Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Much of our activity for the past year has been focused on post-transcriptional processing of viral RNAs. We knew from previous work that the LEF-4 subunit of RNA polymerase was a capping enzyme with RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. We also knew that this protein was essential for late transcription and viral replication. We wanted to know whether it was needed for its enzymatic activities or simply because it was a subunit of the RNA polymerase. To test this hypothesis we constructed a virus with a deletion of the lef-4 gene. As expected the virus was not able to replicate or express late genes. We then tested whether mutant versions of lef-4 that specifically lacked either the RTPase or GTase activity could rescue the mutants. We found that RTPase mutants could rescue, but GTase could not. We then followed up this seemingly contradictory result. We wondered whether the RTPase function was dispensable because the virus encodes another
gene with a similar function that could potentially compensate. We then made a double mutant virus that lacked both RTPase functions and found that it could also replicate. We also found that the structure of viral RNA caps was not affected by the loss of these two enzymes. We are currently trying to determine whether host enzymes are compensating using RNA silencing. These experiments are complicated by the fact that host processing is needed early in infection, so timing of inhibition is considerably more critical than is normally the case for RNAi. In addition we have been working on an annotation of baculovirus genome sequences. This will allow us to more easily identify conserved genes and specific amino acid residues within genes of interest.
PARTICIPANTS: My postdoctoral trainee Lillian Li has worked on this project for the past three years. This is an excellent opportunity for her. She has gained considerable experience in many areas of biochemistry, virology and genetics.
TARGET AUDIENCES: The baculovirus database will be an invaluable asset for the community.
Impacts
The importance of the first project is related to the evolution of mRNA capping systems in eukaryotes. These enzymes differ between higher and lower eukaryotes. Baculoviruses are the only genomes that are known to contain both types of enzymes and knowledge of their essential nature may help us to better understand the relationship between them. The second project will be a tremendous resource for the baculovirus community. Many different groups are engaged in genome projects, but there is no single repository of sequences that is easily accessible and allows for simple manipulation of gene sequences.
Publications
- No publications reported this period
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Progress 11/14/01 to 11/13/07
Outputs
OUTPUTS: Much of our activity for the past year has been focused on post-transcriptional processing of viral RNAs. We knew from previous work that the LEF-4 subunit of RNA polymerase was a capping enzyme with RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. We also knew that this protein was essential for late transcription and viral replication. We wanted to know whether it was needed for its enzymatic activities or simply because it was a subunit of the RNA polymerase. To test this hypothesis we constructed a virus with a deletion of the lef-4 gene. As expected the virus was not able to replicate or express late genes. We then tested whether mutant versions of lef-4 that specifically lacked either the RTPase or GTase activity could rescue the mutants. We found that RTPase mutants could rescue, but GTase could not. We then followed up this seemingly contradictory result. We wondered whether the RTPase function was dispensable because the virus encodes another
gene with a similar function that could potentially compensate. We then made a double mutant virus that lacked both RTPase functions and found that it could also replicate. We also found that the structure of viral RNA caps was not affected by the loss of these two enzymes. We are currently trying to determine whether host enzymes are compensating using RNA silencing. These experiments are complicated by the fact that host processing is needed early in infection, so timing of inhibition is considerably more critical than is normally the case for RNAi. In addition we have been working on an annotation of baculovirus genome sequences. This will allow us to more easily identify conserved genes and specific amino acid residues within genes of interest.
PARTICIPANTS: My postdoctoral trainee Lillian Li has worked on this project for the past three years. This is an excellent opportunity for her. She has gained considerable experience in many areas of biochemistry, virology and genetics.
TARGET AUDIENCES: The baculovirus database will be an invaluable asset for the community.
PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The importance of the first project is related to the evolution of mRNA capping systems in eukaryotes. These enzymes differ between higher and lower eukaryotes. Baculoviruses are the only genomes that are known to contain both types of enzymes and knowledge of their essential nature may help us to better understand the relationship between them. The second project will be a tremendous resource for the baculovirus community. Many different groups are engaged in genome projects, but there is no single repository of sequences that is easily accessible and allows for simple manipulation of gene sequences.
Publications
- Li Y, Guarino LA: Roles of LEF-4 and PTP/BVP RNA triphosphatases in processing of baculovirus late mRNAs. J Virol 2008, 82:5573-5583.
