Characterization of two abundant mRNAs of Autographa californica nuclear polyhedrosis virus present late in infection

Cytoplasmic poly(A)(+) RNA isolated from Spodoptera frugiperda cells late after infection with Autographa californica nuclear polyhedrosis virus (30-40 hr pi) was fractionated according to size on denaturing methyl mercury gels. Two major RNA species (1.4 kb and 0.75 kb) and several minor RNA species were detected by ethidium bromide staining. The predominant RNA species of about 1.4 kb was considered to be polyhedrin mRNA because (1) in vitro translation of the RNA, which was eluted from methyl mercury gels, yielded a polypeptide of MW 33K, which comigrated with polyhedrin. (2) When poly(A)+ RNA was fractionated on a sucrose column and then translated in vitro, the distribution and abundance profiles of a 33K polypeptide product and of 1.4-kb RNA were similar. (3) The 33K polypeptide made in vitro and purified polyhedrin gave rise to similar patterns of peptides when digested with S. aureus V8 protease. The polyhedrin mRNA (1.4 kb) hybridized to BamHI-F and HindIII-V AcNPV DNA fragments and hybridization selection with BamHI-F AcNPV DNA yielded a 33K polypeptide, which comigrated with polyhedrin. The second RNA species (0.75 kb in size) hybridized to overlapping EcoRI-P and HindIII-Q regions of the AcNPV genome and translated into a methionine deficient polypeptide of MW = 8K. It was synthesized in large quantities late in the infection and appeared to be coordinately expressed with polyhedrin in infected cells. The 8K polypeptide was detected as early as 15 hr pi and was still synthesized at 60 hr pi.     

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.     

A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment

A diagnostic method is described for the identification and differentiation of nucleopolyhedrovirus (NPV) pathogens of Helicoverpa species (Lepidoptera: Noctuidae) isolated from the environment. The method is based on the polymerase chain reaction (PCR) used in conjunction with restriction fragment length polymorphism (RFLP) analysis and comprises three parts. The first part describes procedures for obtaining PCR quality viral DNA from individual diseased H. armigera cadavers recovered during bioassay analyses of soil and other types of environmental sample. These procedures were modified from standard techniques used for the routine purification and dissolution of NPV polyhedra and provided an overall PCR success rate of 95% (n=60). The second part describes the design of several sets of PCR primers for generating DNA amplification products from closely and distantly related NPVs. These PCR primers were designed from published DNA sequence data and from randomly cloned genomic DNA fragments isolated from a reference H. armigera SNPV (HaSNPV) isolate. The final part of the method describes how specific PCR products when digested with specific restriction endonuclease enzymes, can be used to generate diagnostic DNA profiles (haplotypes) that can be used both to identify heterologous NPVs e.g. Autographa californica MNPV and related viruses, and to differentiate genotypic variants of Helicoverpa SNPV. In the latter case, only two PCR products and four restriction digests were required to differentiate a reference set of 10 Helicoverpa SNPV isolates known to differ 0.1--3.5% at the nucleotide level. The diagnostic method described below marks the second part of a two-phase quantitative-diagnostic protocol that is now being applied to a variety of ecological investigations. In particular, its application should lead to a significant improvement in our understanding of the distribution and population genetics of Helicoverpa SNPVs in the Australian environment, as well as providing a sound basis for the design of pre- and post-release monitoring systems for genetically enhanced bioinsecticides. It is also likely that this method can be adapted readily to the study of other insect pathogen associations important economically.     

Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system

The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection ofSpodoptera frugiperda (Sf9) cells with infectious DNA fromAutographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.     

Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia Coli

The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase. It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host. A clone was serendipitously isolated that expressed in E. coli a protein of approximately 120 kDa in size at high levels. DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF. Polyclonal antibodies raised against this protein purified from E. coli were used for immunological analysis. The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots. In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells. The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus. The replicating viral DNA and the polymerase were colocalized in these globular sites. Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.     

