Trapping solid aerosols with optical tweezers: a comparison between gas and liquid phase optical traps.

We demonstrate a method for the optical trapping of solid aerosol particles. Suspension of silica particles in ethanol allows their delivery to the trapping volume using a commercial medical nebulizer. The ethanol quickly evaporates, leaving the solid particles trapped in air. We use the technique to make comparisons between aerosol and colloid tweezing through power spectra analysis of the particle's positions fluctuations for identical particles trapped in a water or air suspending medium.

Optical guiding of aerosol droplets.

We characterize the ability of Gaussian and Bessel beams to guide water, ethanol and dodecane aerosol droplets. Droplets produced from a nebuliser source are trapped using radiation pressure and then by varying the beam power are controllably guided in a vertical direction. The use of a zeroth-order Bessel beam, which has a non-diffracting thin core, is shown to improve guiding distances over a comparable Gaussian beam by more than three times with guiding distances of up to 2.75mm for dodecane droplets. We discuss the applications for this work in the context of tools for optically manipulating airborne particles.

Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus.

The occlusion derived form of baculovirus is specially adapted for primary infection of the host midgut epithelium. As such, the virion must contain the proteins essential for host range determination and initiation of infection. Because knowledge of virion composition is a prerequisite for functional investigation, this study used a combination of techniques to identify the proteins present within or associated with the occlusion-derived virus (ODV) virion. Thirty-one proteins, including proteins known to be essential for viral DNA replication, were identified with confidence. An additional 13 proteins were identified by using one of the three techniques. A comparison of gene conservation among the ODV proteins encoded in the 16 sequenced baculoviridae genomes is presented. With knowledge of the composition of ODV, it is now possible to target proteins and study their role(s) during primary infection.

Identification of BV/ODV-C42, an Autographa californica nucleopolyhedrovirus orf101-encoded structural protein detected in infected-cell complexes with ODV-EC27 and p78/83.

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

Effects of deletion and overexpression of the Autographa californica nuclear polyhedrosis virus FP25K gene on synthesis of two occlusion-derived virus envelope proteins and their transport into virus-induced intranuclear membranes.

Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559-8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (DeltaFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25K(polh)). In contrast, no significant changes were observed in the levels of E25 among wt-, DeltaFP25K-, and FP25K(polh)-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the DeltaFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25K(polh) mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both DeltaFP25K- and FP25K(polh)-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in DeltaFP25K-infected cells suggests that the pathway of E66 trafficking to the inner nuclear membrane and intranuclear microvesicles is specifically regulated and must be influenced by factors that do not control the traffic of E25.

Mutations within the Autographa californica nucleopolyhedrovirus FP25K gene decrease the accumulation of ODV-E66 and alter its intranuclear transport.

Previous reports indicate that mutations within the Autographa californica nucleopolyhedrosis virus FP25K gene (open reading frame 61) significantly reduce incorporation of enveloped nucleocapsids into viral occlusions. We report that FP25K is a nucleocapsid protein of both the budded virus (BV) and occluded virus (ODV), and we describe the effects of two FP25K mutations (480-1 [N-terminal truncation] and FP-betagal [C-terminal fusion]) on the expression and cellular localization of ODV-E66 and ODV-E25. Significantly decreased amounts of ODV-E66 are detected in cells infected with 480-1 or FP-betagal viral mutants, even though during FP-betagal infection, steady-state levels of ODV-E66 transcripts remain unchanged. While ODV-E66 is normally detected in intranuclear microvesicles and ODV envelopes by 24 h postinfection (p.i.), ODV-E66 remains cytosolic throughout infection in cells infected with 480-1 virus (up to 96 h p.i.), and its intranuclear localization is not detected until 96 h p.i. in cells infected with the FP-betagal mutant virus. The nuclear localization of ODV-E25 is not affected during infection by the FP-betagal mutant; however, its trafficking is significantly delayed during infection by the 480-1 mutant. Temporal Western blot analyses of cell lysates show that both 480-1 and FP-betagal mutant virus infections result in altered accumulation patterns of several structural proteins, including gp67, BV/ODV-E26, and the major capsid protein p39. In addition to BV/ODV-E26, ODV-E66 and gp67 may interact with FP25K, and ODV-E25 and p39 may also be components of a protein complex containing ODV-E66 and FP25K. Together, these data suggest that FP25K and its associated protein complex(es) may play an important role in the targeting and intracellular transport of viral proteins during infection.