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Progress 01/01/06 to 12/31/06
Outputs
The baculovirus research has continued to focus on the LEF-4 gene of the baculovirus Autographa californica nuclear polyhedrosis virus. We unexpectedly found that the RNA triphosphatase function of this protein is not essential for viral infection. This is surprising because this enzymatic function is essential in all other systems in which it has been found. To further examine this finding, we are also working on another viral protein that is predicted to have an overlapping function. This protein is also not essential for virus replication. Now we are examining the intracellular results of these mutations on the processing and function of viral mRNAs. Our research on mRNA processing in baculoviruses has taken us in a new direction, which is RNA processing in SARS virus. This virus belongs to a different virus family, called the coronaviridae. While SARS was noted for its pathogenicity to humans, other members of this family are significant because of diseases caused
in poulty. We are investigating the function of several SARS genes that are involved in mRNA processing. We believe that these proteins are important in the synthesis of viral RNAs and may also be involved in repressing the host immune response.
Impacts
The LEF-4 results are significant because they are totally unexpected in molecular biology. Our future results will be very interesting as we try to determine the biological effects of these mutations. The SARS virus results are also quite interesting. We have found that the heteromeric structure of the viral protein influences its function. We are now expanding our research on SARS to include two additional mRNA capping enzymes.
Publications
- Knebel-Morsdorf, D. I. Quadt, Y. Li, L. Montier, and L. A. Guarino. 2006. Expression of baculovirus late/very late genes depends on LEF-4, a component of the viral RNA polymerase whose guanyltransferase function is essential. J. Virol. 80:4168-73.
- Bhardwaj, K., J. Sun, A. Holzenburg, L.A. Guarino, C.C. Kao. 2006. RNA recognition and cleavage by the SARS coronavirus endoribonuclease. J. Mol. Biol. 361:243-256.
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Progress 01/01/05 to 12/31/05
Outputs
During the past year, our work has been focused on the LEF-4 gene of the baculovirus Autographa californica nuclear polyhedrosis virus. We showed that LEF-4 was essential for viral replication by RNA interference and bacmid knockout technology. Silencing of LEF-4 in wildtype virus-infected cells suppressed expression of structural genes, while early expression was unaffected, demonstrating its essential role in late gene expression. The lef-4 mutant bacmid was transfected into insect cells and no viral progeny was produced. Co-transfection with wildtype lef-4 plasmid restored normal replication, but plasmid encoding a guanyltransferase-deficient version failed to rescue. We are further examining another function of LEF-4, which is RNA triphosphatase.
Impacts
These results are significant because we are trying to build a complete picture of the proteins that transcribe baculovirus late genes. Further, our preliminary data suggest that the RNA triphosphatase function is not required for viral replication. This is an unexpected result and will be pursued further.
Publications
- Mistretta, T.-A. and Guarino, L.A. 2005. Transcriptional activity of baculovirus very late factor-1. J. Virol. 79;1958-1960.
- Knebel-Morsdorf, D. I. Quadt, Y. Li, L. Montier, and L. A. Guarino. 2006. Expression of baculovirus late/very late genes depends on LEF-4, a component of the viral RNA polymerase whose guanyltransferase function is essential. J. Virol., in press.
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Progress 01/01/04 to 12/31/04
Outputs
During the past year, our work has been focused on two projects involving the characterization of baculovirus proteins that affect late gene expression. This is an ongoing area of investigation for us. We have previously shown that baculoviruses encode their own RNA polymerase, which is used for the transcription of late genes. Now we are in the process of characterizing the viral factors that interact with the RNA polymerase. The first project concerned the viral protein VLF-1 and its affect on template specificity. Purified RNA polymerase specifically recognizes and transcribes late and very late templates with equal affinities. We showed that addition of VLF-1 to purified RNA polymerase increased template preference for the very late promoter by a factor of 10. To determine which promoter sequences were necessary for this effect, we constructed chimeric templates, and showed that VLF-1 action was dependent on the A+T-rich sequence downstream of the polyhedrin
promoter. A manuscript describing this work was published last year. Now we are involved in additional experiments to further define the DNA sequences within the promoter that are necessary for response as well as identification of specific amino acid residues needed for VLF-1 action. The other factor we studied is the transcription factor LEF-5. This protein was expected to function as an elongation factor, but our studies showed that it functioned during the elongation phase. During the last year, we have attempted to determine whether it was involved in escape from abortive initiation. To this end, we developed an assay to analyze the synthesis of abortive products, but found that LEF-5 acted at an earlier step of initiation. We are now developing assays to study these earlier steps. My lab has recently started working on an entirely new research area that concerns the development of antivirals against SARS coronavirus. This interest grew out of previous research in my lab
concerning the function of a baculovirus-encoded methyltransferase After the SARS outbreak two years ago, we decided to extend our work on this protein to the SARS ortholog. The SARS work was further extended to include two other proteins predicted to function in RNA processing. One manuscript was published on this project last year.
