Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.

In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar.

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

Analysis of p74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo.

In nature, nuclear polyhedrosis viruses (NPV) are transmitted when susceptible insect larvae ingest viral occlusion bodies (OB). These dissociate in the alkaline environment of the midgut and release encapsulated virions (PDV) which bind to midgut epithelial cells and initiate an infection. A previous study showed that expression of the Autographa californica NPV (AcMNPV) p74 gene during replication is essential for the production of infectious OB. A set of p74 deletion and overexpression recombinants was used for the production and screening of monoclonal antibodies, and for an investigation of gross cytopathology and localization of p74. No differences in virus structure or morphogenesis were observed in infected cells when the p74 gene of AcMNPV was deleted, even though the infectivity of OB harvested from the cells was abolished when they were fed to Trichoplusia ni larvae. Mutant OB released virus particles and degraded insect peritrophic membrane as in infections with wild-type virus; in addition, virions purified from mutant OB were infectious when injected into the haemocoel of T. ni larvae. Western blot analysis confirmed that p74 was associated with the PDV and could not be detected in the budded form virion phenotype. The polypeptide was readily degraded by treatment of purified PDV with proteinase K, in the presence and absence of detergent, and could be extracted from PDV by a non-ionic detergent treatment. The data are consistent with p74 being a structural polypeptide of the PDV phenotype, most probably as a component associated with the outside surface of the virion envelope. Its presence is shown to be essential for primary infection of midgut cells of insect larvae.

Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes.

We examined the role of the Autographa californica nucleopolyhedrovirus (AcMNPV)-encoded chitinase in virus pathogenesis in Trichoplusia ni larvae. In conjunction with the AcMNPV-encoded cathepsin, it promotes liquefaction of the host in the latter stages of infection. Insects infected with virus mutants lacking either the chitinase A gene (chiA) or cathepsin gene (cath) remained intact several days after death. However, if both viruses were used to infect insects, liquefaction of the host was restored. Chitinase was readily detected in AcMNPV-infected insects using a chitinase-specific antibody, but it was absent from insects infected with a chiA deletion mutant (AcchiA-). The chitinase was also detected in polyhedra purified from AcMNPV-infected insects but not in those from AcchiA-. However, polyhedra derived from a virus lacking an intact chiA were no less effective in initiating an infection in second instar T. ni larvae than those of the unmodified AcMNPV. It was also demonstrated that the virus chitinase retained high levels of activity between pH 3.0 and 10.0. In contrast, chitinases isolated from Serratia marcescens, although active under acidic conditions, rapidly lost activity above pH 7.0 illustrating that despite 57% sequence identity, the two proteins have distinct enzymic activities.

Characterization of the baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus p10 gene indicates that the polypeptide contains a coiled-coil domain.

The DNA sequence and transcription pattern of the p10 gene from the spruce budworm baculovirus Choristoneura fumiferana multicapsid nuclear polyhedrosis virus (CfMNPV) were analysed. The CfMNPV p10 gene codes for a protein 81 amino acids in length: this is the shortest p10 peptide identified thus far. A novel characteristic of the CfMNPV p10 gene is that it contains tandem late initiation sites (TAAG) having different temporal transcription patterns. Comparative analysis of CfMNPV p10 and the amino acid sequences of other p10 gene products showed that they each appear to have a similar N-terminal structure: an amphipathic alpha-helical terminus which condenses as coiled-coil multimers. Another feature of the p10 N terminus is that the central region of the coiled-coil domain resembles a bend or hairpin loop and could fold into a hairpin or form a bent rod structure. The condensation of p10 monomers to coiled-coil multimers is likely to be a step leading to the formation of p10 fibrous bodies in infected cells.

Identification and characterization of the v-cath gene of the baculovirus, CfMNPV.

The v-cath gene of the Autographa californica multi-nucleocapsid nuclear polyhedrosis virus (AcMNPV) encodes a cathepsin L-like proteinase which plays a role in the liquefaction of host tissues during a viral infection [1]. We have identified a homologous gene in the spruce budworm virus, Choristoneura fumiferana MNPV (CfMNPV). The CfMNPV v-cath gene is 74% identical to AcMNPV v-cath at the nucleotide sequence level and 80% identical at the level of predicted amino acid sequence. Transcription analysis of the CfMNPV v-cath gene revealed that it is expressed late in infection and that transcription initiates within the consensus baculovirus late-promoter motif.

Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) contains a 966 bp ORF that encodes a papain type cysteine proteinase with cathepsin L-like characteristics. Using Western blot analysis of infected cell extracts we showed that v-cath proteinase has 35.5 kDa and 32 kDa precursor forms which are processed to a 27.5 kDa mature form in a manner characteristic of papain and cathepsin L. V-cath proteinase activity was greatest under acidic conditions (pH 5.0) and was reduced in the presence of the cysteine proteinase inhibitors, leupeptin and E64. Urea, a known enhancer of cathepsin L activity, also enhanced v-cath proteinase activity. AcMNPV v-cath proteinase was detected post-mortem in tissues of insects infected with wild-type (wt) virus. Insects infected with a v-cath deletion mutant did not become flaccid after death as is normally observed with wt AcMNPV infections. These findings indicate a link between v-cath activity and degradation of host tissues during virus pathogenesis.

The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.

The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence data confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.

Molecular analysis of archael flagellins: similarity to the type IV pilin-transport superfamily widespread in bacteria.

Ultrastructural, biochemical and genetic evidence has shown that the flagella and flagellin proteins from members of the archaea are distinct from their bacterial counterparts. The most important evidence is the sequence dissimilarity between archael and bacterial flagellins. We report here similarity between archael flagellins and members of the bacterial type IV pilin-transport superfamily. In addition to sequence similarity, the archael flagellins and the type IV pilin-transport superfamily share an unusual signal sequence cleavage site and may have functional parallels. This relationship has important implications for the assembly and biogenesis of archael flagella.

Nucleotide sequence of the p74 gene of a baculovirus pathogenic to the spruce budworm, Choristoneura fumiferana multicapsid nuclear polyhedrosis virus.

Polypeptide p74 has been found to be essential for production of virulent occlusion bodies of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). Hybridization with AcMNPV-derived probes has led to the location of the p74 gene in the spruce budworm virus, Choristoneura fumiferana MNPV. Sequence data indicate that CfMNPV p74 is 73% identical to AcMNPV at the nucleotide level and 77% identical at the amino acid level. Elements of predicted secondary structure are also conserved.

Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus.

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.

Identification of p74, a gene essential for virulence of baculovirus occlusion bodies.

DNA sequencing of the HindIII-P fragment of the baculovirus Autographa californica nuclear polyhedrosis virus downstream of a major late protein, p10, revealed the presence of an open reading frame (ORF) 1935 nucleotides in length and in opposite polarity to p10. The gene product is considered essential for virus virulence in Trichoplusia ni larvae since infection with occlusion bodies from a mutant, Ac228z, in which portions of adjacent carboxy-termini from peptides p74 and p10 were deleted, failed to kill larvae, whereas virus with deletions in p10 alone were as infectious to larvae as wild-type virus. The ORF has the potential to code for a polypeptide of 645 amino acid residues (Mr 73,819) and was designated p74. Time course analysis of RNA from infected cells using primer extension assays suggested that the gene's promoter was weak and was most active at 16-20 hr postinfection. The transcription initiation site of the RNA was located at -90/-91 bases upstream of the start codon. The p74 gene was cloned into a baculovirus expression vector and a recombinant virus was produced which overexpressed the p74 protein.

Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus.

Monoclonal antibodies with specificity for the abundant envelope surface glycoprotein (gp67) of Autographa californica nuclear polyhedrosis virus (AcMNPV) were used to screen a lambda gt11 expression library of AcMNPV DNA fragments. The gp67 gene was mapped to the left end of the EcoRI H fragment in a right-to-left orientation on the consensus map of AcMNPV. A 2.1-kilobase transcript which hybridized to the region was first detected in cell extracts at 2 h postinfection; it peaked in abundance at 18 h postinfection and thereafter was present at lower levels. The nucleotide sequence of the region was determined, and a 1,590-nucleotide open reading frame flanked by an AT-rich sequence was identified that could encode a polypeptide with 529 amino acid residues (molecular mass of 60,167 daltons). Computer analysis indicated that the peptide possesses two hydrophobic regions near the N and C termini as well as six potential N-linked glycosylation sites. We suggest that following cleavage of a signal peptide, the polypeptide undergoes further processing and becomes anchored at its C terminus in the virus envelope. The final seven amino acid residues at the C terminus contain basic amino acids and may have a role in virion assembly.

A cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion/deletion mutants.

