James M. Slavicek

A Gene for an Extended Phenotype

An insect virus gene controls the behavior of the dying host to increase dispersion of the virus. Manipulation of host behavior by parasites and pathogens has been widely observed, but the basis for these behaviors has remained elusive. Gypsy moths infected by a baculovirus climb to the top of trees to die, liquefy, and “rain” virus on the foliage below to infect new hosts. The viral gene that manipulates climbing behavior of the host was identified, providing evidence of a genetic basis for the extended phenotype.

Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from the microbial pesticide Gypchek

Abstract Two Lymantria dispar nuclear polyhedrosis virus isolates, 5–6 and A2-1, differing in the phenotypic characteristic of the number of viral occlusions in infected cells, were obtained from a production lot of the microbial pesticide Gypchek and several of their replication properties were investigated and compared. Budded virus titer produced in cell culture, polyhedral inclusion body production in cell culture and in vivo, the number of virions present within occlusion body cross sections, and potency determinations suggest that isolate 5–6 is a few polyhedra plaque variant and that A2-1 is a many polyhedra wild-type isolate.

Both Lymantria dispar nucleopolyhedrovirus enhancin genes contribute to viral potency.

ABSTRACT 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 anenhancin gene (E1) in the Lymantria disparmultinucleocapsid 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 secondenhancin 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 fromTrichoplusia ni GV, Pseudaletia unipuncta GV,Helicoverpa armigera GV, and Xestia c-nigrumGV. Northern analysis of viral RNA indicated that the E2gene 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 bothenhancin genes (E1delE2del). Theenhancin 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 anE1 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.

The Lymantria dispar Nucleopolyhedrovirus Enhancins Are Components of Occlusion-Derived Virus

ABSTRACT Enhancins are metalloproteinases, first identified in granuloviruses, that can enhance nucleopolyhedrovirus (NPV) potency. We had previously identified two enhancin genes (E1 and E2) in the Lymantria dispar multinucleocapsid NPV (LdMNPV) and showed that both were functional. For this study, we have extended our analysis of LdMNPV enhancin genes through an immunocytochemical analysis of E1 and E2 expression and localization. E1 and E2 peptide antibodies recognized proteins of ∼84 kDa and 90 kDa, respectively, on Western blots of extracts from L. dispar 652Y cells infected with wild-type virus. The 84- and 90-kDa proteins were first detected at 48 h postinfection (p.i.) and were present through 96 h p.i. E1 and E2 peptide antibodies detected E1 and E2 in polyhedron extracts, and the antibodies were shown to be specific for E1 and E2, respectively, through the use of recombinant virus strains lacking the enhancin genes. E1 and E2 were further localized to occlusion-derived virus (ODV). The enhancins were not found in budded virus. Immunoelectron microscopy indicated that E1 and E2 were present in ODV envelopes and possibly in nucleocapsids. Fractionation studies with several detergents suggested that the enhancins were present in ODV envelopes in association with nucleocapsids. In contrast, enhancins in granuloviruses are located within the granulin matrix. The presence of LdMNPV enhancins within ODV provides a position for the proteins to interact directly on the peritrophic membrane as ODV traverses this host defense barrier.

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.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD(50) in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

Pathogenesis of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar and mechanisms of developmental resistance.

Lymantria dispar has a long historical association with the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), which is one of the primary population regulators of L. dispar in the field. However, host larvae exhibit strong developmental resistance to fatal infection by LdMNPV; the LD50 in newly moulted fourth instars is 18-fold lower than in the middle of the instar (48-72 h post-moult). Using a recombinant of LdMNPV expressing lacZ, we examined the key steps of pathogenesis in the host to explore mechanisms of developmental resistance. At the midgut level, we observed reduced primary midgut infections in mid-fourth instars, indicating increased sloughing of infected cells. Additional barriers were observed as the virus escaped the midgut. Mid-fourth instars had higher numbers of melanized foci of infection associated with the midgut, apoptotic tracheal epidermal cells and haemocytes, and reduced numbers of infected haemocytes later in infection. Our results show that the co-evolutionary relationship between L. dispar and LdMNPV has resulted in both midgut-based and systemic antiviral defences and that these defences are age-dependent within the instar. This age-related susceptibility may contribute to how the virus is maintained in nature and could influence management of L. dispar by using the virus.

Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix.

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.

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.

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.