Impacts
The experiments on VLF-1 are significant because they address a central question in baculovirology, which is the regulation of very late gene expression. This group of genes is unique to the baculoviruses, and a better understanding of the regulation of these proteins will contribute to basic virology, and will help us design better baculovirus expression vectors and agents of biological control. The results on LEF-5 are significant because we are trying to build a complete picture of the proteins that transcribe baculovirus late genes. Further, this protein is similar to a cellular protein, yet apparently has a unique function The SARS virus research is exciting because it is a human pathogen with the potential to cause intermittent outbreaks. The research ongoing in my lab may aid in the development of antivirals.
Publications
- Guarino, L.A. 2004. The baculoviruses. Nature Encyclopedia of Life Sciences. In press.
- Bardwaj, K., Guarino, L.A., and Kao, C. 2004. The SARS Coronavirus nsp15 protein is an endoribonuclease that prefers manganese as cofactor. J. Virol. In press.
- Mistretta, T.-A. and Guarino, L.A. 2004. Transcriptional activity of baculovirus very late factor-1. J. Virol. In press
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Progress 01/01/03 to 12/31/03
Outputs
During the past year, our work has been focused on three areas. The first characterization of baculovirus proteins that affect late gene expression. This is an ongoing area of investigation for us. We have previously shown that baculoviruses encode their own RNA polymerase, which is used for the transcription of late genes. Now we are in the process of characterizing the viral factors that interact with the RNA polymerase. The first factor we have characterized is the protein LEF-6. This factor increases the rate of transcription and acts at the step of initiation. To further understand the mechanism of its action, we constructed a series of alanine substitution mutants. Amino acids were selected for mutagenesis, either because they were highly conserved among other LEF-6 proteins or because they were conserved in a yeast methyltransferase with significant homology to LEF-6. The assay results revealed that all of the selected residues were essential for function.
The second project concerned the viral protein VLF-1 and its affect on template specificity. Purified RNA polymerase specifically recognizes and transcribes late and very late templates with equal affinities. We showed that addition of VLF-1 to purified RNA polymerase increased template preference for the very late promoter by a factor of 10. To determine which promoter sequences were necessary for this effect, we constructed chimeric templates, and showed that VLF-1 action was dependent on the A+T-rich sequence downstream of the polyhedrin promoter. The third project involves the post-transcriptional processing of viral RNAs. Viral RNAs are methylated on the cap and on the penultimate ribose. We identified a putative methyltransferase was identified by sequence homology. This protein, called MTase I was expressed in insect cells and purified to homology. The protein bound S-adenosylmethionine consistent with its classification as a methyltransferase. MTase I added a methyl
group to capped RNA. Analysis of the capped structure revealed that the methyl group was added to the ribose of the first transcribed base, indicating that MTase I was a nucleoside (2'O)-methyltransferase.
Impacts
The results on LEF-6 are significant because we are trying to build a complete picture of the proteins that transcribe baculovirus late genes. This is the last of the proteins that was left to be characterized. This should allow us to develop a completely reconstituted transcription complex, which has never been done for a DNA-dependent RNA polymerase from a eukaryotic virus. The experiments on VLF-1 are significant because they address a central question in baculovirology, which is the regulation of very late gene expression. This group of genes is unique to the baculoviruses, and a better understanding of the regulation of these proteins will contribute to basic virology, and will help us design better baculovirus expression vectors and agents of biological control. The methyltransferase results are significant due to their impact on our understanding of the mechanisms of baculovirus transcription. In addition, this work has led to a collaboration with Cheng
Kao at Texas A&M University on the mechanism of capping of SARS coronavirus. SARS virus encodes a protein with significant homology to MTase I. Our work on the baculovirus enzyme has provided a framework for further studies of RNA capping in coronavirus.
Publications
- Wu, X. and L.A. Guarino. 2003. Autographa californica nuclear polyhedrosis virus orf-69 encodes a RNA cap (nucleoside-2'-O)-methyltransferase. J. Virol 77: 3430-3440.
