Analysis of the C-polyhedrin genes from different geographical isolates of a type 5 cytoplasmic polyhedrosis virus.

The C-polyhedrin genes of two different geographic isolates of a type 5 cytoplasmic polyhedrosis virus (CPV) were cloned. A CPV infecting Orgyia pseudotsugata (OpCPV), isolated in the Pacific Northwest of the U.S.A., and a CPV infecting Heliothis armigera, isolated in South Africa, were studied. Both genes were found to be 883 nucleotides in length and encoded a predicted protein of 246 residues (M(r) of 28,890). Comparison of the nucleotide sequences of these two viruses with another type 5 geographic isolate, infecting Euxoa scandens (EsCPV; isolated in Eastern Canada), showed that there were only 17 nucleotide differences among the three genes. The only nucleotide variation that had an effect on the encoded protein was a deletion of nucleotide 774 in the gene of EsCPV. The deletion introduces a frameshift mutation resulting in the alteration of the carboxyl-terminal amino acid sequence. Sequence alignment of the OpCPV C-polyhedrin showed little homology to a type 1 CPV (infecting Bombyx mori) or with analogous proteins (N-polyhedrins) from two baculoviruses infecting O. pseudotsugata. Interestingly, most of the conserved residues between the N- and C-polyhedrins were either basic or aromatic amino acids.

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].

Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata.

The gene encoding the 39-kDa major structural protein (p39) of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) was sequenced and transcriptionally mapped, and its expression was examined at various times postinfection. By Northern hybridization, primer extension, and S1 nuclease analysis, we identified p39 mRNAs of approximately 2600 nt. By primer extension analysis, we identified two major sets of transcripts which initiated around -48 and -96 nt upstream of the translation start codon. The transcription start sites were located within the conserved baculovirus late gene consensus sequence, ATAAG, which is duplicated in the p39 5' flanking region. In OpMNPV-infected Lymantria dispar cells, the p39 mRNAs were expressed abundantly at 24 and 36 hr p.i. but were present in lower quantities at 48 hr p.i. The p39 gene contained an open reading frame of 1053 nt which encodes a predicted protein of 351 amino acids with an estimated molecular weight of 39.5 kDa. Three repeats of the amino acid sequence Ala-Pro-Ala-Ala-Pro were identified at the C-terminus of the predicted p39 protein.

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.

The genome of the multicapsid baculovirus of Orgyia pseudotsugata: restriction map and analysis of two sets of GC-rich repeated sequences.

Five cosmids containing inserts that comprise the complete genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata were mapped with four restriction enzymes (Bg/II, ClaI, SstI, XhoI). From these cosmid maps, composite maps of the complete genome were constructed for each restriction enzyme. A region containing repeats of the sequence GGC downstream of the polyhedrin gene was used to probe the genome. It cross-hybridized with a region which, upon sequence analysis, was found to be a highly repetitive GC-rich region of nearly 500 nucleotides. The two GC-rich regions appeared to be evolutionarily unrelated.

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.

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.

Nucleotide sequencing and transcriptional mapping of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus p10 gene.

A 32P-labeled cloned DNA fragment (AcMNPV HindIII-Q) containing one of the repeated sequences and a portion of the p10 gene from Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to probe Southern blots containing restriction endonuclease digests of Orgyia pseudotsugata muticapsid nuclear polyhedrosis virus (OpMNPV) DNA. A single 3.6-kb fragment, OpMNPV HindIII-Q, was hybridized. The OpMNPV HindIII-Q fragment was cloned into pUC-18, mapped with restriction endonucleases, and reprobed with the AcMNPV HindIII-Q fragment. A small region of ca. 700 bp, near the left end of the cloned fragment, was cross-hybridized. DNA sequencing in this region revealed an open reading frame of 279 bp which shares detectable homology with the p10 gene of AcMNPV. The sequences downstream from the p10 gene in both viruses also contain long open reading frames which share homology. Northern blot analysis of RNA from OpMNPV infected O. leucostigma cells was used to define the temporal and spatial organization of transcripts from this region. S1 analysis of both termini of the major p10 mRNA indicates nontranslated regions of 52-53 bases at the 5' end and 175 bases at the 3' end. The 5'-mRNA start site was located within a 12-nucleotide sequence which is conserved in all late hyperexpressed baculovirus genes.

