G.S. Beaudreau

A baculovirus polyhedral envelope-associated protein: genetic location, nucleotide sequence, and immunocytochemical characterization

Using a polyclonal mouse antiserum produced against purified virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV), two immunoreactive lambda gtII clones were identified which contained nonoverlapping insert DNAs which mapped to a single open reading frame (ORF) in the HindIII-M fragment. Analysis of nucleotide sequence data indicates that this ORF encodes a protein with a MW of 32.4 kDa. A trpE-p32 gene fusion containing the entire p32 ORF was constructed, and the fusion protein was purified and used to immunize rabbits. Western blot analysis and immunofluorescence studies using the anti-TrpE-p32 antiserum detected a polyhedra-derived virus (PDV)-associated protein of 32 kDa at 24 hr postinfection (hr p.i.). The protein was observed in the cytoplasm and nucleus at 24 hr p.i. and became concentrated in the cytoplasm late in infection. Western blot analysis and immunofluorescent microscopy of polyhedra solubilized under various conditions indicated that p32 is associated with the polyhedral envelope. The predicted amino acid sequence for p32 showed 58% amino acid identity with the predicted amino acid sequence for an ORF (ORF 3) in a similar region of the genome of the MNPV of Autographa californica (AcMNPV). The solubility properties of the p32 protein and reciprocal immunoblotting experiments indicate the OpMNPV p32 gene encodes a protein which is homologous to the polyhedral envelope-associated phosphoprotein of AcMNPV, pp34, recently reported by M.A. Whitt and J.S. Manning [(1988) Virology 163, 33-42].

p39, a major baculovirus structural protein: immunocytochemical characterization and genetic location.

A series of monoclonal antibodies was produced against proteins from polyhedra-derived virions of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). Two of these antibodies (214 and 236) reacted with a protein of 39 kDa on Western blots of electrophoretically separated OpMNPV viron proteins derived from both budded and polyhedra-derived virions. This protein appears to be a major component of both BV and PDV. One of the p39 antibodies was used to characterize p39 synthesis in infected Lymantria dispar cells by using Western blots and immunofluorescent staining. The p39 protein was detected by immunofluorescence microscopy at 24 hr postinfection. By 48 hr, p39 was detected primarily in cell nuclei with little or no detectable staining of the cytoplasm. The two MAbs were used to identify three immunoreactive clones from a lambda gt11 expression library of OpMNPV DNA. By hybridization of insert DNA from three lambda gt11 clones to blots of restriction digests of OpMNPV genomic DNA, the location of the 39-kDa gene was mapped on the OpMNPV genome. Using the lambda gt11 insert DNAs and the monoclonal antibodies, the p39 genes and proteins of OpMNPV and Autographa californica NPV (AcMNPV) were shown to be closely related in size, DNA sequence, and antigenicity. One of the p39 monoclonal antibodies cross-reacted with a host cell protein associated with the condensed chromosomes present during mitosis.

Characterization of baculovirus p10 synthesis using monoclonal antibodies

A series of monoclonal antibodies were produced against virion proteins of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). Four of these antibodies reacted with a protein of 14 kd on Western blots of electrophoretically separated OpMNPV virion proteins. These antibodies were used to identify immunoreactive clones from a lambda gt11 expression library of OpMNPV DNA. By hybridization of insert DNA from the lambda gt11 clones to blots of digests of OpMNPV genomic DNA, and by sequencing the ends of the lambda gt11 inserts, these clones were shown to contain a portion of the p10 gene. The regions containing epitopes recognized by the four monoclonal antibodies were located using fusion proteins made from selected portions of the p10 reading frame in a trpE vector. One of the p10 antibodies was used to characterize p10 synthesis in infected Lymantria dispar cells by using Western blots and immunofluorescent staining. The p10 protein was detected with immunofluorescent microscopy at 14 hr postinfection and by 20 hr it formed intensely staining cytoplasmic structures. On Western blots of infected cells, two forms of p10 (of about 14 and 15 kd) were observed. One of the p10 monoclonal antibodies showed a strong cross-reaction with cytoskeletal structures in uninfected insect cells and rat fibroblasts.

N-terminal polyhedrin sequences and occludedBaculovirus evolution

SummaryA phylogenetic tree for occluded baculoviruses was constructed based on the N-terminal amino acid sequence of occlusion body proteins from six baculoviruses including three lepidopteran nuclear polyhedrosis viruses (NPVs), [two unicapsid (Bombyx mori andOrgyia pseudotsugata) and one multicapsid (Orgyia pseudotsugata)]; one granulosis virus (Pieris brassicae); and NPVs from a hymenopteran (Neodiprion sertifer) and a dipteran (Tipula paludosa). Amino acid sequence data for theB. mori NPV were from a report by Sere-bryani et al. (1977) and that for theO. pseudotsugata NPVs were reported previously by us (Rohrmann et al. 1979). The other N-terminal amino acid sequences are presented in this paper. The phylogenetic relationships determined based on the molecular evolution of polyhedrin were also investigated by antigenic comparisons of the proteins using a solid phase radioimmune assay. The results indicate that the lepidopteran NPVs are the most closely related of the above group of viruses and are related to these viruses in the following order:N. sertifer NPV,P. brassicae granulosis virus, andT. paludosa NPV. These data, in conjunction withBaculovirus distribution and evidence concerning insect phylogeny, suggest that theBaculovirus have an ancient association with insects and may have evolved along with them.