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Progress 01/01/02 to 12/31/02
Outputs
Our work has been focused on four areas. The first is the purification of the baculovirus protein PP31, which is essential for viral late gene expression. We purified PP31 from infected insect cells by a combination of heparin affinity, cation exhange chromatography, and gel filtration. We were able to purify 5 mg of PP31 95 mg of starting material confirming that PP31 is one of the most abundant viral proteins. DNA binding assays were done to compare the binding affinity of PP31 to single-stranded and double-stranded DNA, and we found that PP31 bound both DNAs with equal affinities. Then we tested the effect of adding PP31 to in vitro transcription assays with purified baculovirus RNA polymerase. This resulted in a strong inhibition of transcription. This indicates that the viral RNA polymerase was not able to displace PP31. The second series of experiments is related to the role of LEF-12 in late transcription. This protein is essential for transient expression of
late genes. We constructed a mutant virus that was unable to express LEF-12 by inserting the b-galactosidase gene into the lef-12 open reading frame. Mutant virus was viable and single-step growth curves indicated that final yields of virus were not reduced by the absence of LEF-12. Furthermore, pulse-labeling of infected cells revealed that LEF-12 mutant viruses entered the late phase, and synthesized late proteins at levels equivalent to those of wildtype virus-infected cells. Western blot analysis showed that LEF-12 was not synthesized in cells infected with mutant virus. In wild type virus-infected cells, LEF-12 was not detected until 18 hours post infection, and accumulation of LEF-12 peaked at 24-36 hours postinfection. LEF-12 was not detected in cells treated with aphidicolin, indicating that DNA replication was essential for its expression, and so it should be classified as a late gene. The third project focused on the characterization of the viral protein LEF-5. This protein
has homology with a eukaryotic transcription elongation protein. We tested whether LEF-5 had this function in vitro, but our experiments revealed that LEF-5 was an initiation factor, not an elongation factor. The fourth project involves the post transcriptional processing of viral RNAs. The RNAs should be methylated on the cap and on the penultimate ribose. A putative ribose methyltransferase was identified by sequence homology. This protein ORF69 was expressed in insect cells and purified to homology. We found that the protein bound S-adenosylmethionine consistent with its classification as a methyltransferase, and furthermore showed that the protein added a methyl group to capped RNA. We also showed that ORF69 was not essential for replication of virus in tissue culture.
Impacts
The PP31 results are significant because we previously showed that the viral RNA polymerase is composed of only 4 viral subunits. Some of our colleagues have expressed reservations regarding this result because 9 viral proteins are known to be required for transient late expression. Our result provides an explanation for these conflicting results. The in vivo template is probably coated with proteins like PP31 and is not naked like the in vitro template. These other viral proteins could then be responsible for removing the proteins that inhibit transcription activity. Thus our characterization of PP31 provides an assay to analyze the functions of additional viral factors in late transcription. Our results on the essential nature and temporal expression of LEF-12 raises interesting questions regarding its role in late gene expression. Since it is not essential for viral replication, it cannot be essential for late gene expression in the context of virus infection even
though it is required for transient late gene expression. Also it is not expressed until after late genes are expressed. We hypothesize that LEF-12 is involved in posttranscriptional regulation, and we are continuing to work on this problem. The LEF-5 experiments are interesting because they suggest that a protein homologous to an elongation factor is an initiation factor. This role has been proposed for the eukaryotic homolog, but not yet proven. Thus, our experiments may provide a clue to novel functions for eukaryotic transcription factors.
Publications
- Guarino, L.A., T. Mistretta, W. Dong. 2002. DNA binding activity of the late transcription factor PP31. Virus Research 90:187-195.
- Guarino, L.A., T. Mistretta, W. Dong. 2002. Baculovirus LEF-12 is not required for viral replication. J. Virol. 76:12032-12043. Guarino, L.A., W. Dong, and J. Jin. 2002. In vitro activity of baculovirus LEF-5. J. Virol. 76:12663-12675.
- Wu, X. and L.A. Guarino. 2003. Baculovirus AcORF-69 encodes an RNA methyltransferase. J. Virol. In review.
