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L E Volkman

Publications and source records attributed to L E Volkman.

At least 19 recordsLinked to original sources
PubMed2002

Autographa californica M nucleopolyhedrovirus ProV-CATH is activated during infected cell death.

V-CATH, a cathepsin L-like cysteine protease encoded by the baculovirus Autographa californica M nucleopolyhedrovirus, has been shown to play an essential role in host liquefaction. Similar to cellular cathepsin L, V-CATH is synthesized as an inactive proenzyme and is activated by cleavage of the propeptide. Previous studies indicated that removal of the propeptide was rapid, occurring as soon as the protein could be detected by Western blot, 22 h postinfection. We found, however, that these results reflected artifactual processing of the proenzyme. When the protease inhibitor E-64 was used to prevent this aberration, we found that proV-CATH accumulated in infected cells and activation did not begin until the onset of cell death, at approximately 80 h postinfection. Western blot analysis of fractions of live and dead cells isolated by fluorescence-activated cell sorting revealed that mature V-CATH was found only in dead cells. The regulation of activation of proV-CATH, therefore, was quite different from that of cellular cathepsins. Acridine orange staining revealed that lysosome integrity was lost in dead cells, an occurrence that could lead to the activation of proV-CATH by lysosomal proteases.

Animals↗
PubMed2001

Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea.

Autographa californica M nucleopolyhedrovirus (AcMNPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between AcMNPV pathogenesis in Heliothis virescens and Helicoverpa zea, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of H. zea, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in H. virescens. Further, H. zea hemocytes, unlike those of H. virescens, were highly resistant to AcMNPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes-by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci-together with the host's melanization response, formed the basis of H. zea's resistance to fatal infection by AcMNPV.

Animals↗
PubMed2000

Autographa californica M nucleopolyhedrovirus chiA is required for processing of V-CATH.

Infection of permissive insect hosts by the baculovirus Autographa californica M nucleopolyhedrovirus results in liquefaction, a pathogenic effect that enhances the dispersal of progeny virions. Two viral gene products-a protease, V-CATH, and a chitinase, chiA-have been shown to be required for liquefaction to occur. It has been generally accepted that the primary functions of these proteins is to degrade the proteinaceous and chitinous components of the host cadaver, respectively. We have generated suggestive evidence, however, that chiA may also serve as a molecular chaperone for proV-CATH, the precursor of V-CATH. When cells were infected with virus lacking a functional chiA gene, proV-CATH failed to undergo processing in vivo and in vitro and formed insoluble aggregates in the endoplasmic reticulum of infected cells. Thus, expression of chiA may be required for the proper folding of the nascent V-CATH polypeptide in the endoplasmic reticulum. Identical results were obtained when tunicamycin was used to block N-linked glycosylation in cells infected with wildtype virus, suggesting that the putative chiA/V-CATH interaction is mediated by N-linked oligosaccharides.

Animals↗
PubMed2000

Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production.

Autographa californica M nucleopolyhedrovirus (AcMNPV) is the prototypical member of the NUCLEOPOLYHEDROSIS: genus of the BACULOVIRIDAE:, a family of large, double-stranded DNA viruses that are highly diverse. Nucleocapsid morphogenesis of AcMNPV and others in the NUCLEOPOLYHEDROVIRUS: genus takes place within the nuclei of infected host cells. Previously, we showed that filamentous actin (F-actin) is essential for this process to occur in AcMNPV-infected cells, an unprecedented finding for a DNA virus that replicates within the nucleus. Because of the fundamental importance of this requirement to our understanding of virus-host interactions, and because of the diversity of viruses included within the Nucleopolyhedrovirus genus, we were compelled to determine whether the replication of other nucleopolyhedroviruses was also F-actin dependent. We report here that progeny virus production of six other lepidopteran nucleopolyhedroviruses, representing both phylogenetic groups I and II within the genus, is also F-actin dependent. The six viruses studied (Spodoptera frugiperda MNPV, Bombyx mori NPV, Orgyia pseudotsugata MNPV, Lymantria dispar MNPV, Anticarsia gemmatalis MNPV and Helicoverpa zea SNPV) were unable to produce progeny in the presence of either cytochalasin D or latrunculin A, two actin-binding agents that interfere with F-actin-dependent processes but differ in their modes of action. F-actin-dependent progeny morphogenesis, therefore, appears to be a characteristic common among viruses in this genus that have lepidopteran hosts.

