INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Baculoviruses are viruses that are specific to insects and other invertebrates and include two genera, the nucleopolyhedroviruses (NPVs) and the granuloviruses (GVs). Both groups of viruses produce virions that are similar in both structure and pathology of infection, though NPVs have members that can package virions singularly or in groups and GVs can package virions only singularly (8, 10). Another distinction between these viruses is that NPVs produce occlusions that contain many viral particles and GV occlusions contain a single viral particle. Sequence analysis of NPV and GV genomes revealed that the NPVs and GVs contain many homologous genes (15, 16, 24).

Enhancins are proteins first found in GV occlusion bodies that have the ability to enhance the infection of some NPVs. Also referred to as virus-enhancing or synergistic factors, enhancins were first identified and isolated by Tanada (34, 37; see reference 35 for a review). The enhancin protein of Pseudaletia unipuncta GV (PuGV) was found to enhance the infection of P. unipuncta (PuNPV) only when both the PuGV protein and PuNPV were inoculated orally, indicating that the midgut is the site of the enhancin activity (38). The enhancin protein purified from the Trichoplusia ni GV (TnGV) enhanced Autographa californica multinucleocapsid NPV (AcMNPV) infection in Helicoverpa zea, Spodoptera exigua, P. unipuncta, and T. ni by 2- to 14-fold, depending on the host species (41). At present, enhancin genes have been sequenced in four GVs, Helicoverpa armigera GV (HaGV) (30), PuGV (30), TnGV (14), and Xestia c-nigrum GV (XcGV) (16). The first GV genome to be completely sequenced, XcGV, was found to have four different enhancin genes (16). In contrast, the Plutella xylostella GV genome lacks an enhancin gene (15).

Two functions have been proposed for enhancins, enhancement of virus-host cell fusion and disruption of the peritrophic membrane. The PuGV enhancin was thought to facilitate the uptake of virus particles by increasing the number of virus-membrane fusion events (23, 35, 36, 39). A specific binding site of the TnGV enhancin has been found in the midgut brush border membrane of P. unipuncta but not in the brush border membranes of T. ni, H. zea, or S. exigua, all of which demonstrate a response to the TnGV enhancin when infected with AcMNPV (41). The TnGV enhancin was found to damage the peritrophic membrane lining the larval midgut, exposing the gut wall to viral infection (7). Bioassay results of T. ni larvae infected with AcMNPV and various concentrations of the TnGV enhancin demonstrated that the major effect of enhancin appears to be an increase in infection efficiency caused by the disruption of the insect peritrophic membrane (11). This enhancin was later found to be a metalloproteinase, which degrades mucin, a major protein constituent of the peritrophic membrane (25, 40).

The Lymantria dispar MNPV (LdMNPV) is a baculovirus pathogenic to L. dispar, the gypsy moth, a forest and urban tree-defoliating pest in the northeastern and some midwestern states. The genome of LdMNPV is significantly larger than that of most other baculoviruses. The genome of the sequenced strain of LdMNPV is 161,046 bases (24), in contrast to 133,894 bases for AcMNPV (2), 128,413 for Bombyx mori NPV (12), 131,403 bases for H. armigera single nucleocapsid NPV (6), 131,990 bases for Orgyia pseudotsugata MNPV (1), and 135,611 bases for S. exigua MNPV (19). LdMNPV contains several unique open reading frames (ORFs) as well as ORFs with homology to GV ORFs. The first GV homolog found in LdMNPV was the enhancin 1 (E1) gene, which was also the first enhancin gene found in NPVs (5). The LdMNPV E1 gene product exhibits 29% amino acid identity to the sequenced enhancin genes of TnGV, PuGV, and HaGV and contains a conserved zinc-binding domain characteristic of metalloproteases. E1 gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter.

A viral isolate lacking a functional E1 gene was constructed to investigate enhancin function. Potency analysis revealed that viral strain without the enhancin 1 gene was approximately 2- to 3-fold less potent than wild-type viruses, suggesting that the LdMNPV enhancin affects viral potency (5). A second enhancin gene (E2) was recently identified in LdMNPV when the entire genome of isolate 5-6 was sequenced (24). Five other NPVs have been completely sequenced, but no enhancins have been found (AcMNPV [2], B. mori NPV [12], H. armigera NPV [6], O. pseudotsugata MNPV [1 ], and S. exigua MNPV [19]). In this study, we characterized the E2 gene and determined the effect of the enhancin genes both individually and collectively on viral potency.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Cells, virus, and insects. L. dispar 652Y cells were grown as monolayers at 27°C in Goodwin's IPL-52B medium (JRH Scientific) supplemented with 6.25 mM glutamine (Gibco-BRL) and 10% fetal bovine serum (Atlanta Biologicals). The LdMNPV isolate A21-MPV (31) served as the parental line for all subsequent viruses. A recombinant virus (E1cat), generated in a previous study (5), that contains the chloramphenicol acetyltransferase (cat) gene was also used in this study. L. dispar egg masses were obtained from the U.S. Department of Agriculture's Animal and Plant Health Inspection Service rearing facility at Otis Air Force Base (Otis, Mass.). Larvae were reared on gypsy moth diet (ICN) (3).