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Progress 01/01/01 to 12/31/01
Outputs
Baculoviruses are popular eukaryotic expression vectors, and have been used to produce hundreds of different proteins for basic research and pharmaceutical applications such as medical therapeutics, diagnostics, vaccines, and drug discovery. This system works because of the high-level activity of the viral-encoded RNA polymerase, which transcribes viral late and very late genes. The focus of my research is on the mechanisms that regulate baculovirus gene expression, particularly those viral factors that influence levels of late gene expression. We have previously shown that the viral RNA polymerase is a complex of four viral proteins. This four-subunit polymerase has specific promoter-recognition, catalytic, and mRNA capping, and polyadenylation activities. Our major goal for the past year was to develop baculovirus RNA polymerase as a biotech tool to be used for the production of capped and polyadenylated mRNAs in vitro. We collaborated with Epicentre Technologies
(Madison, WI), who hoped to market his project. We used three different strategies for expression. The first was to express the subunits separately and then reconstitute the complex. This was not successful because three subunits were insoluble when expressed in E. coli. The second strategy was to express all four subunits in each cell so that the complex could assemble in vivo. This yielded soluble enzyme. We also tried to denature and refold inclusion bodies, but this was also not successful. We also collaborated with Roche Molecular Biochemicals to test whether The Rapid Translation System (RTS) could be used for expression of RNApol. This is a preparative-scale instrument for coupled transcription and cell-free translation. The advantage of this system is that it allows expression of proteins that cannot be expressed in vivo due to problems with toxicity. Unfortunately we discovered that the RTS system will not work for RNApol because one of the subunits has ATPase activity. Our
second specific aim was to define the promoter specificity of baculovirus RNA polymerase. In order for baculovirus RNA polymerase to be useful as a research tool, it must have a high degree of promoter specificity and not recognize cryptic promoter motifs within target genes. We constructed new transcription templates that contained three additional promoter motifs. We could detect low level transcription at very high ratios of enzyme to template, but not within a normal range of use. Baculovirus RNA polymerase, with its ability to synthesize, cap, and polyadenylate RNA, has the potential for a to be a useful biotech tool. Unfortunately, it's just the nature of recombinant protein expression: there are no rules because every protein is unique, and so solutions have to be found empirically, which takes time.
Impacts
It is important to understand the mechanisms that control baculovirus gene expression. Recombinant baculoviruses have been constructed for use as biological control agents to control specific insect pests. These viruses express insect toxins, which are potentially dangerous. Also, baculoviruses are commonly used as expression vectors for the production of pharmaceuticals and for basic research.
Publications
- McDougal, V.V., and L.A. Guarino. 2001. DNA and ATP Binding Activities of the Baculovirus DNA Helicase P143. J. Virol. 75:7206-7209.
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Progress 01/01/00 to 12/31/00
Outputs
This last year we made significant progress on two areas of the temporal regulation of viral gene expression. We showed that the baculovirus RNA polymerase has two additional enzymatic activities, it can terminated RNA transcription at oligoU and it can polyadenylate terminated transcripts. This is important because it indicates that baculovirus RNA polymerase is unique, only a four subunit RNA polymerase yet it has all the funtions of the much larger eukaryotic transcription machinery. In addition, we showed that the baculovirus protein P143 is an helicase. This had been predicted for years, but previous attempts to prove this had failed. Subsequent mechanistic experiments indicate that P143 unwinds DNA by an inchworm mechanism.
Impacts
We have filed a patent application for the use of baculovirus RNA polymerase as a biotech tool. In addition, a Small Business Technology Transfer Grant was funded from the NIH to develop this technology.
Publications
- McDougal, V. and L.A. Guarino. 2000. The Autographa californica nuclear polyhedrosis virus p143 gene encodes a DNA helicase. J. Virol. 74:5273-5279.
- Jin, J., and L. Guarino. 2000. 3? end formation of baculovirus late transcripts. J. Virol. 74:8930-8937.
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Progress 01/01/99 to 12/31/99
Outputs
The focus of my research is on the mechanisms that regulate viral gene expression, particularly those viral factors that influence levels of late gene expression. We have previously shown that the RNA polymerase which transcribes baculovirus late genes is a complex of four viral proteins. This four-subunit polymerase has both specific promoter-recognition, catalytic, and mRNA capping activities. This past year we turned our attention to the mechanisms of 3'-end processing (Jin and Guarino, 2000). In vitro assays revealed that 3' ends were not formed by a cleavage mechanism as commonly believed, but by termination at T-rich regions followed by polyadenylation. Termination and polyadenylation are intrinsic properties of the baculovirus viral RNA polymerase. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism of termination. We also wanted to characterize the viral DNA replication proteins because late gene expression is absolutely dependent
upon DNA replication. Therefore, we purified baculovirus DNA polymerase (DNApol) to homogeneity (McDougal and Guarino, 1999; Hang and Guarino, 1999). DNApol was active in polymerase assays on singly-primed M13 template. DNApol is highly processive and has moderate strand displacement activity. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. E. Coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand-displacement in this assay. Additional studies were conducted on the baculovirus P143 protein, which is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143 (McDougal and Guarino, 2000). We found that ATPase activity co-purified with P143 protein
during purification and also during gel filtration at high salt. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated four-fold by the addition of single-stranded DNA. The ATPase activity of P143 had a Km of 60 mM and a turnover number of 4.5 molecules of ATP hydrolyzed/sec/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.
