Identification of additional genes that influence baculovirus late gene expression.

We were unable to confirm transient late gene expression using constructs of 18 genes that had been reported to support Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) late gene expression when transfected into Spodoptera frugiperda cells [Lu, A., and Miller, L. K. (1995). J. Virol. 69, 975-982]. Three genes (orf66, orf68, and orf41) were included, all or in part, in the constructs used in that study, but they had not been independently tested. Therefore we investigated these and neighboring orfs for their influence on late gene expression. We found that orf41 was required for late gene expression and that sequences within orf45 appeared to be required for the expression of orf41. Although orf66 and orf68 did not appear to affect late gene expression, orf69 stimulated expression. orf69 was found to have high homology to recent entries in GenBank from a variety of organisms. In addition, it was found that orf121, which was shown to be involved in early gene expression, and the viral homolog of pcna did not influence late gene expression.

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.

AcMNPV late expression factor-5 interacts with itself and contains a zinc ribbon domain that is required for maximal late transcription activity and is homologous to elongation factor TFIIS.

The late expression factor-5 gene (lef-5) of Autographa californica multinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione-S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However, all deletions tested involving the N-terminal 194 aa significantly reduced LEF-5:LEF-5 interaction. LEF-5 or LEF-5 deletion mutants were transfected into Sf-9 cells with the full complement of genes required for baculovirus late transcription. All deletion clones tested reduced expression of a beta-glucuronidase (GUS) reporter gene under control of the late vp39 capsid promoter. Amino-acid sequence analysis of LEF-5 identified a previously unreported domain within the C-terminal 32 aa that is homologous to the zinc ribbon domain of RNA polymerase II elongation factor IIS (TFIIS) from a variety of taxa. Molecular modeling of the putative LEF-5 Zn ribbon using the NMR data available for the Zn ribbon of TFIIS suggested that this domain could fold into a Zn ribbon structure similar to TFIIS. Alanine scanning mutagenesis of amino acids predicted to be important for functioning of the LEF-5 ribbon structure significantly reduced LEF-5 activity in transient expression assays. Mutations changing the amino acids predicted to coordinate Zn2+ caused a reduction in activity similar to that when the domain was eliminated completely.

Production of early expressed parasitism-specific proteins in alternate sphingid hosts of the braconid wasp Cotesia congregata.

Parasitism of Manduca sexta larvae by the braconid wasp Cotesia congregata or injection of C. congregata polydnavirus (CcPDV) causes numerous alterations in host physiology, including developmental arrest, abrogation of host immunity, and the production of three abundant early expressed proteins (EP1, EP2, and EP3) that are secreted in large amounts into the host's hemolymph. Here we compare the levels of these proteins present in the hemolymph of three other sphingid species that vary in their compatibility for C. congregata. Hyles lineata was found to be permissive for C. congregata and EP1, EP2, and EP3 were present in larval hemolymph at levels comparable to those found in hemolymph from parasitized M. sexta larvae. By contrast, the lowest levels of EP proteins were found in hemolymph from parasitized Pachysphinx occidentalis larvae and this species was found to be completely refractory, since C. congregata eggs were invariably encapsulated. Parasitism of Sphinx vashti by C. congregata resulted in moderate levels of EP production. While the observed immune response was incomplete and some encapsulation of C. congregata eggs and/or larvae was observed, low numbers of S. vashti nevertheless were able to complete their development and emerge as adults. Thus, a correlation was established between host compatibility and induction of synthesis of the three parasitism-specific proteins, although the linkage between quantitative levels of EP production and the extent of encapsulation was variable.

Differential stability of baculovirus late transcription complexes during initiation and elongation.

An in vitro system for baculovirus late transcription was developed that allows comparison of conditions that affect transcription initiation and elongation. A series of synthetic promoters was constructed based on the baculovirus late p6.9 promoter. The modified promoters were designed with a cytidine-free region downstream of the late promoter in order to yield paused transcripts of defined lengths in the absence of CTP. Transcription was found to be more efficient from a supercoiled template than from a linear template for this promoter. The stalled transcription complex remained competent and could be elongated in the presence of a full set of nucleotides. This made it possible to separately test the effects of heat treatment and inhibition by sarkosyl and heparin on initiation and elongation. Elongation complexes were more resistant than initiation complexes to each of these treatments. Furthermore a 1-2 mM MgCl2 concentration is critical for optimal initiation, but elongation can proceed in the presence of MgCl2 concentrations as high as 20 mM.

The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome.

The nucleotide sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and the Autographa californica MNPV were found. These include the presence in OpMNPV of three complete iap gene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses.

An abundantly expressed hemolymph glycoprotein isolated from newly parasitized Manduca sexta larvae is a polydnavirus gene product.

Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp C. congregata. CcPDV virions are present in large numbers in the oviducts of C. congregata and injected with eggs into the hemocoel of M. sexta larvae during parasitization. Injection of sucrose density purified virions into nonparasitized larvae causes several of the parasitism-induced alterations in the physiology of host larvae that occur as a result of natural parasitism, including the synthesis of novel hemolymph proteins and abrogation of the host's immune response against the developing parasites. One of these proteins, early-expressed protein 1 (EP1), is a 190-kDa molecule which constitutes up to 5% of the total hemolymph protein by 24 hr following oviposition by the wasp. Using N-terminal sequence data for EP1 to construct primers for use in the polymerase chain reaction, we amplified and cloned a cDNA corresponding to the gene encoding EP1. This cDNA hybridized to DNA of the CcPDV genome, but not to DNA isolated from M. sexta larvae, suggesting that EP1 is a CcPDV gene product. A cDNA clone was isolated from an expression library generated from RNA extracted from newly parasitized M. sexta larvae. Sequence analysis of the cDNA clone revealed the presence of an open reading frame of 819 bp encoding a protein of 30.7 kDa. In vitro transcription/translation of the cDNA clone produced a protein of approximately 31 kDa, which was immunoprecipitated by EP1-specific polyclonal antiserum generated against purified deglycosylated EP1. EP1-like sequences also were amplified from male wasp genomic DNA, suggestive of integration of EP1-like sequences in the genome. This report constitutes the first evidence that a specific protein isolated from a parasitized host insect is a wasp polydnavirus gene product.