Northern blot analysis and primer extension mapping of transcripts. Infected Ld652Y cells were harvested at various times postinfection (P.I.). Cytoplasmic RNA was isolated as described by Friesen and Miller (9). Poly(A) RNA was isolated from the cytoplasmic RNA by the PolyATtract mRNA isolation system (Promega), separated on a 1.2% agarose-formaldehyde gel, and then transferred to NitroPlus nitrocellulose transfer membrane (MSI). Northern blots were performed as described by Mahmoudi and Lin (26). Thirty-base oligonucleotides (complementary to bp 61158 to 61187 for the E1 gene or bp 156708 to 156737 for the E2 gene [24]) were end labeled with [-³²P]ATP (NEN, Boston, Mass.) and used as strand-specific probes to detect the transcript of interest. Blots were imaged with the Storm 860 phosphorimager (Molecular Dynamics).

In vitro transcription and translation of E2 gene. Plasmid pDB184 (see below) was digested with SstI, and a 2.8-kbp fragment containing the E2 gene was subcloned into the SstI site of pBluescript to generate pE2SstI. The E2 protein was expressed from pE2SstI by the TNT quick coupled transcription-translation system (Promega). The expressed protein was labeled with ³⁵S-EasyTag express protein labeling mix (NEN). Reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.

Computer programs used for sequence comparison. The amino acids of known enhancins available in GenBank were aligned using the Clustal W program (http://www2.ebi.ac.uk/clustalw/). Highlighted comparisons of proteins were produced using the Boxshade program (available at http://www.ch.embnet.org/software/BOX_form.html). An unrooted phylogenetic tree of known enhancin proteins was constructed using the PAUP 3.1 program (33). Bootstrap analysis with 100 replicates was performed to assess the integrity of the phylogeny generated.

Construction and verification of recombinant viruses. An E2 gene transplacement vector was constructed to generate an A21-MPV recombinant strain lacking the E2 gene. A vector containing the E2 gene was constructed by restricting A21-MPV cosmid 15 (4) with BglII and then religating the fragments. Because there is a single BglII restriction site within the SuperCos 1 vector (Stratagene), a plasmid was generated, pDB184, containing ca. 6.0 kbp of A21-MPV viral DNA (ca. bp 154.2 to 160, Fig. 1B). A second plasmid with a lacZ gene insertion in the E2 gene was made as follows. Plasmid pDB184 was restricted with BstXI. A 3.8-kbp XbaI-BamHI fragment containing the HSP70lacZ gene marker was isolated from plasmid p210.1 (provided by American Cyanamid, Princeton, N.J.). The ends of both of the fragments were end filled with T4 DNA polymerase, and the fragments were ligated together to form pDB186. To create a plasmid with a deletion in the E2 gene, plasmid pDB184 was then restricted with PvuII and BglII and with EcoR47III and EcoRI. The 1.7-kbp PvuII-BglII and 2.2-kbp EcoR47III-EcoRI fragments were isolated. These fragments were subcloned into the pBluescript vector (Stratagene) BamHI and EcoRI sites. The resulting plasmid, pDB185, has a 3.9-kbp insert missing 2.1 kbp (701 amino acids) within the E2 gene.

