrovirus) families. J. Virol. 76:5301-5304.
- Mikhailov, V., and G. Rohrmann. 2002. Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures. BMC Molecular Biology 3:14.
- Pearson, M. N., R. L. Q. Russell, and G. F. Rohrmann. 2002. Functional analysis of a conserved region of the baculovirus envelope fusion protein, LD130. Virology 304:81-88.
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Progress 01/01/01 to 12/31/01
Outputs
Baculoviruses have achieved widespread use as expression vectors because of their ability to overexpress genes cloned under certain of their late promoter elements and are being intensively studied for development of a new generation of insecticides. The long-term objective of the program outlined in this proposal is to understand the means by which baculoviruses replicate their DNA and how they enter cells and initiate infection. This research focuses on the following objectives: i) The mechanisms of DNA replication and the enzymes and other proteins that are involved in this process. ii) The function of a baculovirus fusion protein called ld130 that is involved in viral entry into cells. iii) The roles of homologs of this fusion protein a baculoviruses which use a different fusion protein but encode homologs of ld130. We have made considerable progress in the characterization of the replication factors LEF-1 and LEF-2 and demonstrated that LEF-1 functions as a DNA
primase where LEF-2 may be involved in interacting with LEF-1 and DNA. We have also demonstrated that the baculovirus fusion protein, ld130 is related to the predicted envelope protein of a group of insect retroviruses. In addition we have demonstrated that the homologs of ld130 that are encoded by virus that use a different fusion protein are not active in fusion.
Impacts
This research has provided major information on the function of proteins involved in viral replication and cell fusion. It has also resulted in major insights into virus evolution.
Publications
- Rohrmann ,G.F and P. A. Karplus. 2001. Relatedness of baculovirus and gypsy retrotransposon envelope proteins. BMC Evolutionary Biology, 1, 1.
- Pearson, M. N., R. Russell, and G. F. Rohrmann. 2001. Characterization of a baculovirus encoded protein that is associated with infected-cell membranes and budded virions. Virology. 291:22-31.
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