Impacts
It is important to understand the mechanisms that control baculovirus gene expression. Recombinant baculoviruses have been constructed for use as biological control agents to control specific insect pests. These viruses express insect toxins, which are potentially dangerous. Also, baculoviruses are commonly used as expression vectors for the production of pharmaceuticals and for basic research.
Publications
- Hang. X. and L.A. Guarino. 1999. Purification of AcNPV DNA polymerase from infected insect cells. J. Gen. Virol., 80: 2519-2523.
- McDougal, V. and L.A. Guarino. 1999. Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement. J. Virol, 73:4908-4918.
- McDougal, V. and L.A. Guarino. 1999. The Autographa californica nuclear polyhedrosis virus p143 gene encodes a DNA helicase. J. Virol, in press.
- Jin, J., and L. Guarino. 2000. Termination and polyadenylation of baculovirus late transcripts. Submitted for publication.
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Progress 01/01/98 to 12/31/98
Outputs
During the past year, we made significant progress on our characterization of the mechanisms that control late viral gene expression. We purified the viral RNA polymerase to homogeneity. We showed that it was a complex of four proteins and demonstrated that all four proteins were encoded by the virus. This complex of four protein has both catalytic activity and promoter recognition activity. Furthermore we showed that one of these proteins had both guanylyltransferase and RNA triphosphatase activity. This indicates that baculoviruses encode not just their own RNA polymerase, but also their own capping enzymes. We are extending these results to look at 3' processing of viral RNAs.
Impacts
(N/A)
Publications
- Guarino, L.A., B. Xu, J. Jin, and W. Dong. 1998. A viral-encoded RNA polymerase purified from baculovirus infected cells. J. Virol: 772:7985-7991.
- Jin, J., W. Dong, and L. Guarino. 1998. The LEF-4 subunit of baculovirus RNA polymerase has 5'-RNA triphosphatase and ATPase activities. J. Virol. 72: 10011-10019.
- Guarino, L.A., J. Jin, and W. Dong. 1998. Guanylyltransferase activity of the LEF-4 subunit of baculovirus RNA polymerase. J. Virol 72: 10003-10010.
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Progress 01/01/97 to 12/31/97
Outputs
The goal of this project for the last year was to purify active transcription complexes from infected cell extracts prepared at 36 hr post infection. We expected that the baculovirus late transcription complex would consist of multiple separable factors, similar to other eukaryotic transcription systems. However, when we attempted to purify transcription factors and RNA polymerase activity, we found that these functions always copurified, indicating that they were assembled into stable complexes. Then we purified the very late transcription complex by four successive chromatographic steps. This complex has both promoter recognition and RNA polymerase activity. This transcription complex contains four proteins. One of these proteins was identified by immunochemical analyses as LEF-8, a viral protein which contains a motif conserved among RNA polymerases. The three other proteins were identified by amino acid sequence analysis and were shown to be LEF-9, LEF-4, and p47.
A paper describing the purification has been submitted for publication. We have constructed viruses that overexpress these four proteins both individually and together. We have developed purification schemes for these proteins and are currently attempting to analyze the functions of the individual subunits. Thus far, we have determined that LEF-4 encodes both guanylatransferase and RNA triphosphatase activities. These are two of the functions required for mRNA capping. A manuscript describing these functions of LEF-4 is in preparation.
Impacts
(N/A)
Publications
- Ross, L. and L.A. Guarino. 1997. Cylcoheximide inhibition of baculovirus early gene expression. Virology 232: 105-113.
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Progress 01/01/96 to 12/30/96
Outputs
This past year, we continued our research on the characterization of viral proteins involved in the regulation of viral gene expression. We focused on the viral protein pp31 which is known to be required for late gene expression. pp31 associates with the virogenic stroma, the site of viral DNA replication and transcription in infected cells. pp31 binds DNA and possibly serves to anchor viral DNA to the virogenic stroma. We showed that nuclear targeting of viral DNA and, therefore its function, is modified by phosphorylation by viral-encoded protein kinases. In addition, we mapped the nuclear targeting and DNA binding domains of pp31.