Actins↗
PubMed1999

Nuclear F-actin is required for AcMNPV nucleocapsid morphogenesis.

During nucleocapsid assembly, filamentous actin (F-actin) colocalizes with the major capsid protein of Autographa californica M nucleopolyhedrovirus (AcMNPV) within nuclei of infected lepidopteran host cells. Cytochalasin D (CD) disrupts actin filaments and prevents assembly of progeny AcMNPV, suggesting that nuclear F-actin is essential for nucleocapsid morphogenesis. Direct proof for this hypothesis was provided by the demonstration that two AcMNPV recombinants engineered to express either wild-type- or CD-resistant actin at equivalent rates were differentially sensitive to CD. The AcMNPV requirement for nuclear F-actin is unique among intracellular pathogens and may constitute a significant host range factor.

Actins↗
PubMed1999

Multiple nucleocapsid packaging of Autographa californica nucleopolyhedrovirus accelerates the onset of systemic infection in Trichoplusia ni.

Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a beta-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality ( approximately 70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.

Animals↗
PubMed1998

Actin binding and nucleation by Autographa california M nucleopolyhedrovirus.

The budded form of Autographa californica M nucleopolyhedrovirus enters permissive cells via adsorptive endocytosis. Shortly after nucleocapsid penetration into the cytoplasm, thick actin cables form, which frequently project toward the nucleus. These actin cables are transient structures, formed in association with viral nucleocapsids prior to viral gene expression and concomitant with nucleocapsid transport to the nucleus. In this paper we report that nucleocapsids are capable of nucleating actin polymerization in vitro in a concentration-dependent manner. Two viral-encoded capsid proteins, p39 and p78/83, were found to bind actin directly and therefore could be involved in the observed acceleration of actin polymerization. When nucleocapsids were added to actin in the presence of cytochalasin D, actin polymerization was reduced to levels below those obtained with actin and cytochalasin D alone, suggesting that the nucleocapsids bound to the pointed ends of actin filaments. Finally, treatment of infected cells with the myosin inhibitor 2,3-butanedione monoxime delayed nucleocapsid transport to the nucleus. We postulate that upon entering the cytoplasm, AcMNPV nucleocapsids induce the polymerization of actin cables, which, in conjunction with a myosin-like motor, facilitate their transport to and/or into the nucleus.

Actins↗
PubMed1998

AcMNPV pathogenesis and developmental resistance in fifth instar Heliothis virescens.

Autographa californica M nucleopolyhedrovirus carrying a lacZ reporter gene was used to study pathogenesis and developmental resistance in fifth instar Heliothis virescens. Compared to fourth instar larvae, the progression of infection proceeded much more slowly in fifth instar larvae, and developmental resistance in orally inoculated larvae was much more sudden and severe. The resistance occurred within the first 6 h of the fifth instar and was partially relieved by the optical brightener M2R. Inclusion of M2R in oral inocula not only increased mortality levels, but also increased both the percentages of insects expressing lacZ and the numbers of foci 24 h after infection. This early developmental resistance was not observed in insects infected intrahemocoelically, indicating that it was mediated by the midgut and/or the tracheal system servicing the midgut. Another less severe resistance was encountered in insects intrahemocoelically infected 36 to 48 h postmolt. This later resistance was partially relieved by methoprene indicating that it was, in part, hormonally mediated. Fifth instar insects fatally infected prior to 12 h postmolt never pupated, but pupation increasingly occurred in insects fatally infected 12 to 48 h postmolt. This pattern of larval/pupal death was consistent with viral inactivation of the ecdysone commitment peak.