The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase.

Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells. Since few data are available on the cell cycle of insect cells, the first task was to define Sf9 cell cycle kinetics. The cell cycle phase distribution of Sf9 cells grown in suspension culture was determined to be evenly distributed (29% of the cells in G1, 33% in S, and 36% in G2/M phase), with the duration of G1 and S phases both being about 6 h and the combined duration of G2/M phase being about 8 h. When Sf9 cells were infected with AcMNPV (Autographa californica nuclear polyhedrosis virus), approximately 84% of the cells were arrested in G2/M phase by 18-24 h p.i. Concomitant with the viral-induced arrest in G2/M phase, high levels of both cdc2-associated histone H1 kinase activity and cyclin B protein were detected. By 24 h p.i. cyclin B was no longer detected; however, cdc2-associated histone H1 kinase activity remained throughout the infection. These data suggested that early in infection, cyclin B/cdc2 complex may be used to regulate the transition from G2 to M phase, but prolonged arrest may be due to a protein(s) encoded by AcMNPV. DNA hybridization analysis showed that the maximal rate of viral DNA replication occurred before G2/M arrest. We noted that viral DNA replication still occurred late in infection, when the majority of the cells were arrested in G2/M phase. Since cellular DNA replication normally does not occur during G2 or M phase, experiments were designed to determine if viral DNA replication could occur even when host cell DNA replication was arrested. Sf9 cells were arrested and "frozen" at the boundary of G1/S phase using 5-fluoro-2'deoxyuridine (FdUrd) treatment and then infected with AcMNPV In the blocked, infected cells, viral DNA replication was detected; however, cellular DNA remained at steady-state levels. These results suggested that cellular DNA replication was not necessary for viral DNA replication and show that viral DNA replication was not significantly inhibited by FdUrd treatment. It was a surprise to detect viral DNA replication when the host cells were "frozen" at G1/S phase. We wanted to determine if the viral infection was progressing to the stage of progeny virus production. Our data showed that progeny budded virus (BV) and virus-induced intranuclear microvesicles were produced in the frozen, infected cells; however, the intranuclear microvesicles had an unusual structure. They were irregular in shape and thickened compared to those observed in a normal infection. Very few enveloped nucleocapsids were visible in the nucleus of the frozen, infected cells and the occluded-derived virus envelope proteins, ODV-E66 and ODV-EC27, were not detected by Western blot analyses. Since the cells were sustained at the boundary of G1 and S phases for the duration of this experiment, the decreased amount of enveloped ODV in the nucleus could be due to several factors, including decreased levels of proteins expressed from late genes, aberrant microvesicles, or the necessity of G2/M phasing of the infected cell for efficient production and maturation of intranuclear microvesicles. These data indicate that AcMNPV infection results in cell cycle arrest in G2/M phase and this arrest may be due to a viral-encoded protein(s) that has cdc2-associated kinase activity. (ABSTRACT TRUNCATED)

Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes.

The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV). Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.). The amount of protein increased by 16 h p.i. and remained at high levels throughout infection. By 36 h p.i. several additional immunoreactive proteins were detected which migrated at approximately 18 kDa and remained through 96 h p.i. Western blot analysis of purified virus envelope and nucleocapsid preparations revealed that both the 26- and 18-kDa proteins are structural proteins of the envelope of BV and ODV. Immunoelectron microscopy performed at a time when only the 26-kDa species of the protein was present confirmed that the protein located to ODV envelope. The protein was named BV/ODV-E26 to designate incorporation into viral progeny, envelope location, and apparent molecular weight. Studies designed to follow localization of BV/ODV-E26 demonstrated that early in infection, the protein was incorporated into cytoplasmic vesicles and by 16 h p.i., BV/ODV-E26 was detected in the nucleus associated with virus-induced intranuclear microvesicles and ODV envelope. Coimmunoprecipitation and yeast two-hybrid assays showed that BV/ODV-E26 and FP25K were capable of interacting with each other to form a complex and coimmunoprecipitation assays indicated that cellular actin was a third component of this complex. Together, these data suggest that FP25K and cellular actin may participate in the regulation, or movement through the cell, of baculovirus proteins and/or virus nucleocapsids.