Impacts
(N/A)
Publications
- Broussard, D.R., L.A. Guarino, and D.L. Jarvis. 1996. Dynamic phosphorylation ofAutographa californica nuclear polyhedrosis virus pp31. J. Virology 70: 6767-6774.
- Broussard, D.R., L.A. Guarino, and D.L. Jarvis. 1996. Mapping functional domains in Autographa californica nuclear polyhedrosis virus pp31. J. Virology 222: 318-331.
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Progress 01/01/95 to 12/30/95
Outputs
This past year, we continued our research on the development of an in vitro DNA replication system for baculoviruses. We purified the baculovirus single-stranded DNA binding protein and showed that it was encoded by the lef-3 gene which is essential for DNA replication. In addition, we purified viral DNA polymerase and showed that the enzyme was highly processive. We purified two additional DNA replication proteins and are currently characterizing their functions. We completed work on the characterization of the viral IEI protein, the major transcription factor required for early transcription. We have also continued our work on the purification of baculovirus RNA polymerase. These results were very exciting as they indicate that the viral RNA polymerase is unusual for a eukaryotic organism. The structure of the polymerase is more similar to bacterial polymerases than eukaryotic ones. These studies should aid in the understanding of virus-host interactions in
lepidopteran systems.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
During the past year, we completed a description of a phospholipid anchor that attaches ubiquitin to baculovirus particles. This is a novel type of posttranslational modification, and we now want to determine whether this type of modification is found in other systems. If it is unique to insects, then it is possible that the responsible enzymes could be targeted as a specific means of insect control. We continued our research on the development of an in vitro DNA replication system for baculoviruses. In addition, we purified baculovirus RNA polymerase. These results were very exciting as they indicate that the viral RNA polymerase is unusual for a eukaryotic organism. The structure of the polymerase is more similar to bacterial polymerases than eukaryotic ones. These studies should aid in the understanding of virus-host interactions in lepidopteran systems.
Impacts
(N/A)
Publications
- GUARINO, L.A., and W. DONG. 1994 Functional dissection of the Autographa californica nuclear polyhedrosis virus enhancer element hr5. Virology 200; 328-335.
- SCHNEIDER, C., K. WEISSHART, I. GILBERT, L.A. GUARINO, I, DORNREITER, and E. FANNING. 1994. Species-specific functional interactions of DNA polymerase (alpha)-primase with SV40 T antigen require SV40 origin DNA. Mol. Cell Biol. 14: 3176-.
- YOO, S., and L.A. GUARINO. 1994. The Autographa californica nuclear polyhedrosis virus ie2 gene encodes a transcriptional regulator. Virology 202:746-753.
- YOO, S., and L.A. GUARINO. 1994. Functional dissection of the Autographa californica nuclear polyhedrosis virus ie2 gene product. Virology 202:164-172.
- GONG, M. and L.A. GUARINO. 1994. The apoptotic suppressor P35 increases expression of a delayed early gene of Autographa californica nuclear polyhedrosis virus. Virology 204:38-44.
- REILLY, L.M. and L.A. GUARINO. 1994. The pk-1 gene of Autographa californica nuclear polyhedrosis virus encodes a protein kinase. J. Gen. Virol. 75: 3007-3014.
- GUARINO, L.A., G. SMITH, and W. DONG. 1995. Ubiquitin is attached to membranes of baculovirus particles by a novel type of phospholipid anchor. Cell, in press.
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Progress 01/01/93 to 12/30/93
Outputs
During the past year, we completed two projects on the elucidation of the mechanisms of action of two viral proteins IE1 and IE2. These proteins regulate the expression of early genes of the virus Autographa californica nuclear polyhedrosis virus (AcNPV). IE1 was shown to be a phosphoprotein whose activity is dependent upon a specific phosphorylation state. IE2 was shown to be a transcriptional activator, and a series of mutants was constructed in order to determine which motifs were essential for activity. A third project was developed relating to DNA replication of baculoviruses in insect cells. A fourth project was developed on the regulation of baculovirus late gene expression. These studies should aid in the understanding of virus-host interactions in lepidopteran systems.
Impacts
(N/A)
Publications
- CHOI, J.C., and GUARINO, L.A. 1994. A viral transactivator, IE1, of Autographa californica nuclear polyhedrosis virus is expressed as multiple phosphorylated forms during viral infection. Submitted for publication.