Cyclosporin A inhibits vaccinia virus replication in vitro

Summary The effect of the immune modulator, Cyclosporin A (CsA) on vaccinia virus replication has been examined in cell cultures. In the present study we report that CsA is anti-viral towards vaccinia virus. Viral yield was inhibited by more than 97% after 24 h postinfection in the presence of 16 µM to 40 µM CsA. An analysis of the infectious cycle in greater detail revealed that CsA did not effect the total level of [³⁵S] methionine incorporation into vaccinia infected cells. However, both early and late viral gene expression were inhibited by CsA. Late viral protein synthesis appeared to be more sensitive to the drug. At least one late viral polypeptide of approximately Mr 38 000 was virtually undetected up to 8 h postinfection in the presence of 40 µM CsA. Host protein synthesis which is normally inhibited by the virus was not turned off until very late in infection. Viral DNA replication was also inhibited by the addition of CsA at levels comparable to those observed for late protein synthesis.     

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.     

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV.     

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbp knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.     

HearSNPV orf83 encodes a late, nonstructural protein with an active chitin-binding domain

The ORF83 (ha83) of Helicoverpa armigera nucleopolyhedrovirus (HearSNPV) was characterized during the present study. Sequence analysis and chitin-binding assay revealed that Ha83 contained an active chitin-binding domain. Northern blot and Western blot analyses demonstrated that ha83 was expressed as a late gene and encoded a nonstructural protein of HearSNPV. Ha83 gene was transcribed beginning at 12h post-infection in infected Helicoverpa zea cells (HzAM1). Western blot analysis using a rabbit derived polyclonal antibody showed the product of ha83 in infected cells was a 20 kDa protein, in tune with the theoretical size of 18.8 kDa. The protein was first detected in the cytoplasm of infected HzAM1 cells at 12h p.i., and was transported later into the nucleus during infection.     

Characterization of Spodoptera litura multicapsid nucleopolyhedrovirus 38.7 k protein, which contains a conserved BRO domain

Homology analysis revealed that Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) 38.7 k protein has 22-83% amino acid identities with Ecotropis obliqua NPV, Mamestra configurata MNPV, Helicoverpa armigera SNPV, H. zea SNPV, S. exigua MNPV and S. littoralis MNPV 38.7 k proteins. Analysis of the relationship of these 38.7 k proteins indicated that they contain a conserved BRO-N domain, and SpltMNPV and SpliMNPV 38.7 k proteins also contain a motif found in all known viral and prokaryotic single-strand DNA binding proteins. RT-PCR results showed that SpltMNPV 38.7 k gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter motif (ATAAG). Western blot analysis revealed that the 38.7 k was expressed in infected S. litura cells as a 41 kDa form and this protein distributed in the nucleus of infected cells. Using a histone extraction protocol, SpltMNPV 38.7 k could be detected in the histone H1 fraction. Micrococcal nuclease treatment released SpltMNPV 38.7 k protein from the chromatin fraction, suggesting that its involvement in nucleosome structures. Furthermore, column chromatography using DNA-cellulose showed that SpltMNPV 38.7 k protein interacted with nucleic acids. It was proposed that SpltMNPV 38.7 k might function as a DNA-binding protein.     

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.     

Molecular characterization of genes in the GP41 region of baculoviruses and phylogenetic analysis based upon GP41 and polyhedrin genes.

A newly sequenced Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) gp41 gene was used to reconstruct the phylogeny for gp41 by comparison with Autographa californica MNPV, Bombyx mori MNPV, Helicoverpa zea single nucleopolyhedrovirus (SNPV), Lymantria dispar MNPV, Orgyia pseudotsugata MNPV and Spodoptera frugiperda MNPV. The 3.5 kb fragment of the AgMNPV gp41 region not only contained the gp41 gene but also three other open reading frames that had significant homology with the very late factor (vlf-1) of baculoviruses, AcMNPV ORF78, AcMNPV ORF79, and one partial open reading frame homologous to AcMNPV ORF81. The reconstructed phylogenetic tree of baculovirus gp41 genes compared with the polyhedrin gene tree produced similar topologies. Two other phylogenetic trees were reconstructed based on either combined gp41 and polyhedrin nucleotide sequences (total evidence) or combined evolutionary histories of both genes (strict consensus tree). The former had an identical tree topology as the gp41 gene tree alone, and the latter lost resolution in the branch of AcMNPV and BmMNPV. Mutation rate analysis showed the gp41 gene had a higher nucleotide substitution rate than the polyhedrin gene, implying that the polyhedrin gene may have a different selection constraint than the gp41 gene. Both genes have nonsynonymous/synonymous substitution values close to 0.1, similar to other DNA viruses.