Animals↗
PubMed1997

Evidence for rolling circle replication of Autographa californica M nucleopolyhedrovirus genomic DNA.

Autographa californica M nucleopolyhedrovirus (AcMNPV) is a large ds DNA virus restricted to larval lepidopteran insect hosts. Using field inversion gel electrophoresis and digestion with a restriction enzyme which cuts the AcMNPV genome once, we detected multiple unit-length genome fragments from replicating viral DNA. Our data suggest that AcMNPV replicates in a head-to-tail manner via rolling circle replication.

Animals↗
PubMed1997

Nucleopolyhedrovirus interactions with their insect hosts.

It is clear from this brief review that our understanding of the molecular cross-talk between insects and their baculovirus pathogens is still very limited. Studies in cell culture have taught us a great deal about the basic baculovirus molecular machinery and how it is regulated, and in many cases this information has been predictive of what occurs in infected insects. Frequently, however, studies in cell culture do not adequately predict the infection process in insect hosts, as demonstrated by viral mutants (some of which were discussed in this review) that behave identically to wild-type virus in cell culture but differ markedly in larvae. More baculovirus studies, therefore, need to be conducted in vivo if we are to improve our understanding of the complex interactions between baculoviruses and their hosts. Conducting baculovirus studies in insects (or at least in primary cell culture) also offers the opportunity to address questions that reach beyond the baculovirus community in significance. For example, almost all of our knowledge of viral fusion mechanisms comes from infection of cells in culture where the pH is neutral or acidic and the temperature is constant at 27 degrees or 37 degrees C. An answer to the question of how the ODV envelope fuses with the microvillar membrane of columnar epithelial cells in the highly alkaline midgut environment at low temperatures will not only be important for an improved understanding of baculovirus infection in the natural world, but will also constitute a new chapter on viral entry mechanisms. Similarly, the answer to the question of how baculovirus nucleocapsids move basally within microvilli promises to involve factors and/or a mechanism not yet described by cell biologists, and so will constitute a valuable contribution to both baculovirology and cell biology. There are many more such examples of biological mechanisms that can be uniquely explored within the context of baculoviruses and their insect hosts, some of which have been highlighted in this review. As more and more young investigators realize the importance of combining a knowledge of virology, molecular technology, and insect biology, however, many of the outstanding mysteries will be solved.

Animals↗
PubMed1997

Copper treatment increases recombinant baculovirus production and polyhedrin and p10 expression.

Treatment with 2 mM CuSO4 was used to induce a Drosophila melanogaster metallothionein (Mtn) promoter that had been cloned into a recombinant baculovirus. Careful study revealed that the Mtn promoter functioned as an inducible, if somewhat "leaky" promoter within the context of baculovirus-infected cells. In the process of generating a recombinant-baculovirus, it was discovered that post-transfection treatment with copper resulted in a 10-fold increase in the production of recombinant virus. This effect on virus production was specific to transfection, as treatment of infected cells with copper did not increase the production of virus. Treatment of infected cells with copper did, however, extend the period of expression of the polyhedrin and p10 proteins by at least 12 h. These findings have practical applications for the production of recombinant baculoviruses and the subsequent expression of foreign proteins using baculovirus expression vectors.

Animals↗
PubMed1996

Actin binding and proteolysis by the baculovirus AcMNPV: the role of virion-associated V-CATH.

