A comparison of the adaptations of strains of Lymantria dispar multiple nucleopolyhedrovirus to hosts from spatially isolated populations.

The adaptation of pathogens to either their hosts or to environmental conditions is the focus of many current ecological studies. In this work we compared the ability of six spatially-distant Lymantria dispar (gypsy moth) multiple nucleopolyhedrovirus (LdMNPV) strains (three from eastern North America and three from central Asia) to induce acute infection in gypsy moth larvae. We also sequenced the complete genome of one Asian (LdMNPV-27/0) and one North American (LdMNPV-45/0) strain which were used for bioassay. We found that all of the North American virus strains, with the exception of one, demonstrated higher potency than the Asian virus strains, either in North American (Lymantria dispar) larvae or, in Asian (Lymantria dispar asiatica) larvae. Complete genome sequencing revealed two gene deletions in the LdMNPV-27/0 strain: the virus enhancin factor gene (vef-1) and the baculovirus repeated orf gene (bro-p). These deletions were not seen in the LdMNPV-45/0 strain nor in other American strains available in archiving systems. We also found deletions of the bro-e and bro-o genes in LdMNPV-45/0 strain but not in the LdMNPV-27/0 strain. The phylogenetic inference with an alignment of the 37 core gene nucleotide sequences revealed the close relationship of the LdMNPV-45/0 strain with other American strains accessed in GenBank (Ab-a624 and 5-6) while the LdMNPV-27/0 strain was clustered together with the LdMNPV-3054 strain (isolated in Spain) instead of predicted clustering with LdMNPV- 3029 (isolated in Asia). Our study demonstrated that first, different LdMNPV isolates from the same metapopulations of L. dispar exhibit little or no difference in the degree of virulence towards host larvae and second, that locality of host population is not an important driver of LdMNPV virulence. Virulence of LdMNPV is determined only by viral genetics. The genetic differences between North American and Central Asian virus strains are discussed.

Both Lymantria dispar nucleopolyhedrovirus enhancin genes contribute to viral potency.

Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified an enhancin gene (E1) in the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133-8140, 1997). Inactivation of the E1 gene product within the viral genome lowered viral potency by an average of 2.9-fold. A second enhancin gene (E2) was identified when the entire genome of LdMNPV was sequenced (Kuzio et al., Virology 253:17-34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the LdMNPV E1 protein as well as the enhancins from Trichoplusia ni GV, Pseudaletia unipuncta GV, Helicoverpa armigera GV, and Xestia c-nigrum GV. Northern analysis of viral RNA indicated that the E2 gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the E2 gene (E2del) and a second with deletion mutations in both enhancin genes (E1delE2del). The enhancin gene viral constructs were verified by Southern analysis and shown not to produce enhancin gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce an E1 gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both LdMNPV enhancin genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency.

Identification of a non-LTR retrotransposon from the gypsy moth.

A family of highly repetitive elements, named LDT1, has been identified in the gypsy moth, Lymantria dispar. The complete element is 5.4 kb in length and lacks long-terminal repeats. The element contains two open reading frames with a significant amino acid sequence similarity to several non-LTR retrotransposons. The first open reading frame contains a region that potentially encodes a polypeptide similar to DNA-binding GAG-like proteins. The second encodes a polypeptide resembling both endonuclease and reverse transcriptase sequences. All members of the LDT1 element family sequenced thus far have poly-A tails or A-rich tails of 12-18 nucleotides in length, but lack a poly-A addition signal in the expected location. The amplification of retrotransposon insertion junction regions in different gypsy moth individuals indicates that polymorphisms exist at some of the insertion sites, suggesting that this element is or was, until recently, capable of transposition.

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV).

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.

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.

Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion.

In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.

Characterization of the replication cycle of the Lymantria dispar nuclear polyhedrosis virus.

The life cycle of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) was characterized through analysis of budded virus (BV) release, the temporal formation of polyhedra, the temporal transcription pattern of representative early, late, and hyper-expressed late genes, and the onset of DNA replication in the Ld652Y cell line. Transcripts from the LdMNPV immediate early gene G22 were detected 4 h post infection (h p.i.). The late and hyper-expressed late p39 capsid and polyhedrin genes were initially transcribed at approximately 20 and 24 h p.i., respectively. Viral DNA replication initiated at approximately 18-20 h p.i. Budded virus was released from infected cells between 24 and 36 h p.i., and polyhedra were first detected at approximately 48 h p.i.

Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus.

A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear polyhedrosis virus to date. The LdMNPV enhancin gene is located between 67.6 and 70.1 kbp on the viral genome. Northern and primer extension analyses of viral RNAs indicate that the enhancin gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The LdMNPV enhancin exhibits 29% amino acid identity to the enhancin proteins of the Trichoplusia ni, Pseudaletia unipuncta, and Helicoverpa armigera GVs. All four proteins contain a conserved zinc-binding domain characteristic of metalloproteases. A recombinant virus (enhancin::cat) was constructed in which the LdMNPV enhancin gene was inactivated by insertion mutagenesis in order to ascertain the effect of the enhancin protein on viral potency. The bioassay results indicate that disruption of the enhancin gene in the LdMNPV results in a reduction in viral potency.

Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25K FP gene may be caused by DNA replication errors.

We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J. M. Slavicek, N. Hayes-Plazolles, and M. E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants exhibited the traits of few polyhedra (FP) mutants; however, no large DNA insertions or deletions that correlated with the appearance of the FP phenotype were found. In this study, we have characterized several of the PF mutants at the phenotypic and genetic levels. Genetic techniques were used to group the mutations in the LdMNPV PF mutants to the same or closely linked genes. Wild-type viruses were recovered after coinfection of L. dispar 652Y cells with certain combinations of PF mutants. These viruses were analyzed by restriction endonuclease analysis and found to be chimeras of the original PF mutants used in the coinfections. Marker rescue experiments localized the mutations in one group of PF isolates to the region containing the LdMNPV 25K FP gene. The mutations in these PF mutants were identified. Four of five of the LdMNPV FP mutants contain small insertions or deletions within the 25K FP gene. The fifth LdMNPV FP mutant analyzed contained a large deletion that truncated the C terminus of the 25K FP gene product. All of the deletions occurred within the same potential hairpin loop structure, which had the lowest free energy value (most stable hairpin) of the five potential hairpin loop structures present in the 25K FP gene. One of the insertion mutants contained an extra base within a repetitive sequence. These types of mutations are likely caused by errors that occur during DNA replication. The relationship between the types of mutations found within the LdMNPV 25K FP gene and DNA replication-based mutagenesis is discussed.

Characterization of the Lymantria dispar nucleopolyhedrovirus 25K FP gene.

The Lymantria dispar nucleopolyhedrovirus (LdMNPV) gene encoding the 25K FP protein has been cloned and sequenced. The 25K FP gene codes for a 217 amino acid protein with a predicted molecular mass of 24870 Da. Expression of the 25K FP protein in a rabbit reticulocyte system generated a 27 kDa protein, in close agreement with the molecular mass predicted from the nucleotide sequence. The gene is located between 40.3 and 40.8 map units on the viral genome. It is transcribed in a counterclockwise direction with respect to the circular map at late times during the infection cycle from a consensus baculovirus late promoter. The LdMNPV and Autographa californica nucleopolyhedrovirus (AcMNPV) 25K FP proteins exhibit 52% amino acid identity with several regions showing greater than 75% identity. Homologues to the AcMNPV orf59 and orf60 were also identified upstream (with respect to the genome) of the 25K FP gene in LdMNPV and exhibit 52% and 45% amino acid identity, respectively.

Identification and characterization of a RAPD-PCR marker for distinguishing Asian and North American gypsy moths.

The recent introduction of the Asian gypsy moth (Lymantria dispar L.) into North America has necessitated the development of genetic markers to distinguish Asian moths from the established North American population, which originated in Europe. We used RAPD-PCR to identify a DNA length polymorphism that is diagnostic for the two moth strains. The polymorphism maps to an autosomal locus with codominant Mendelian inheritance. DNA sequence analyses of the Asian and North American forms enabled development of locus-specific primers so that this marker, designated FS-1, will be useful for strain identification under varying conditions in different laboratories.

Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted M(r) of 22,000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M(r) of 24,000, in close agreement with the molecular mass predicted from the nucleotide sequence. G22 is not significantly homologous to any known protein, nor is a G22 homologue present in the Autographa californica MNPV (AcMNPV). Temporal expression studies indicated that the G22 gene was transcribed at readily detectable levels in the presence of cycloheximide. Transcripts were detected immediately after the virus adsorption period and throughout the infection cycle. The early transcriptional start sites of G22 map to a sequence that resembles a subset of RNA polymerase II promoters/start sites that are found upstream of Drosophila melanogaster developmental and retrotransposon genes which lack TATA box motifs. Several consensus late baculovirus promoter/mRNA start site sequences (ATAAG) were identified upstream of the G22 gene start codon.

Identification and characterization of the ecdysteroid UDP-glucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

We have located, cloned, sequenced and characterized the ecdysteroid UDP-glycosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. (LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) disrupts the hormonal balance of the host larva by galactosylating ecdysone, which prevents moulting. The location of the LdMNPV egt gene, determined by hybridization analysis using a cloned coding segment of the AcMNPV egt gene, was mapped to between 79.1 and 80.2 map units on the viral genome. This region contains an open reading frame of 1464 nucleotides capable of encoding a 55K polypeptide. This predicted protein exhibits a 42% amino acid identity with the AcMNPV egt polypeptide. Transcripts of the egt gene were analyzed by Northern blot and primer extension. The egt gene is transcribed from approximately 12 to 48 h, and maximally at about 16 h post-infection. Transcription occurred in the presence of aphidicolin, a viral DNA synthesis inhibitor, but not in the presence of cycloheximide, a protein synthesis inhibitor. Therefore the LdMNPV egt gene is classified as a delayed early gene. The egt gene is transcribed in a clockwise direction with respect to the circular map, and transcription initiates at a single site. Comparisons between the two baculoviral egt proteins and mammalian UDP-glucuronosyltransferases reveal areas which are conserved between the mammalian and baculoviral genes, as well as areas that are only conserved in the viral egt proteins. The LdMNPV protein sequence appears to include a signal peptide, which would allow the protein to be secreted into the haemolymph.

Identification and characterization of a protein kinase gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from Drosophila melanogaster, and homology to several other protein kinases from yeasts, mice, and bovines. The homology suggests that vPK is a serine/threonine protein kinase as defined by Hanks et al. (S.K. Hanks, A.M. Quinn, and T. Hunter, Science 241:42-52, 1988). Temporal expression studies indicate that vPK is expressed throughout the infection cycle beginning at 4 h postinfection, first as a delayed-early gene and subsequently as a late gene. Sequence analysis and primer extension reactions confirm the presence of distinct early and late transcription initiation regions. Expression of vPK with a rabbit reticulocyte system generated a 31-kDa protein, which is in close agreement with the predicted size of 32 kDa from the amino acid sequence. Phosphorylation activity of in vitro-expressed vPK was demonstrated by using calf thymus histones.

Temporal analysis and spatial mapping of Lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides.

Genomic expression of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was studied. Viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through Northern analysis. Sixty-five distinct transcripts were identified in this analysis. Most viral transcripts were expressed late in infection, and originated from throughout the viral genome. Viral polypeptides expressed in infected 652Y cells were labeled with [35S]methionine and identified by autoradiography after separation by SDS polyacrylamide gel electrophoresis. Viral protein synthesis was found to occur in a sequential manner. Four proteins were identified in the early phase of viral replication (4-12 h p.i.), 24 proteins in the intermediate phase (12-24 h p.i.), and 5 proteins during the late phase (greater than 24 h p.i.). Cytoplasmic RNAs were isolated from LdMNPV infected cells at 16, 24, and 48 h p.i., and used for hybrid selections with overlapping DNA fragments that covered the entire LdMNPV genome. The selected RNAs were translated in vitro, and 61 distinct viral polypeptides were identified and their genomic origins mapped. Temporal and spatial transcription and translation maps of the LdMNPV genome were generated with these data, and the expression pattern of the LdMNPV genome was compared to that of the Autographa californica nuclear polyhedrosis virus.

A karyophilic signal sequence in adenovirus type 5 E1A is functional in Xenopus oocytes but not in somatic cells.