Bioassay analysis of viruses. Fourth-instar L. dispar larvae were infected by injection of 5 µl of budded virus for each test virus (titer of about 5 × 10⁶ tissue culture-infective doses per ml). Larvae were placed on fresh food and allowed to feed until death. Polyhedra were then isolated from insects. The concentration of occluded virus required to kill 50% of the test larvae (LC₅₀) and the mean time to kill 50% of the test larvae (LT₅₀) for all viruses were determined by the droplet feeding method developed by Hughes et al. (18) as described by Slavicek et al. (32) on first-day, second-instar L. dispar larvae. Larvae were maintained at 27 ± 1°C at a photoperiod of 14 h light-10 h dark during the bioassay. LC₅₀ values were calculated using Polo-PC (29), and LT₅₀ values were determined by the ViStat 2.1 program (17). Statistical comparisons of bioassay data were performed using the StatView program from Abacus Concepts.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Characterization of E2 gene. The two enhancin genes in LdMNPV are located at kbp 60.3 to 62.6 (E1) and kbp 155.8 to 158.2 (E2) in the genome (Fig. 1A) (24). Analysis of the sequence upstream of the E2 gene revealed a potential baculovirus late promoter sequence, TTAAG, beginning 28 bp upstream of the enhancin gene start codon. Transcription of the E1 gene in LdMNPV (5) and of the HaGV enhancin (30) begins within consensus baculovirus late promoters (TTAAG and TTAAG, respectively, at the underlined nucleotides). All of the other enhancin genes identified to date in TnGV, PuGV, and XcGV also contain baculovirus late promoters. To determine the transcriptional start site of the E2 gene transcript, primer extension reactions were performed with a 25-base oligonucleotide. Transcription initiated at the first T residue within the consensus baculovirus late promoter sequence TTAAG (data not shown). No polyadenylation signal was identified immediately downstream of the E2 gene. Two consensus polyadenylation sites (AATAAA) are located 280 and 294 bp downstream of the stop codon in the E2 gene. These sites lie between the end of homologous repeat 8 and the bro-p gene. Recent studies on the mechanism of 3'-end formation of baculovirus late transcripts, using an in vitro assay, indicate that 3' ends are formed by termination after transcription of a T-rich region, as opposed to a cleavage-polyadenylation mechanism using a consensus polyadenylation site (21). An earlier study of polyadenylation sites of late transcripts indicated that the sites occurred after a T-rich region a variable distance downstream of consensus polyadenylation sites (42), which may suggest that the consensus site has a role in the formation of late-transcript 3' ends.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Genomic location of the two LdMNPV enhancin genes.E1 is located at bp 60268 to 62619, and E2 is located from bp 155812 to 158178 based on the sequence generated by Kuzio et al. (24). Several other LdMNPV genes are also indicated (A). The enlarged map of plasmid pDB184 indicates the Ca. 6 kb surrounding and encoding the E2 gene (kbp 155.8 to 158.2). The location of the E2 ORF and restriction sites used in subcloning of the gene are shown (B).

Identification and temporal analysis of E2 gene transcripts. The temporal expression of the E2 gene transcript was investigated in Ld652Y cells infected with isolate A21-MPV. A primary transcript of approximately 3.8 kb and a secondary transcript of approximately 4.1 kb were found using a strand-specific oligonucleotide probe at late times postinfection 48, 72, and 96 h p.i. (Fig. 2A). No transcripts were evident at the end of the 1-h infection period or at 24 h p.i. The 4.1-kb transcript is more clearly seen in Fig. 8. The lengths of E2 gene transcripts are longer than expected if the most proximal polyadenylation sites are used. With these sites, transcripts of approximately 2.9 kb would be generated. Further downstream, past the bro-p gene, are two consensus polyadenylation sites, and downstream from these sites are several T-rich areas. An E2 gene transcript terminating near these sites would be approximately 3.9 kb in length, which is the approximate transcript length observed.

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Temporal analysis of the enhancin 2 gene transcripts. Cells were infected with A21-MPV or E2del, and cytoplasmic RNA was isolated at the indicated hours postinfection. Poly(A) RNA was isolated from the cytoplasmic RNA, and 1 µg was separated by formaldehyde-agarose gel electrophoresis, blotted, and probed with a strand-specific oligonucleotide complementary to positions 156708 to 156737 in the E2 ORF. RNA from uninfected cells was used as a mock infection sample. RNA size standards (in kilobases) are indicated on the left.

Characteristics of E2 protein and comparison to other enhancins. The E2 gene could encode a 788-amino-acid protein with a predicted molecular mass of 88,408 Da. A rabbit reticulocyte-coupled transcription-translation system was used to express E2 to determine if the gene did encode an expressed protein of the predicted size. Plasmid E2SstI, which contained the E2 gene under the control of the T7 promoter, was used to express the E2 gene, and Bluescript Sk+ was used as a negative control. A number of bands were produced by pE2SstI, though a major band was seen at 88 kDa, which corresponds to the expected size of the E2 protein (Fig. 3, lane 2). The E2 protein is similar in size to the LdMNPV E1 protein, which contains 783 amino acids. Both LdMNPV enhancin proteins are considerably smaller than the known GV enhancins, most of which range in size from 856 to 902 amino acids. The E1 protein in XcGV is of intermediate size, with a length of 824 amino acids.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Analysis of expression of the enhancin 2 gene. The enhancin 2 gene was expressed in a rabbit reticulocyte transcription-translation system, and the proteins were separated by SDS-PAGE. Lane 1, Bluescript SK+; lane 2, pE2Sstl, expressing E2. Positions of molecular mass standards are indicated to the left, and the position of the enhancin protein is indicated to the right.

