On the origin of the polyhedral protein of the nuclear polyhedrosis virus of Autographa californica

The purpose of this study was to investigate the origin of the polyhedral protein of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), one of the best characterized viruses of the family Baculoviridae. The present knowledge of the properties of the nuclear polyhedrosis virus of Autographa californica, are described in chapter 1.One of the most striking features of nuclear polyhedrosis viruses is that singly or multiply enveloped virus particles become occluded into protein crystals during their multiplication cycle in the nucleus of their invertebrate host cells. Because of their shape these crystals are called polyhedra. The formation of occlusion bodies is not restricted to the nuclear polyhedrosis viruses. Several other virus families exhibit this phenomenon. The similarities and differences between the occlusion bodies of different virus families are also described in chapter 1 to provide basic information for the subsequent summary of arguments that have been used in favour of either the hypothesis that the occlusion body protein is produced by the host as a defence mechanism against the virus, or the hypothesis that it is produced by the virus to protect itself against environmental influences.Although previous investigations have provided indirect evidence that the polyhedral protein might be of viral origin, our approach resulted in direct proof that this protein is coded for by the viral genome. By isolating the messenger RNA for polyhedral protein and demonstrating that this mRNA is of viral origin, we were able to solve this more than 30 year old problem.To start with a genetically pure virus preparation, and to avoid as much as possible the formation of the so-called few polyhedra variant (Section 1.2.3.), we have plaque-purified the virus (Chapter 2). Minor variants which were present in the original preparation were removed by this procedure.Our attemps to isolate the mRNA for AcNPV polyhedral protein have moved along various experimental pathways. Our first approach is reflected in chapter 3, which describes the isolation of polysomes from healthy and infected cells. As AcNPV polyhedral protein is a very abundant protein late in the infection cycle (the whole nucleus eventually becomes filled with polyhedra), one would expect that the mRNA for this protein would also be present in large amounts. Consequently, a certain size class of polysomes would be favoured in infected cells when compared to the polysomal size distribution of healthy cells. No significant differences, however, were detectable between the polysomal profiles of healthy and 24 hr infected cells when analysed on isokinetic sucrose gradients. As a consequence no new abundant mRNA size class would be expected to be translated by polysomes from 24 hr infected cells, although new mRNAs may be present in the existing size classes.However, some interesting characteristics were discovered when the optimum conditions for the isolation of polysomes were determined.- Intact polysomes could only be detected when cycloheximide was added to the cell culture medium while the addition of RNase inhibitors did not increase the yield of polysomes. Run-off is the most satisfactory explanation for the effect of these chemicals on the yield of polysomes.- The yield of polysomes was increased by using a lysis buffer solution with a high pH (pH 9.0). As high pH values are also infavourable for lysosomal RNases, this observation includes the additional advantage of minimizing any residual RNase activity.Some characteristics which had been observed before in vertebrate systems were also observed in our invertebrate system. They include:- The existence of two populations of 40 S particles when ribosomes are analysed under low salt conditions (Chapter 3: fig. 4A, 7A, 10A, 10B, 11).- The demonstration of initiation complexes in sucrose gradient profiles of polysomes from cycloheximide treated cells (Chapter 2: fig. 9).- The difference in sedimentation behaviour of ribosomes and their subunits under various conditions (Chapter 3: fig. 10).These observations have been described before in vertebrate cell systems but have not yet been reported in any invertebrate system.Another approach in the isolation of the mRNA for AcNPV polyhedral protein, which involved the immunoprecipitation of polyhedral protein synthesizing polysomes, is also described in chapter 3. Polysomal breakdown, which occurred during the necessary purification procedure of the polysomes, probably was responsible for the negative results obtained with this technique.As polysomes were induced by the incubation of cycloheximide, which inhibits protein-, but not mRNA synthesis, the possibility remained that a pool of mRNA existed in the cytoplasm for which no ribosomes were available. In that case, size differences in cytoplasmic mRNA populations from healthy and 24 hr infected cells might be detectable after all. When poly(A) containing RNA from healthy and 24 hr infected cells was analysed on sucrose gradients, the size of the mRNA populations corresponded well to that expected on the basis of the size of the polysomes. No new mRNA large enough to code for AcNPV polyhedral protein could be detected in the mRNA population from 24 hr infected cells (Chapter 4).Isolation and analysis of (on AcNPV-DNA-cellulose selected) viral RNA isolated from 24 hr infected cells resulted in the detection of two minor, and one predominant RNA species with a molecular weight of 240,000 dalton (Chapter 5). This corresponds well to the minimum size expected for the mRNA for AcNPV polyhedral protein (See discussion chapter 3).In vitro translation of preparatively isolated viral RNA resulted in the synthesis of a protein which comigrated with purified AcNPV polyhedral protein on polyacrylamide gels (Chapter 6). Immunoprecipitation of the translation products followed by polyacrylamide gel electrophoresis demonstrated that the 30,000 dalton translation product represented the AcNPV polyhedral protein.No translatable RNA could be isolated from healthy cells by selection on AcNPV-DNA-cellulose, indicating that the mRNA for polyhedral protein is a virus- induced mRNA. As there is some aspecific hybridization in our hybridization procedure the theoretical possibility remains that the AcNPV polyhedral protein is translated from the less than 12.5 % host RNA which may still be present in the viral mRNA preparation. Several arguments plead against this hypothesis.- Size analysis of the viral mRNA population reveals only one mRNA with the right size for AcNPV polyhedral protein. This RNA is present in a quantity which is much greater than 12.5 %.- No attempt could be made to optimize the hybridization - and subsequent washing - and elution procedure. As no preliminary selection was made to remove ribosomal and transfer RNA, the rather high percentage of nonspecific hybridization most probably is a reflection of badly removed host ribosomal and transfer RNA rather than badly removed host mRNA.Selection of mRNA for AcNPV polyhedral protein with restriction endonuclease fragments of AcNPV DNA may show that some fragments are and other fragments are not capable of selecting the mRNA for AcNPV polyhedral protein. This would definitely prove that non-specifically hybridized host mRNA is not responsible for the in vitro synthesis of AcNPV polyhedral protein. This technique will localize at the same time the gene coding for AcNPV polyhedral protein on the physical map of the AcNPV DNA.Selection of viral mRNAs by means of hybridization followed by in vitro translation provides an excellent way of analysing the time course and size distribution of viral mRNA and protein synthesis in systems (such as ours) where host macromolecular synthesis is not shut off completely during the viral replication cycle. Defects in the viral replication cycle, such as the unability of AcNPV to synthesize polyhedral protein in Bombyx mori cells, can be classified as a pre- or a posttranscriptional event from the absence or presence of the mRNA for AcNPV polyhedral protein. The same approach would be useful in characterizing temperature sensitive mutations which result in the absence of polyhedra production at the restrictive temperature.