The nucleotide sequence of the Pieris brassicae granulosis virus granulin gene.

Two overlapping restriction fragments containing the Pieris brassicae granulosis virus (GV) granulin gene were cloned into plasmids. The regions containing the coding region and the 5' and 3' flanking regions were subcloned into M13 and sequenced. The nucleotide sequence data were compared to those for the granulin gene from the Trichoplusia ni GV and the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus (AcMNPV). The amino acid sequences derived from these DNA sequences indicated that the two GV proteins are more closely related to each other (77% amino acid homology) than either is to the AcMNPV (about 53% amino acid homology for either GV). The N-terminal region shows the greatest degree of variation between these proteins. Highly conserved amino acid sequences were identified between the two GVs and were also found between NPVs. Certain of these conserved regions are shared between GVs and NPVs while others are not.

Cloning and sequencing of the granulin gene from the Trichoplusia ni granulosis virus.

A restriction fragment containing the granulin gene from the Trichoplusia ni granulosis virus was located in a blot of viral genomic DNA using a cloned polyhedrin gene as a probe. This fragment was cloned, mapped, subcloned, and the sequence of the coding region and 5' and 3' flanking regions was determined. Although granulin is very similar in size to nuclear polyhedrosis virus polyhedrins, the N-terminal region of granulin demonstrated a high degree of variability with the first 60 amino acids only 28% homologous to the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin sequence. Between amino acid 60 and the carboxyterminus at amino acid 248, the sequence was very similar (64%) to polyhedrin sequences. Overall the nucleotide and amino acid sequences were 58 and 53%, respectively, related to those of AcMNPV. No introns were evident in the gene.

Delta endotoxin of Bacillus thuringiensis subsp. israelensis.

From Bacillus thuringiensis subsp. israelensis, a proteinase-resistant protein was purified which exhibited toxicity to larval mosquitoes and cultured mosquito cells, lysed erythrocytes, and was lethal to mice. To extract the protein, a sporulating culture of B. thuringiensis subsp. israelensis was treated with alkali, neutralized, and incubated with trypsin and proteinase K. It was then purified by gel filtration and DEAE column chromatography. Up to 240 micrograms of toxic protein was purified from 1 g (wet weight) of culture pellet. Two closely related forms of toxic protein were obtained: the 25a and 25b proteins. The two forms comigrated near 25,000 daltons in a sodium dodecyl sulfate-polyacrylamide gel, were serologically related, and showed similar partial protease digestion profiles, but were distinguishable by DEAE chromatography and nondenaturing polyacrylamide gel electrophoresis. Protein sequencing data indicated the 25b protein lacked the two amino acids at the amino terminus of the 25a protein. A Western blot enzyme-linked immunosorbent assay of alkali-solubilized proteins that were not treated with proteases suggested the toxic 25a and 25b proteins were proteolytically derived from a larger molecule of about 28,000 daltons. Alkali-solubilized proteins from an acrystalliferous strain of B. thuringiensis subsp. israelensis and from B. thuringiensis subsp. kurstaki failed to cross-react with antibodies to the 25a protein.

Conservation of genome organization in two multicapsid nuclear polyhedrosis viruses.

The genome of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata was mapped by examining overlapping HindIII fragments from cosmid clones which had been constructed from partial HindIII digests of viral DNA. Five OpMNPV cosmid clones containing fragments encompassing the entire OpMNPV genome were hydridized to blots of DNA from the multicapsid nuclear polyhedrosis virus of Autographa californica. The hybridization pattern indicated that the genomes of these viruses are similarly organized.

Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses.

Monoclonal antibodies were produced to polyhedrins from Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) and single-capsid nuclear polyhedrosis virus (OpSNPV). Although the polyhedrins are closely related, antibodies were selected which allowed differentiation between the two viruses. In an indirect enzyme-linked immunosorbent assay, purified OpMNPV and OpSNPV polyhedrins could be detected by specific monoclonal antibodies at concentrations as low as 2 and 5 ng/ml, respectively. The antibodies were also capable of identifying their homologous polyhedrin in extracts of infected insects. These antibodies would be useful for monitoring production of the viral insecticide, TM Biocontrol-1, which by license must contain only OpMNPV, and to confirm that insect mortality after aerial spraying with this insecticide is attributable to OpMNPV infection.

Induction of endogenous avian tumor virus gene expression by pyrrolizidine alkaloids.