Infection of larvae by Autographa californica M nuclear polyhedrosis virus (AcMNPV) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. In Spodoptera frugiperda tissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. The first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. Cable formation requires release of the budded virus (BV) nucleocapsid from the endosome, but does not require new protein synthesis, suggesting that the nucleocapsid contains the activity necessary to induce cable formation. We have identified two distinct BV-associated actin-targeting activities. The first, a nucleocapsid-associated actin-binding activity, enabled actin copelleting and may also induce actin polymerization and cable formation. The second activity, associated with the nucleocapsid and envelope fractions of BV, was a protease that specifically degraded actin. This protease was identified as V-CATH, a cathepsin L-like protease that is a product of the AcMNPV v-cath gene.

Actins↗
PubMed1995

Developmental resistance in fourth instar Trichoplusia ni orally inoculated with Autographa californica M nuclear polyhedrosis virus.

Larvae of lepidopteran insects commonly become increasingly resistant to baculovirus infections as they age. The mechanism responsible for this development resistance is not known, but the phenomenon does not occur if the viral inoculum is administered intrahemocoelically instead of orally, which is the natural route of infection. This observation indicates that the factors mediating developmental resistance are operative during infection of the primary target tissue, the larval midgut, and not during subsequent systemic infection. To learn more about the mechanism of developmental resistance, we orally inoculated four cohorts of fourth instar Trichoplusia ni larvae with a recombinant of Autographa californica M nuclear polyhedrosis virus expressing a reporter gene. While these cohorts differed only by a few hours in age, we found increasing resistance to infection in successively older cohorts. By assessing the presence and location of infected cells at intervals during the first 48 hr after inoculation, we identified two key factors relevant to the resistance pattern among the developmental cohorts. These factors were: (i) an age-dependent rate of establishing and/or sloughing infected midgut cells and (ii) the ability of fourth instar T. ni to completely clear infection of the midgut epithelium by ecdysis to the fifth instar.

Animals↗
PubMed1995

Comparative pathogenesis of Autographa californica M nuclear polyhedrosis virus in larvae of Trichoplusia ni and Heliothis virescens.

We compared early viral pathogenesis and dose-mortality relationships for larvae of two highly susceptible hosts, Trichoplusia ni and Heliothis virescens, using a construct of AcMNPV containing the lacZ reporter gene. Larvae were inoculated either as newly molted fourth instars (4(0)) or 15 hr after the molt (4(15)). In 4(0)-inoculated larvae, first lacZ expression was detected in the midgut epithelium of T. ni at 4 hr postinoculation (hpi) compared to 18 hpi in H. virescens, and systemic infections were initiated from tracheole cells servicing the midgut epithelia beginning at 12 and 20 hpi, respectively. The longer viral tenure within the midgut and the slower progression of systemic infections within H. virescens ultimately contributed to its longer time to death. For 4(0)-inoculated H. virescens, proportions of lacZ-expressing larvae increased from 18 hpi until the onset of the molt to the fifth instar at 36 hpi; at this time point, the proportion of signaling insects equaled the final larval mortality. Viral infections within the midgut epithelium of H. virescens were lost during the molt to the fifth instar. Dose-mortality relationships suggested that the peritrophic membrane provided little protection from AcMNPV infections for either species.

Analysis of Variance↗
PubMed1995

Proteolysis of p6.9 induced by cytochalasin D in Autographa californica M nuclear polyhedrosis virus-infected cells.

Cytochalasin D, a fungus-derived compound that interferes with actin polymerization, inhibits Autographa californica M nuclear polyhedrosis virus production in infected Spodoptera frugiperda (IPLB-Sf-21) cells. Cytochalasin D appears to inhibit nucleocapsid morphogenesis by interfering with nucleoprotein packaging. We were interested in determining, therefore, whether the drug affected the synthesis or processing of p6.9, the major core protein involved in nucleoprotein packaging. We found that cytochalasin D had no effect on the synthesis, phosphorylation, or dephosphorylation of p6.9, but that it induced the proteolysis of p6.9, an effect which could account for the inhibition of nucleocapsid morphogenesis. We also determined that the cytochalasin D-induced proteolysis of p6.9 was reversible upon removal of the drug, even in the absence of protein synthesis.

Actins↗