The role of the Autographa californica nuclear polyhedrosis virus p10 gene in viral cytopathology and morphogenesis was examined using classes of p10 deletion mutants with and without lacZ (beta-galactosidase) gene fusion. Mutant-infected cells did not form the fibrillar cytoplasmic and nuclear structures normally observed late in infection with wild-type (wt) virus, and the cells failed to lyse even at 2 weeks post-infection. Based on wt and mutant cytopathology, we suggest lysis may be facilitated by stepwise exhaustion of the host nuclear membrane, and may require a function resident in the carboxy region of p10; this portion of the molecule is also essential for formation of the p10-rich fibrillar bodies. Additional changes in cytopathology were correlated with the level of p10/LacZ fusion protein expression. The insertional mutant designated Ac229, which encodes 51 N-terminal amino acids of p10 fused to LacZ, caused intranuclear accumulation of granular structures at sites corresponding to the fibrillar bodies of wt viral infections. Occlusion body membranes, which associate with the fibrillar bodies in wt infections, were also formed in mutant virus-infected cells. However, membranes did not associate with occlusion bodies in Ac229 infections, and were aberrantly attached to occlusion bodies in cells infected with mutants having simple p10 deletions (represented by Ac231). Loss of the outer membrane increased sensitivity of the occlusion bodies to disruption by physical stress; a partially attached membrane afforded some protection from disruption.

Nucleotide sequence of the p10 polypeptide gene of Autographa californica nuclear polyhedrosis virus.

A portion of the Autographa californica nuclear polyhedrosis virus genome lying within the EcoRI-P region of the physical map has been sequenced. Previous studies employing Northern blotting and in vitro translation had shown that this region encoded the gene for a "late" 8-10K polypeptide [D. Z. Rohel, M. A. Cochran, and P. Faulkner, Virology 124, 357-365, (1983); G. E. Smith, J. M. Vlak, and M. D. Summers, J. Virol.45, 215-225, (1983)]. The 1313-base pair region contained an open reading frame for a polypeptide of 93 amino acid residues of MW 10,138. The predicted amino acid composition based on the DNA sequence lacked the amino acids methionine, cysteine, tryptophan, tyrosine, and histidine and was similar in composition to that of an abundant nonstructural polypeptide, p10, purified from infected cell extracts. S1 mapping located the origin of transcription 64 by upstream from the translation start codon. A possible stem and loop structure was detected within the untranslated leader sequence. Examination of the base sequence revealed the presence of TATAAT and CAATAT elements upstream from the transcription start site and potential polyadenylation signals lying downstream from the polypeptide termination codon. Computer analysis revealed other potential open reading frames within and extending from the ends of the sequenced region.

Regions of repeated DNA in the genome of Choristoneura fumifefana nuclear polyhedrosis virus.

The genome of the nuclear polyhedrosis virus of the spruce budworm, Choristoneura fumiferana (CfMNPV), was shown, by Southern blot hybridization, to contain regions of repeated DNA sequences. These homologous regions have been located at four sites on the physical map of CfMNPV. In addition, the DNA at one of these sites is expanded, possibly via tandem duplication, in a fraction of the total viral DNA population.

Genomic variants of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus containing specific reiterations of viral DNA.

Restriction endonuclease analysis of DNA from isolates of a temperature-sensitive mutant, ts.8, of Autographa californica nuclear polyhedrosis virus revealed the presence of genomic variants. ts8 type A plaque isolate DNA had altered restriction patterns while ts8 type B plaque isolate DNA restriction patterns were similar to wild-type viral DNA. The alterations in ts8 type A included an extra 1.35 kilobase pair (kbp) EcoRI fragment, and a series of extra Hind111 and XhoI restriction fragments present in decreasing molar ratios with increasing molecular weight.Southern blot and DNA-DNA cross-blot analysis indicated that the ts8A extra restriction fragments were homologous to viral sequences from the 90% region of the genome. The t&A extra restriction fragments appeared to result from an insertion of repeated viral DNA sequence at the EcoRI P-B junction. The higher molecular weight ts8A Hind111 and XhoI submolar fragments appeared to have increasing copy numbers of the 1.35 kbp EcoRI-EcoRI repeat-unit flanked by authentic EcoRI fragment P and B sequences. Approximately 57% of the ts8 plaques isolates analyzed were type A and 43% were type B genomic variants. Although type A and type B genomic variants appeared to be stable in high multiplicity of infection passage experiments, lo-20% of plaque isolates in stock homogeneity experiments converted from one type to another. The genomic alterations did not appear to have any observable effect on the temperature-sensitive phenotype of the mutant.