View larger version (224K): [in this window] [in a new window]   FIG. 4.   Aligned amino acid sequences of known baculovirus enhancin genes. Black and gray areas indicate positions with 70% or greater sequence identity or similarity, respectively, among all enhancins. The enhancin sequences aligned include those of LdMNPV, HaGV, PuGV, TnGV, and the four from XcGV. Asterisks indicate the area containing the HEXXH conserved zinc-binding domain of metalloproteases.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Phylogenetic tree of known baculovirus enhancins. The most parsimonious tree for known baculovirus enhancins was constructed with the heuristic search algorithm of PAUP. Numbers above the lines in roman type indicate phylogenetic distance, and italic numbers below the lines indicate the frequency of a given cluster after bootstrap analysis (100 replicates).

Construction and verification of recombinant viruses. To address the role of the LdMNPV E2 in viral potency, a recombinant virus was constructed that lacked the E2 gene. In addition, to determine if one enhancin gene could compensate for the lack of the other, a virus lacking both enhancin genes was generated. The E2 gene (2,364 nucleotides in length) was subcloned from the genome into a plasmid, pDB184 (Fig. 1B). A lacZ gene was inserted into the BstXI site of pDB184, at nucleotide 66 within the E2 gene ORF, and used to construct the intermediate virus, E2lacz. This virus was then recombined with a plasmid containing a deletion of E2 gene nucleotides 66 to 2168 to produce the virus E2del (Fig. 6). A lacZ gene was inserted into the NarI sites of the E1 gene (2,349 nucleotides in length) at nucleotides 1309 and 1492, and the resulting plasmid, pE1lacz, was recombined with the E2del virus to generate the virus E1laczE2del. This virus was recombined with a plasmid containing a deletion of E1 gene nucleotides 26 to 1492 to produce the virus E1delE2del. To confirm the effect of deletion of both enhancin genes, a virus was constructed in which the E1 gene was replaced. Plasmid pDB126, which contains the E1 gene, was recombined with E1laczE2del5AA to give E1repE2del (Fig. 6). The identity of the recombinant viruses was verified by restriction enzyme and Southern blot analysis (data not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Schematic representation of A21-MPV and enhancin recombinant viruses. Arrows show the location and direction of ORFs in the regions of the E1 and E2 genes (24). The shaded ORFs are those where the enhancin gene has been either deleted or disrupted. The cat gene is indicated by a black box, and the lacZ gene is indicated by a striped box. Expression of the enhancin gene products is indicated for each virus on the right.

View larger version (42K): [in this window] [in a new window]   FIG. 7.   Northern blot analysis of enhancin gene transcripts from cells infected with recombinant viruses. Cells were infected with A21-MPV, E1cat, E2del, and E1delE2del AE2 viruses. Poly(A) RNA was isolated from the cytoplasmic RNA, and 1µg was separated by formaldehyde-agarose gel electrophoresis, blotted, and probed with a strand-specific oligonucleotide for the enhancin 1 gene (A) or enhancin 2 gene (B). RNA from uninfected cells was used as a control (lane mock). Positions of RNA size standards (in kilobases) are indicated on the left.

Bioassay analysis. The biological activity of the recombinant enhancin viruses was determined through bioassay of L. dispar larvae. A previously constructed recombinant virus (5) containing a cat gene insertion in the E1 gene was also used in the bioassays. The potency of the E2del virus was found to be consistently less than that of the control virus in three separate bioassays (Table 1). The decrease in potency ranged from 1.2-to 2.9-fold. In agreement with our previous findings, the E1cat recombinant virus exhibited a drop in potency compared to the control virus (Table 1). In this study, the decrease ranged from 1.6- to 3.8-fold, which is in good agreement with the range of 1.4-to 4.0-fold reported in the previous study (5). These results indicate that enhancin 2 had an effect on viral potency and that both enhancin genes encode functional proteins that increase viral potency. Analysis of the time-mortality response showed similar killing speeds for the E2del, E1cat, and A21-MPV viruses (Table 1). Taken together, these results indicate that the enhancins are functioning at the level of viral entry into the insect host and do not affect systemic transmission. E2del, E1cat, and A21-MPV exhibited essentially identical rates of budded virus formation (data not shown), which indicates that the enhancin genes are not involved in viral replication. In addition, this finding indicates that a random mutation in a gene involved in DNA replication did not occur in the recombinant viruses. In contrast to the present findings, a decrease in the survival time of 50% of larvae (ST₅₀) was noted in bioassays of mid-fourth-instar T. ni larvae infected with AcMNPV in combination with the TnGV enhancin protein (11). Larvae that showed the same mortality with virus and with virus and enhancin had a lower ST₅₀ in the larvae infected with virus and enhancin. Perhaps a dual activity is possible for some enhancin proteins, or the impact on ST₅₀ was a function of the experimental conditions employed.