- SCHNEIDER, C., WEISSHART, K., GILBERT, I., GUARINO, L.A., DORNREITER, I. and FANNING, E. 1994. Species specific interactions of DNA polymerase (alpha)-primase with SV40 T antigen require SV40 origin. submitted for publication.
- YOO, S., and GUARINO, L.A. 1994. The Autographa californica nuclear polyhedrosis virus ie2 gene encodes a transcriptional regulator. submitted for publication.
- YOO, S., and GUARINO, L.A. 1994. Functional dissection of the Autographa californica nuclear polyhedrosis virus ie2 gene product. submitted for publication.
- GUARINO, L.A., and DONG, W. 1993 Functional dissection of the hr5 Autographa californica nuclear polyhedrosis virus enhancer element hr5. Virology, in press.
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Progress 01/01/92 to 12/30/92
Outputs
During the past year, we have focused our efforts on two areas of the molecular biology of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The first area concerns the regulation of viral gene expression. To better understand the factors that control expression of delayed early genes, we constructed a series of linker scan mutations in the promoter of the 39K gene that is regulated by dual delayed early and late promoters. This allowed us to define the nucleotide sequences that control expression of this gene throughout infection. An analysis of the structure and function of the IE-1 gene was performed by construction of C- and N-terminal mutants of this protein. From this series of experiments we were able to assign the functional domains of this protein. An analysis of the viral ubiquitin gene resulted in the discovery of a unique type of phospholipid modification of protein. The role of this modification in the life cycle of the virus is
currently under investigation.
Impacts
(N/A)
Publications
- GUARINO, L.A. and M.W. SMITH. 1992. Regulation of delayed early gene transcription by dual TATA boxes. J. Virol. 66: 3733-3739.
- KOVACS, G.R., J. C. CHOI, L.A. GUARINO, and M.D. SUMMERS. 1992. Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate early-1 transcriptional regulatory protein. J. Virol 66: 7429-7437.
- GUARINO, L.A., W. DONG, B. XU, D.R. BROUSSARD, R.W. DAVIS, and D.L. JARVIS. 1992. The baculovirus phosphoprotein pp31 is associated with the virogenic stroma. J. Virol. 66: 7113-7120.
- HUYBRECHTS, R., L.A. GUARINO, M. VAN BRUSSEL, and V. VULSTEKE. 1992. Nucleotide sequence of a transactivating Bombyx mori nuclear polyhedrosis virus immediate early gene. Biochim. Biophys. Acta. 1129: 328-330.
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Progress 01/01/91 to 12/30/91
Outputs
During the past year, we have focused our efforts on the elucidation of the mechanism of action of two viral factors that regulate the expression of early genes of the virus Autographa californica nuclear polyhedrosis virus (AcNPV). The DNA sequence of one factor, the IEN gene, was determined and the predicted amino acid sequence indicates that the encoded protein has several sequence motifs common to transcriptional regulators. Several mutants have been constructed in order to determine whether these motifs are essential for activity. Analysis of the second factor, IE1, indicated that its activity was mediated through binding to the baculovirus enhancer elements. Construction of IE1 deletion mutants and subsequent analysis indicated that the DNA binding region was localized in the C-terminus of the protein, while the transactivating domain was localized to the N-terminus. Progress was also made in a third project in the lab relating to the identification of viral
kinase genes. Identification of this gene is important because of the central role of kinases in the post-translational regulation of transcription factors. These studies should aid in the understanding of virus-host interactions in lepidopteran systems.
Impacts
(N/A)
Publications
- CARSON, D.D., SUMMERS, M.D. and GUARINO, L.A. 1991. Transient expression of the AcMNPV Immediate early gene IEN is regulated by three viral elements. J. Virol 65:945-951.
- CARSON, D.D., SUMMERS, M.D. and GUARINO, L.A. 1991. Molecular analysis of a baculovirus regulatory gene. Virology, 182: 279-286.
- GUARINO, L.A., and DONG, W. 1991. Transient expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene. J. Virol. 65:3676-3680.
- KOVACS, G.R., GUARINO, L.A., and SUMMERS, M.D. 1991. Transcriptional regulatory properties of the baculovirus AcMNPV IE1 AND IE0 gene products. J. Virol: 5281-5288.
- KOVACS, G.R., GUARINO, L.A., GRAHAM, B.L. and SUMMERS, M.D. 1991. Identification of spliced baculovirus RNAs expressed late in infection. Virology, 185-633-643.
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