The carboxy-terminal five amino acids of the adenovirus type 5 E1A gene product are necessary and sufficient for this protein to become localized in the nuclei of somatic cells. In this report, we demonstrate that E1A contains a second nuclear localization signal, which resides within residues 140 to 185 and which functions in Xenopus oocytes but not Xenopus or mammalian somatic cells. These data demonstrate the cell-type-specific utilization of a nuclear localization signal.

Rapid turnover of adenovirus E1A is determined through a co-translational mechanism that requires an aminoterminal domain.

The product of the adenovirus E1A 13S mRNA can both stimulate and repress the expression of certain viral and cellular genes. As with several other regulatory proteins, E1A has a short half-life, approximately 40 min. Although this short half-life is observed in cells expressing the E1A gene, it is not the case with cells injected with E1A protein, where its half-life is very long, generally greater than 15 h. We have sought to reconcile these apparent differences in E1A stability. Using Xenopus oocytes, we find that E1A exhibits its characteristic short half-life when it is synthesized from injected mRNA while it has a very long half-life when it is injected as a protein synthesized originally in Escherichia coli or reticulocyte lysates. In order to delineate the amino acids responsible for rapid E1A turnover, several deletion mRNAs were constructed, injected into oocytes, and E1A half-life determined. Carboxyl-terminal deletions and an internal deletion of residues 38-86 failed to increase the half-life of E1A. In contrast, amino-terminal deletions of 70 and 14 residues resulted in very stable E1A proteins (t1/2 greater than 20 h). Furthermore, deletion of the second amino acid, an arginine, resulted in a stable E1A protein. The amino-terminal region of E1A was able to induce the rapid turnover of a normally stable protein, beta-globin, in oocytes injected with an E1A-globin chimeric mRNA. This E1A-induced instability of globin was abolished, however, when the protein was first synthesized in reticulocyte lysates and then injected into oocytes. The amino-terminal region of E1A is also important in governing halflife in adenovirus-infected HeLa cells. These results demonstrate that the half-life of E1A is established cotranslationally through a mechanism involving sequences within the amino-terminal 37 residues.

Heterogeneity of adenovirus type 5 E1A proteins: multiple serine phosphorylations induce slow-migrating electrophoretic variants but do not affect E1A-induced transcriptional activation or transformation.

The 289-amino-acid product encoded by the adenovirus E1A 13S mRNA has several pleiotropic activities, including transcriptional activation, transcriptional repression, and when acting in concert with certain oncogene products, cell transformation. In all cell types in which E1A has been introduced (except bacteria), E1A protein is extensively posttranslationally modified to yield several isoelectric and molecular weight variants. The most striking variant is one that has a retarded mobility, by about Mr = 2,000, in sodium dodecyl sulfate gels. We have investigated the nature of this modification and have assessed its importance for E1A activity. Phosphorylation is responsible for the altered mobility of E1A, since acid phosphatase treatment eliminates the higher apparent molecular weight products. By using several E1A deletion mutants, we show that at least two seryl residues, residing between residues 86 and 120 and 224 and 289, are the sites of phosphorylation and that each phosphorylation can independently induce the mobility shift. However, E1A mutants lacking these seryl residues transcriptionally activate the adenovirus E3 and E2A promoters and transform baby rat kidney cells to near wild-type levels.

The effect of renal perfusion pressure on renal vascular resistance in the spontaneously hypertensive rat.

Renal hemodynamics and renal vascular resistance (RVR) were measured in the spontaneously hypertensive rat (SHR) and in the normotensive Wistar-Kyoto rat (WKY). In addition, the autoregulatory response and segmental RVR in the SHR were studied after aortic constriction. Mean arterial pressure (MAP) and RVR were higher in the SHR than in the WKY, but renal blood flow (RBF) and glomerular filtration rate were similar in both groups. Measurement of mean afferent arteriolar diameter (AAD) by a microsphere method showed a significantly smaller AAD in SHR (17.7 +/- 0.35 micrometers) than in the WKY (19.5 +/- 0.20 micrometers). This decrease in AAD could account for a 47% increase in preglomerular resistance. Aortic constriction in the SHR, sufficient to reduce renal perfusion pressure from 152 to 115 mmHg, did not alter the AAD. Since RBF and glomerular filtration were also well maintained following aortic constriction, these autoregulatory responses suggest that vessels proximal to the afferent arteriole rather than postglomerular vasculature are primarily involved in the changes on intrarenal vascular resistance in SHR.