Characterization of the Helicoverpa armigera and Pseudaletia unipuncta granulovirus enhancin genes.

Enhancins are baculovirus proteins capable of enhancing infections in insect larvae by other baculoviruses. We have identified the enhancin proteins in four species of granulovirus (GV). In this paper we describe the cloning and sequencing of the enhancin genes of the Pseudaletia unipuncta granulovirus-Hawaiian strain (PsunGV-H) and the Helicoverpa (Heliothis) armigera granulovirus (HearGV). The PsunGV-H enhancin gene is virtually identical to the previously characterized Trichoplusia ni GV (TnGV) enhancin gene. In contrast, a comparison of the predicted amino acid sequences of TnGV enhancin (901 amino acids) and HearGV enhancin (902 amino acids) revealed an overall identity of only 80%, with greater conservation (88%) from amino acids 1-550. Primer extension analysis of enhancin RNAs identified the baculovirus late promoter motif that serves as the transcriptional start site in the HearGV enhancin gene. It is located three nucleotides from the putative enhancin translational initiator codon. RNase protection analysis demonstrated that both read-through and termination occur at the 3' end of the gene. Since a partial open reading frame (ORF) was identified immediately downstream of the 3' end of the enhancin ORF, these data suggested that a sizeable fraction of the enhancin mRNAs may be bi-cistronic and share a common 3' end with a downstream transcription unit.

Cloning and sequence analysis of a p40 structural protein gene of Helicoverpa zea nuclear polyhedrosis virus.

A gene encoding an occluded virion structural protein was isolated from an expression library constructed from the Helicoverpa zea S-type nuclear polyhedrosis virus (NPV) isolate HzS-15 using both polyclonal and monoclonal antibody screening. The gene was located within a Pstl-Sall fragment of the HzS-15 genome spanning from 96.5 to 97.3 m.u. Sequencing analyses revealed a long open reading frame of 927 nucleotides that predicted a protein of 37 kDa in size. Immunoblot analyses using the monoclonal antibody ENV409 demonstrated that the gene corresponded to a 40-kDa protein (p40) in SDS-polyacrylamide gels that was present exclusively in enveloped occluded virions but not in extracellular budded virions or envelope-stripped nucleocapsids. A p40 protein-specific transcript was detected at 16 hr postinfection in HzS-15-infected Hz 1075/UND-K cells and remained until 22 hr. Primer extension analyses demonstrated that the p40 protein-specific transcript started at -49 nucleotides from the ATG start codon within an ATAAG consensus pentamer found in late protein genes of Baculoviruses. The deduced amino acid sequence of the HzS-15 p40 protein gene shared 44 and 45% sequence homology with the p40 proteins of Bombyx mori NPV and Autographa californica NPV, respectively.

Location and nucleotide sequence of the gene encoding the viral enhancing factor of the Trichoplusia ni granulosis virus.

The gene encoding the viral enhancing factor (VEF) of Trichoplusia ni granulosis virus has been cloned from a lambda gt11 expression library, and the complete nucleotide sequence determined. The VEF gene encodes a protein with a predicted Mr of 104K which does not share homology with any previously reported proteins. A possible promoter is located four nucleotides upstream of the initiation codon and represents a consensus baculovirus late promoter (ATAAG). This has been confirmed by the identification of VEF mRNA in Northern blots of infected larvae 6 days but not 3 days post-infection. Using an anti-VEF-TrpE polyclonal antiserum in Western blots of dissolved viral occlusion bodies, related proteins have been identified in both Pseudaletia unipuncta granulosis virus Hawaiian strain (PuGV-H) and Heliothis armigera GV (HaGV), but not in Erinnyis ello GV (EeGV), T. ni singly enveloped nuclear polyhedrosis virus (TnSNPV) or Autographa californica multiply enveloped NPV (AcMNPV). Similar results were obtained with Southern blots of genomic digests. DNA fragments homologous to an internal portion of the VEF gene were found in PuGV-H and HaGV but not in EeGV, TnSNPV or AcMNPV.

Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.

The transposable IFP2 element of Trichoplusia ni was originally isolated as a host DNA insertion in spontaneous FP mutants of Galleria mellonella or Autographa californica nuclear polyhedrosis viruses (NPVs). The termini of IFP2 insertions from five independently isolated FP mutants were sequenced. In all cases IFP2 is flanked by 13-bp terminal inverted repeats and has additional inverted repeats of 19 bp in length located asymmetrically with respect to the ends of the element. Insertion of IFP2 into the viral genome always generated a duplication of the tetranucleotide target site, TTAA. There was an apparent preference for insertion within a 12-bp A + T-rich imperfect palindromic sequence surrounding the target site. Sequence analysis of three independent IFP2 elements revealed an internal domain of 2.475 kb containing an RNA polymerase II promoter region and two large open reading frames. Primer extension analysis of IFP2-specific mRNA positioned the 5' terminus of the transcript. The element is present in DNA isolated from T. ni cell lines TN-368 and TN-5B1, but is not apparent in DNAs isolated from the TN-R2 cell line or our laboratory colony of T. ni larvae, suggesting IFP2 was recently introduced into the T. ni genome.

Transfection of cloned Heliothis zea cell lines with the DNA genome of the Heliothis zea nuclear polyhedrosis virus.

Transfection conditions were optimized for the cloned UND-K derivative of the IPLB-HZ 1075 cell line using the calcium-phosphate co-precipitation technique and the DNA genome of the Heliothis zea S-type nuclear polyhedrosis virus. Optimal efficiencies were obtained using supercoiled viral DNA, and by extending the adsorption period for the diluted precipitate to 12 h. Transfection efficiencies ranging from 0.5 to 1.3 x 10(3) plaque forming units per microgram of supercoiled viral DNA were routinely obtained for UND-K cells and HzS-15 viral DNA. Transfection efficiencies were compared for 10 other cloned Heliothis cell strains and the uncloned parental IPLB-HZ 1075 cell line. The cloned cell strains UND-F, L, and U were incapable of transfection, while UND-I and G were 3 and 131 fold (respectively) less efficient than UND-K. The UND-K cells and the calcium phosphate transfection procedure permit relatively efficient in vitro manipulation of the Heliothis zea NPV virus genome.

Characterization of genotypic and phenotypic variation in plaque-purified strains of HzSNPV Elkar isolate.

Twenty plaque-purified strains of Heliothis zea S-type nuclear polyhedrosis virus (HzSNPV) were characterized based upon restriction endonuclease digests of viral DNAs, structural protein profiles, and larval melanization response. Each of the 20 strains had a unique genotype. Genomic heterogeneity between strains was localized to 4 regions of the HzSNPV genome. Differences in protein profiles of occluded virus were noted for each strain relative to the wild-type isolate. The strains were separated into 3 groups based upon differences in relative virulence and rate of larval melanization upon death. There was no correlation between differences in pathology and specific alterations in either genotype or structural proteins. The genotype of strain HzS-15 was extensively characterized. Physical maps were constructed for the HzS-15 strain and all of the plaque-purified strains.