Objectif
(1) To purify the RNA polymerase complex from virus-infected cells
(2) To produce baculovirus mutants deficient in late gene transcription and map the altered virus gene(s) via marker rescue and DNA sequencing.
(3) Devise an in vitro transcription system to permit the analysis of the role of the purified polymerase components
(4) To produce insertion mutants of AcNPV, by disrupting coding regions with reporter genes such as lacZ.
(1) To purify the RNA polymerase complex from virus-infected cells
The alpha-amanitin-resistant RNA polymerase was purified from Autographa californica nuclear polyhedrosis virus (AcNPV)-infected cells and resolved as a single peak after three rounds of chromatography. Analysis of the proteins present in this peak suggests a composition similar to the previously published data. Unfortunately, the quantities of proteins generated by this method have been insufficient to permit further protein sequencing. To circumvent this problem, we have devised a new method for 'tagging' putative RNA polymerase subunits with well-characterized epitopes. The method requires the maintenance of the AcNPV genome in yeast cells as an episome. While in the yeast cell, the virus genome is manipulated to introduce the epitope coding regions into the gene of interest. To date, two virus genes (lefs 2 and 8) have been modified in this way using a c-myc epitope. Virus DNA has been recovered from the yeast cells and used to transfect insect cells to generate recombinant virus. Subsequent analysis of virus-infected cell extracts has shown that the LEF-2 protein can be detected with c-myc-specific antibody and accumulates in the late phase of virus infection, supporting its role as a component of the RNA polymerase.
(2) To produce baculovirus mutants deficient in late gene transcription and map the altered virus gene(s) via marker rescue and DNA sequencing.
An AcNPV mutant deficient in very late gene transcription and with delayed late gene expression was generated by chemical mutagenesis of virus-infected cells. The mutation within this virus was mapped to lef-2. Further characterization of the virus demonstrated that virus DNA replication was normal in virus-infected cells. Our data suggest that LEF-2 is involved in very late gene expression in AcNPV-infected cells. These results are supported by the accumulation of LEF-2 in the late/very late phase in virus-infected cells. We have also generated four other virus mutants which are deficient in late/very late gene expression. Although mapping studies were initiated, to date we have not identified the genes responsible for the mutation.
(3) Devise an in vitro transcription system to permit the analysis of the role of the purified polymerase components
By establishing an in vitro transcription system for early, late and very late AcNPV transcription, we were able to distinguish between target promoters of the host RNA polymerase II and of the viral-induced alpha-amanitin-resistant RNA polymerase and to study the requirements for their efficient transcription. The early promoters HE65, ME53 and PE38 were efficiently transcribed in uninfected nuclear extracts of either insect cells (Sf21 and Tn368) or mammalian cells (HeLa and BHK21). Activation of the HE65 promoter by the IE1 gene product, as observed by transient expression assays, could not be mimicked in vitro. Furtherstudies suggest that the late gene promoter element of the ME53 gene is used by the alpha-amanitin-resistant RNA polymerase present in AcNPV-infected nuclear extracts 20 hp.i. The construction of hybrid pe38/polyhedrin gene promoters has demonstrated that the replacement of 8 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter by those of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. Based on the sensitivity of our in vitro transcription, we have investigated putative non-viral targets of the viral-induced RNA polymerase. A possible candidate is the control region of mitochondiral DNA which was isolated and sequenced from Spodoptera frugiperda cells. The mitochondrial DNA was transcriptionally active in the presence of alpha-amanitin only in extracts prepared from AcNPV-infected cells at a comparable level to late viral promoters. Transcription initiation was found to occur at TAAG sequences, a motif which is also recognized by late promoters. The mitochondrial DNA fragment includes three TAAG motifs in either direction in an adenine/thymine rich background, however, not all of them are recognized by the alpha-amanitin resistant RNA polymerase. Transient expression studies confirmed that mitochondrial DNA can function as non-viral target since reporter gene activity driven by the mitochondrial DNA fragment was only detectable in AcNPV-infected cells. These results suggest an involvement of host cell enzymes in late transcription in baculovirus-infected cells.
(4) To produce insertion mutants of AcNPV, by disrupting coding regions with reporter genes such as lacZ.
We have performed directed mutagenesis of certain virus genes to establish whether or not they are required for virus replication. Those virus genes which have been targeted include candidates with a role in transcription: alkaline exonuclease, plasmid copy number protein, global transactivator, polynucleotide kinase and ligase protein. Only for the polynucleotide kinase and ligase protein was it possible to produce a replication competent virus. This suggested that the other virus gene products are essential and may have a role in late/very late transcription. To date, however, assays based on transient replication in uninfected cells transfected with plasmid subclones of the virus genome have not supported this idea. We have concluded that the insertion mutant approach to analysing components of the RNA polymerase is not ideal.
Champ scientifique
Thème(s)
Appel à propositions
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CSC - Cost-sharing contractsCoordinateur
OX1 3SR Oxford
Royaume-Uni