N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection. Autoflourescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized as foci to discrete locations within the nucleus. Immunoelectron microscopy confirmed that these foci predominantly contained intranuclear microvesicles and the reporter fusion proteins were also detected in cytoplasmic membranes near the nucleus, and the outer and inner nuclear membrane. Therefore, these defined hydrophobic domains are sufficient to direct native and fusion proteins to induced membrane microvesicles within a baculovirus-infected cell nucleus and the viral envelope. In addition, these data suggest that movement of these proteins into the nuclear envelope may initiate through cytoplasmic membranes, such as endoplasmic reticulum, and that transport into the nucleus may be mediated through the outer and inner nuclear membrane.

The role of the AcMNPV 25K gene, "FP25," in baculovirus polh and p10 expression.

A previous study showed that an Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) 25K mutant produced less polyhedrin protein than wild-type (Jarvis et al., J. Virol. 66, 6903-6911, 1992). In this study, the role of the 25K gene product (AcMNPV ORF 61) in baculovirus gene expression was further investigated. Five different viral 25K mutants expressed lower levels of polyhedrin protein and less CAT activity under the control of the polh promoter compared to wild-type. Polh RNA was equally stable in wild-type and mutant virus-infected cells while the rate of polh transcription was significantly reduced in mutant-infected cells. In comparison, steady-state levels of p10 RNA were not reduced in 25K mutant-infected cells, indicating that the reduction in polh RNA did not reflect a general effect on very late gene transcription. Expression of ie-1, which also appears to influence polh expression (Choi and Guarino, Virology 209, 90-98, 1995), was not influenced by 25K mutation. These results show that the 25K protein is important for maintaining optimal levels of polh transcription by a mechanism that does not involve maintaining ie-1 expression.

Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27.

This paper identifies two structural proteins of the occluded derived viral envelope of Autographa californica nuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid.

Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56.

An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.

Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production.

Serial passage of nuclear polyhedrosis viruses (NPVs) through cultured cell lines results in the appearance of mutants with a complex phenotype referred to as the 'few polyhedra' (FP) phenotype. The altered plaque morphology and reduced occlusion production associated with the FP phenotype have been observed in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) bearing mutations in the gene encoding the 25 kDa protein (25K gene). In this study, we sequenced the 25K genes of four spontaneously occurring AcMNPV FP mutants. These mutants, together with an artificially generated FP mutant (AcFP beta gal, in which the gene for beta-galactosidase is fused in frame with the 25K ORF), were examined at the ultrastructural level to see if they exhibited the reduced virion occlusion and intranuclear envelopment which is associated with the FP phenotype. Observations on Spodoptera frugiperda Sf9 cells infected with the FP mutants revealed that all five mutants were impaired in virion occlusion and intranuclear nucleocapsid envelopment. The 25K mutants were also found to release two- to fivefold more infectious virus (p.f.u.) into the media of infected Sf9 cells. Marker rescue of AcFP beta gal restored wild-type virion occlusion, intranuclear envelopment and levels of infectious virus production.

Biosynthesis and localization of the Autographa californica nuclear polyhedrosis virus 25K gene product.