The pyrrolizidine alkaloids (PA) are toxic compounds which occur naturally in plant species throughout the world. They have been implicated as both carcinogenic and mutagenic agents. An active metabolite of the alkaloids, the pyrrole, which is a strong alkylating agent, is thought to be the toxicant. The naturally occurring alkaloid, jacobine , is able to induce the production of endogenous avian RNA tumor virus particles in cultured chick embryo fibroblasts (CEF). When jacobine was modified to form retronecine it no longer induced virus particles. Conversion of retronecine to its pyrrole resulted in a compound capable of inducing virus particle production. The isobutyryl monoester of retronecine was also able to induce virus particle production, but the isobutyryl monoester pyrrole was unexpectedly inactive as an inducer. This type of viral induction system is useful for studying the effect of modification of the inducer on its biological activity.

Characterization of a cytoplasmic polyhedrosis virus infecting Manduca sexta.

A cytoplasmic polyhedrosis virus (CPV) infecting Manduca sexta was characterized. The genome consisted of ten equimolar RNA segments ranging in estimated molecular mass from 0.7 to 3.84 Mdaltons and with a total estimated molecular mass of 21.75 Mdaltons . The sizes of the individual RNA segments provisionally placed this CPV within the type 8 classification of Payne and Rivers. The polyhedrin protein had an estimated molecular mass of 28 kdaltons . Six virion-associated proteins were observed, ranging in size from 27.1 to 160 kdaltons . The RNA and protein profiles were compared to profiles observed from two other CPVs infecting Orgyia pseudotsugata and Bombyx mori. In addition, the nucleic acid sequence homology, using 32P-labeled cDNA to the total M. sexta CPV genome, demonstrated no homology with the B. mori CPV or the O. pseudotsugata CPV. The M. sexta CPV was transmitted by per os inoculation to O. pseudotsugata larvae.

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.

Size determination of Orgyia pseudotsugata cytoplasmic polyhedrosis virus genomic RNA.

Molecular-weight estimates were determined for the Orgyia pseudotsugata cytoplasmic polyhedrosis virus (CPV) genome. The molecular-weight estimates were obtained from measurements of glyoxal denatured RNA molecules following electrophoresis in 1% agarose gels and electron micrographic contour lengths. These estimates were compared to the minimal coding capacity derived from the size of translation products of selected genomic segments. The results obtained from these different experimental approaches were in sufficient agreement to estimate the genome size at 20.4 x 106 (29.8 kbp) with a segmental range from 0.61 to 3.53 x 10(6) (0.89-5.19 kbp). Two other members of the Reoviridae family were examined, the Bombyx mori CPV and the human reovirus type 3 (Dearing strain). The molecular-weight estimates for these two viruses were 20.3 and 19.1 x 10(6), respectively. These results suggest that the previously published molecular-weight estimates for these various double-stranded RNA genomes were underestimated.

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.

N-Terminal polyhedrin sequences and occluded Baculovirus evolution.

A 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 and Orgyia 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 the B. mori NPV were from a report by Serebryani et al. (1977) and that for the O. 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, and T. paludosa NPV. These data, in conjunction with Baculovirus distribution and evidence concerning insect phylogeny, suggest that the Baculovirus have an ancient association with insects and may havae evolved along with them.

Induction of endogenous viral gene expression by halogenated hydrocarbons.

Endogenous avian RNA tumor virus gene expression was induced in chick embryo cells by the 1,1,1-trichloro-2-, 2-bis(p-chlorophenyl ethane) metabolites bis(chlorophenyl)acetic acid and dichlorodiphenyl dichloroethylene and the antibacterial agent hexachlorophene. Line 100 x 7, C/ABE, chick cell cultures were incubated with each of these chemicals or the known endogenous virus inducer bromodeoxyuridine. At subtoxic levels, all four chemicals induced the production of virus particles as determined by assay for the viral-specific reverse transcriptase and infectivity. Since the endogenous virus produced is of subgroup E and consequently unable to infect surrounding cells of the type C/ABE, amplification of the induced virions was accomplished by infecting the subgroup E susceptible cell line 15B C/C with supernatants from the chemically treated cultures. Fluorescent antibody treatment of line 100 x 7 C/ABE cultures showed approximately 35% to 40% of the cells stained in the chemically treated cell cultures, suggesting that only a portion of the cells were responding to the chemical induction.