In particular we are interested in the proteins that are involved in the synthesis of DNA and also those that are responsible for the production of complete genomes. The specific aims are as follows: 1. Investigations on genome replication and processing. Our current evidence suggests that alkaline nuclease is involved in recombination-dependent replication and is required for the production of full length genomes.
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In particular we are interested in the proteins that are involved in the synthesis of DNA and also those that are responsible for the production of complete genomes.
The specific aims are as follows: 1. Investigations on genome replication and processing. Our current evidence suggests that alkaline nuclease is involved in recombination-dependent replication and is required for the production of full length genomes. In addition, we have evidence that baculovirus DNA is partitioned in the infected cell into packaged and unpackaged DNA with the latter likely being involved in late gene transcription.
We will continue these investigations as follows: a Recombination-dependent replication in another insect cell line. We will examine whether DNA replication appears to be recombination-dependent in Trichoplusia ni cells which have significantly different properties from the Spodoptera frugiperda cells that we are currently employing.
The latter is likely required for very late gene transcription. Using an assay that we have developed, we will investigate these two categories of DNA during infection. Molecular interactions of VLF VLF-1 is related to lambda integrase. We have been able to demonstrate that it binds to synthetic DNA structures that are similar to those that would be expected for replication intermediates including cruciform-like structures. However, we have not been able to identify any enzymatic activity.
The purpose of these investigations will be to identify a the DNA sequences bound by VLF-1 and b the proteins that interact with VLF-1 such that together they have enzymatic activity. Continued characterization of LEF LEF-3 is a multifunctional protein that appears to play a pivotal role in a variety of processes related to baculovirus DNA replication.
In this project we will map the structural and functional domains of LEF These will include domains that interact with helicase, alkaline nuclease and DNA. In addition, we will examine the role of oligomerization in LEF-3 function in terms of DNA annealing, unwinding, and recombination. In this project we will biochemically characterize the properties of DBP. Project Methods 1 Construction of baculovirus knockouts: One of the major advances in investigations of baculovirus molecular biology has been the development of the use of baculovirus genomes incorporated into bacterial artificial chromosomes.
This allows the manipulation of baculovirus genomes in bacteria and facilitates the investigation of lethal knockouts because the manipulation and growth can be accomplished in bacteria.
The effects of the deletion of the genes can then be tested by transfecting the baculovirus genome into insect cells. We have successfully utilized this approach for the investigation of the roles of a number of baculovirus genes in DNA replication.
First of all, we always construct a repaired virus to ensure that the manipulation in bacteria did not produce any secondary mutations. This is accomplished by generating the virus by transfection of the bacmid into insect cells and then doing growth curves with the virus and comparing it to wt virus. If similar titers are generated, it indicates that the process of generating the deletion in bacteria did not result in other effects elsewhere in the genome.
DNA replication is assessed by real time pcr. Many of our mutants are able to replicate DNA to normal levels but are non-infectious because the DNA is not packaged or processed correctly.
In order to prevent shearing of the DNA, this requires in situ processing of DNA in agarose blocks that includes proteinase digestion to release the DNA and then digestion with restriction enzymes. The agarose blocks are then run on the FIGE gels. Overexpression, purification, and characterization of proteins. One of the major methods that we use as a basis for characterization of the biochemical properties of proteins is to overexpress them in recombinant baculoviruses.
This involves cloning the gene of interest into a baculovirus under the control of the polyhedrin promoter which results in high levels of expression. We also normally express the protein as a His tag and HA tag gene fusion. This allows us to readily purify the protein on nickel columns which bind Histidine and also to monitor the presence of the protein using antibodies directed against the HA tag.
Many of the tests that we use focus on the effect of the enzymatic properties on DNA. Proteins that bind DNA can be analyzed by electrophoretic mobility shift assays. We also have investigated that ability of proteins to unwind or anneal DNA molecules using labeling techniques in concern with analysis by gel electrophoresis. We have also used mass spectrometry to determine the size and sequence of fragments of proteins derived from tryptic digests.
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.
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. 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.
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. A Baculovirus alkaline nuclease knock out construct produces fragmented DNA and aberrant capsids. Virology Jin, L. Analysis of the Genome Sequence of an Alpaca Coronavirus. Virology , Vanarsdall, A. A DBP knockout virus was generated and found to be unable to produce budded.
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.
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 , , and 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 , , and 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 , , or 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. 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. Conserved molecular systems of the Baculoviridae.
Invited review. Characterization of the role of baculovirus very late factor-1 in capsid structure and DNA processing. Mikhailov, V.
Virology Pearson, M. Envelope gene capture and insect retrovirus evolution: the relationship between errantivirus and baculovirus envelope proteins. Virus Res. 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. 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. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region residues 28 to that is relatively resistant to proteolysis.
Baculovirus molecular biology
Rohrmann, George F. Abstract This is the 4th edition of a book that was initiated with the annotation of the function of all the genes in the most commonly studied baculovirus, AcMNPV. It has been almost six years since I reviewed this literature. As with the previous editions, this information is then integrated into chapters covering the major processes central to the replication and pathology of baculoviruses. Topics including taxonomy, the application of baculoviruses as insecticides, the molecular basis for the remarkable ability of these viruses to express genes at high levels, and the interrelationships of baculovirus and transposable elements are also covered.
Sajar One of the major methods that we use as a basis for characterization of the biochemical properties of proteins is to overexpress them in recombinant baculoviruses. The rabbit virus is a member of the herpesviridae and is a member of a primate lineage of these viruses. Transfer, incorporation, and substitution of envelope fusion proteins among members of the Baculoviridae, Orthomyxoviridae, and Metaviridae insect retrovirus families. Wei Sheng Wu Xue Bao. Baculovirus Molecular Biology — NCBI Bookshelf The establishment of solid bases to recognize their molscular relationships is necessary to facilitate the generation of new knowledge and the development of better methodologies. Baculoviruses have been exploited for the over expression of foreign gene products, which are not only highly expressed, but are also processed in a manner equivalent to higher eukaryotes and are biologically active. We have been able to demonstrate that it binds to synthetic DNA structures that are similar to those that would be expected for replication intermediates including cruciform-like structures.