Mutations of the AcMNPV 25K gene are associated with the "few polyhedra" phenotype (M. J. Fraser et al., 1983, J. Virol. 47, 287-300; B. Beames and M. D. Summers, 1989, Virology 168, 344-353). Polyclonal antisera was produced and used to investigate the time course of expression and localization of the 25K protein in infected cells. Western blot analysis detected 25K protein in both cytosolic and nuclear extracts from 18-24 hr p.i. through 96 hr p.i. and also in purified viral occlusions, but not in purified virions. Immunogold electron microscopy revealed that 25K protein was predominantly associated with amorphous cytoplasmic structures and to a lesser extent with a more electron-dense structure in the nucleus. Viral occlusions in cell sections were not specifically labeled by 25K antibody. Observations of purified viral occlusions and nuclei prepared for immunogold EM revealed the presence of contaminating amorphous material that was labeled with 25K antibody.

Polydnavirus-facilitated endoparasite protection against host immune defenses.

The polydnavirus of Campoletis sonorensis has evolved with an unusual life cycle in which the virus exists as an obligate symbiont with the parasite insect and causes significant physiological and developmental alterations in the parasite's host. The segmented polydnavirus genome consists of double-stranded superhelical molecules; each segment is apparently integrated into the chromosomal DNA of each male and female wasp. The virus replicates in the nucleus of calyx cells and is secreted into the oviduct. When the virus is transferred to the host insect during oviposition, gene expression induces host immunosuppression and developmental arrest, which ensures successful development of the immature endoparasite. In the host, polydnavirus expression is detected by 2 hr and during endoparasite development. Most of the abundantly expressed viral genes expressed very early after parasitization belong to multigene families. Among these families, the "cysteine-rich" gene family is the most studied, and it may be important in inducing host manifestations resulting in parasite survival. This gene family is characterized by a similar gene structure with introns at comparable positions within the 5' untranslated sequence and just 5' to a specific cysteine codon (*C) within a cysteine motif, C-*C-CC-C-C. Another unusual feature is that the nucleotide sequences of introns 2 in the subfamily WHv1.0/WHv1.6 are more conserved than those of the flanking exons. The structures of these viral genes and possible functions for their encoded protein are considered within the context of their endoparasite and virus strategy for genetic adaptation and successful parasitization.

Transcription, translation, and cellular localization of PDV-E66: a structural protein of the PDV envelope of Autographa californica nuclear polyhedrosis virus.

A late gene encoding a 66-kDa structural protein of Autographa californica nuclear polyhedrosis virus was mapped and sequenced (26.9-29.7 MU). Transcription initiates from two conserved TAAG motifs (-15 and -37) with transcripts detected from 12 to 72 hr pi. The protein is detected in infected Spodoptera frugiperda (Sf9) cells from 24 to 72 hr pi. Western-blot and immunoelectron microscopic data identify this protein as specific for the polyhedra-derived virus (PDV) envelope. This protein has been named PDV-E66 to identify its viral origin, envelope location, and apparent molecular weight. In addition to being a structural protein of the PDV envelope, PDV-E66 is enriched in foci of microvesicles in the nuclei of infected Sf9 cells.

Autographa californica nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles.

Autographa californica nuclear polyhedrosis virus (AcMNPV) infection results in the production of two types of infectious, enveloped viruses. As both of these viral forms play significantly different roles in the virus life cycle, the different functional characteristics in the roles of the virus may be explained, in part, by the protein and lipid composition and source of the viral envelopes. Both viruses utilize different maturation and envelopment strategies: Extracellular virus (ECV) obtains an envelope by budding from the host cell plasma membrane, while the envelope of polyhedra-derived virus (PDV) is obtained within the nucleus of the host cell. There is compelling evidence for differences between ECV and PDV structural proteins; however, no previous study directly compares ECV and PDV purified from the same source and little data are available on the protein and lipid composition of the viral envelopes. This study begins the systematic comparison of ECV, PDV, and their envelopes to target proteins for use as probes to study the molecular and biochemical basis of viral envelopment within the nucleus. AcMNPV ECV and PDV were isolated from infected Spodoptera frugiperda cells and fractionated into their respective envelope and nucleocapsid fractions. The structural protein, glycoprotein, and phosphoprotein composition of ECV, PDV, and their envelope and nucleocapsid fractions are analyzed and compared, and antigens of ECV and PDV viral envelope are identified. A number of structural proteins are different between ECV and PDV. ECV is enriched for proteins at 67, 45, and 35 kDa, while proteins at 89, 70, 60, 50, and 25 kDa are enriched in PDV. At least two proteins, PDV-E66 and PDV-E43, are identified to be specific for the PDV envelope. There are more N-glycosylated proteins in ECV than in PDV, with ECV-specific proteins found at 137, 128, 89, 45, and 40 kDa. PDV glycoproteins are 70, 53, 49, 42, 40, and 31 kDa. Most phosphoproteins of both ECV and PDV are predominantly found in the viral envelopes. The predominant phosphoprotein of ECV is 85 kDa, whereas PDV major phosphoprotein is 36 kDa. This study presents the first report of the phospholipid and fatty acid content of ECV and PDV viral envelopes. The major phospholipid of ECV is phosphatidylserine (50%), while phosphatidylcholine and phosphatidylethanolamine are the major phospholipids of PDV (39 and 30%, respectively). Since PDV is enveloped within the nucleus of the host cell, the PDV phospholipid composition is compared with the phospholipid composition of purified S. frugiperda (Sf9) nuclei and this analysis demonstrates significant differences between these two membrane systems.

Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system.

Foreign secretory pathway proteins are often produced in surprisingly low amounts in the baculovirus/insect cell expression system. One possible reason for this is that heterologous signal peptides might be inefficiently recognized by the insect cell protein translocation machinery. This idea was supported by a recent study showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide (Tessier, D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F., and Vernet, T. (1991) Gene (Amst.) 98, 177-183). We have extended these observations by measuring the effects of different signal peptide and signal peptide-prosequence combinations on baculovirus-mediated expression and secretion of human tissue plasminogen activator (t-PA). Replacement of the native prepropeptide with signal peptides from a lepidopteran insect secretory protein (cecropin B), a major baculovirus structural glycoprotein (64K), or an abundant, highly conserved lumenal protein of the rough endoplasmic reticulum (GRP78/BiP, a 78-kDa glucose-regulated protein/immunoglobulin heavy chain-binding protein), had no significant effect on t-PA expression or secretion. The same results were obtained with the signal peptide from honeybee prepromellitin, which was able to enhance secretion of plant propapain (Tessier et al., 1991 (above)). Similar results were obtained when heterologous signal peptides were combined with the native prosequence or when the intact cecropin B preprosequence was used. Translational initiation at an upstream, in-frame ATT, which could functionally inactivate any signal peptide, did not explain the low efficiency of t-PA secretion. Finally, deletion of the native signal peptide, prosequence, or both, failed to increase t-PA production. These results showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. They also suggested that the inability of insect cells to recognize the processing signals in human t-PA efficiently is probably not the major factor preventing its high level production in this system.

Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family.

For successful parasitization, the female Campoletis sonorensis endoparasitic wasp injects a polydnavirus into its host, Heliothis virescens, during oviposition. Viral gene expression induces immunosuppression and alters development of the host. We report here that three abundantly expressed genes, VHv1.1, WHv1.0, and WHv1.6, describes a polydnavirus "cysteine-rich" gene family which may be important in inducing these host manifestations. These genes have a similar primary gene structure and their proteins contain cysteine motifs characteristic of snail ion-channel ligands, the omega-conotoxins. Like the omega-conotoxins, the intercysteine amino acid residues are hypervariable with only three identical amino acids in all motifs. The conservation of this domain in the three viral genes may reflect an important functional role for these viral proteins in the parasitization of H. virescens. The three genes also contain introns similar in sequence at comparable positions in their 5' untranslated leaders and coding sequences. VHv1.1 contains two cysteine motifs, and each motif is interrupted by an intron at the same position as in the cysteine motifs of WHv1.0 and WHv1.6. Intron 2 sequences of WHv1.0 and WHv1.6 are 92% identical, while the immediately flanking exon sequences encoding the cysteine motifs are only 76% identical. This provides an example of nuclear pre-mRNA introns which are more conserved than flanking exons among members of a gene family.