Genetic relatedness of two nucleopolyhedrosis viruses pathogenic for Orgyia pseudotsugata

Abstract DNA from two nucleopolyhedrosis viruses pathogenic for Orgyia pseudotsugata showed no common patterns when restriction endonuclease fragments of both DNAs were compared by agarose-gel electrophoresis. Furthermore, DNA-DNA hybridization indicated at most 1% homology between DNAs from the two viruses.

DNA Sequence Homology between Autographa californica and Orgyia pseudotsugata Nuclear Polyhedrosis Viruses

Summary To investigate the DNA sequence homology between the multicapsid nuclear polyhedrosis virus of Autographa californica and the multicapsid and single capsid nuclear polyhedrosis virus of Orgyia pseudotsugata, the stringency of hybridization conditions was varied. Thirteen to 25% homology between the multicapsid viruses of A. californica and O. pseudotsugata was detected in 20, 30 and 40% formamide, indicating that fairly stable duplexes were being formed. Under the most stringent conditions in 50% formamide little homology was detected. In contrast to the multicapsid viruses, the single capsid virus of O. pseudotsugata demonstrated about 10% sequence homology in 20% formamide, but these duplexes were unstable and the percentage homology decreased markedly in the higher formamide concentrations. Examination of the specific regions of homology by hybridizing labelled DNA to Southern blots of the virus DNAs revealed that the regions of homology between these viruses were not limited to one region of the genome but were found on a number of restriction fragments.

Comparison of the p26 gene region of two baculoviruses.

A 1.1-kb region of DNA containing the p26 gene of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was sequenced, transcriptionally mapped, and compared to the same region in the MNPV of Autographa californica (AcMNPV). The mRNA start site of the p26 gene occurs about 22 nucleotides downstream from an A/T-rich putative promoter sequence that is highly conserved between AcMNPV and OpMNPV. The p26 mRNA is transcribed through the p26 gene and coterminates with the p10 gene resulting in a mRNA containing copies of both genes. The reading frames of the OpMNPV and AcMNPV p26 genes showed 47% amino acid sequence homology which is clustered in six regions with over 65% amino acid homology. There was a distinct bias toward incorporation of G/C-rich codons in the OpMNPV p26 gene. No DNA homology was observed between the region upstream of the p26 gene in AcMNPV and OpMNPV. In AcMNPV, this region contains the homologous repeated (hr) sequence hr5. Hybridization of a plasmid containing an AcMNPV-repeated sequence (hr5) to Southern blots of the OpMNPV genome indicated that this repeated sequence is lacking in OpMNPV.

Identification, cloning, and R-Loop mapping of the polyhedrin gene from the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata

Polyadenylated RNA was isolated from Orgyia pseudotsugata larvae 8-10 days postinfection with the multicapsid nuclear polyhedrosis virus. This RNA was centrifuged through a sucrose gradient and fractions enriched for polyhedrin mRNA were identified by in vitro translation. Complementary DNA made to this RNA hybridized predominantly to a 5-kb fragment of XhoI-digested viral DNA. This fragment was cloned into the plasmid pACYC177 and mapped with restriction endonucleases. A SalI subclone with a 2.5-kb insert derived from the cloned XhoI fragment was found to select by hybridization only polyhedrin mRNA as determined by the size of the in vitro translation product and its precipitation by anti-polyhedrin antibodies. The orientation of the polyhedrin gene and the region of the insert encoding the N terminus of the polyhedrin protein were determined by DNA sequencing. R-Loop mapping indicated polyhedrin mRNA is 980 +/- 75 bases long and contains about 250 nucleotides not represented in the final protein. The polyhedrin gene had no observable intervening sequences.

Krebs cycle activity of particles from bean seedlings

Abstract A particulate fraction from etiolated seedlings of Phaseolus vulgaris was found to possess oxidative activity corresponding to the Krebs citric acid cycle. When added to preparations of the particles, intermediates of the cycle caused O 2 uptake equivalent to as high as 84 % of theoretical values for total oxidation. Pyruvate was oxidized only when added with a second intermediate of the cycle. In the presence of malonate, succinate accumulated when citrate or a fumarate-pyruvate combination was added as substrate. ATP, DPN, TPN, glutathione, phosphate, and manganous ions were found necessary for maximum activity of the particulate fraction. In addition, magnesium and cytochrome c were added to the incubation mixtures. Evidence is given for the presence of a glutamic-aspartic acid transaminase in the particulate fraction from Phaseolus vulgaris . Terramycin was added to incubation mixtures to inhibit the growth of bacteria, and to establish that the activity studied was not caused by bacteria.

Comparison of sequence diversity in several cytoplasmic polyhedrosis viruses.

Sequence homology among several different cytoplasmic polyhedrosis virus (CPV) types and the human reovirus (type 1) was examined by Northern blot analysis and S, nuclease analysis, using random-primed cDNA probes synthesized from total genomic RNA. The results show no homology among the CPV type 1, Bombyx mori CPV, type 5, Orgyia pseudotsugata CPV, type 8, Manduca sexta CPV and the human reovirus (type 1). However, there was significant homology among three type 5 CPVs, O. pseudotsugata CPV, Euxoa scandens CPV, and Heliothis armigera CPV. The O. pseudotsugata CPV and E. scandens CPV were 43-44% homologous while each was 6-13% homologous with the H. armigera CPVusing stringent conditions of hybridization.