Autographa Californica Multiple Nuclear Polyhedrosis Virus Research Articles

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection.

Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.

Use of the baculovirus system to assemble polyomavirus capsid-like particles with different polyomavirus structural proteins: analysis of the recombinant assembled capsid-like particles.

The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells.

Persistent baculovirus infection results from deletion of the apoptotic suppressor gene p35.

Infection with the wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) results in complete death of Spodoptera frugiperda (Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor gene p35 allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus with p35 mutated or deleted was blocked by stable transfection of p35 in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection of p35 further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.

Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.

Model of a Split‐Flow Airlift Bioreactor for Attachment‐Dependent, Baculovirus‐Infected Insect Cells

AbstractA mathematical model of a split‐flow airlift bioreactor using attachment‐dependent, baculovirus‐infected insect cells is presented. The model is structured to predict the dynamics of human secreted alkaline phosphatase (seAP) production, nonoccluded virus (NOV) production, infected cell density, uninfected cell density, and lysed cell density for a variety of infection conditions in the reactor. Infected cells are divided into subpopulations on the basis of the time of initial infection and spatial location within the reactor. The infection rate is modeled as first order in virus concentration at low virus concentrations but zero order at high virus concentrations. NOV and seAP production in each subpopulation is assumed to be dependent on the age of the culture and degree of confluency. The subpopulation growth rate is assumed to be dependent on dissolved oxygen concentration and cell density. Extracellular degradation of seAP is included. Model parameters were estimated independently from the literature on Trichoplusia ni BTI‐TN‐5B1‐4 cells infected with recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) encoding seAP. Model predictions of seAP production are in reasonable agreement with laboratory scale bioreactor data for multiplicity of infection (MOI) values ranging from 1 to 30. The model predicts that bioreactor protein productivity could be increased significantly by operating at an MOI of 10, an initial cell density of 2.5 × 105 cells/cm2, an area ratio of riser to downcomer of 25:75, and an airflow rate of 100 mL/min.

The Wild-Type Autographa californica Nuclear Polyhedrosis Virus Induces Apoptosis of Spodoptera littoralis Cells

Spodoptera littoralis cells infected with the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) yielded significantly lower budded virus titers than Spodoptera frugiperda-infected cells and produced very low levels of polyhedrin. Relative to AcMNPV-infected S. frugiperda SF9 cells vital DNA replication was severely reduced in Spodoptera littoralis SL2 cells. Microscopic examination of SL2-infected cells revealed progressive cell blebbing starting at 6-8 hr postinfection and culminating in total cell destruction at 24 hr postinfection. The data suggested that AcMNPV-infected SL2 cells undergo apoptosis. The occurrence of an active apoptotic process in the infected cells was confirmed by: (1) observation of fragmentation of the cell nuclei stained with the specific fluorescent dye DAPI (4′,6′-diamidino-2-phenylindole) and (2) the presence of low-molecular-weight DNA oligomers. Neither SL2 cells infected with S. littoralis nuclear polyhedrosis virus (SINPV) nor SF9 cells infected with AcMNPV, respectively, showed nuclear fragmentation or oligonucleosomal ladder formation.

Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus.

Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) contains a 966 bp ORF that encodes a papain type cysteine proteinase with cathepsin L-like characteristics. Using Western blot analysis of infected cell extracts we showed that v-cath proteinase has 35.5 kDa and 32 kDa precursor forms which are processed to a 27.5 kDa mature form in a manner characteristic of papain and cathepsin L. V-cath proteinase activity was greatest under acidic conditions (pH 5.0) and was reduced in the presence of the cysteine proteinase inhibitors, leupeptin and E64. Urea, a known enhancer of cathepsin L activity, also enhanced v-cath proteinase activity. AcMNPV v-cath proteinase was detected post-mortem in tissues of insects infected with wild-type (wt) virus. Insects infected with a v-cath deletion mutant did not become flaccid after death as is normally observed with wt AcMNPV infections. These findings indicate a link between v-cath activity and degradation of host tissues during virus pathogenesis.

P10 genes of Bombyx mori nuclear polyhedrosis virus and Autographa californica multiple nuclear polyhedrosis virus.

EcoR I-P fragment has been cloned from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genomic DNA and used as a probe. 0.5-kb and 1.1-kb fragments including p10 gene from Bombyx mori nuclear polyhedrosis virus (BmNPV) have been hybridized. The p10 ORF was located in the EcoR I-R fragment. Initiation codon ATG of p10 from BmNPV has been mutated by PCR, and the ATG region became a Bgl II site. A novel transfer vector pBmAcPV-1 has been constructed using both the p10 5'-flanking region whose initiation codon ATG has been mutated with BmNPV and the p10 3'-flanking region of AcMNPV. The vector can recombine with not only AcMNPV DNA to express foreign gene in Sf cells, but also BmNPV DNA to express foreign gene in Bm cells. CAT gene was expressed at high level in Bm cells under the control of the mutated p10 promoter of BmNPV.

Production of human alkaline phosphatase, a secreted, glycosylated protein, from a baculovirus expression system and the attachment-dependent cell line Trichoplusia ni BTI-Tn 5B1-4 using a split-flow, air-lift bioreactor.

A split-flow, air-lift bioreactor for the cultivation of insect cells to produce recombinant protein is described. It can be used advantageously with attached cell systems. This bioreactor incorporates two sections: a rise and a downcomer. Trichoplusia ni BTI-Tn 5B1-4 cells are grown on a support material of glass beads or microcarriers placed in the downcomer. This cell line is more productive than other commonly used insect cell lines, but it has the disadvantage of being difficult to use at large volumes since it is not easily adaptable to suspension culture. Adequate oxygen demand is supplied by sparging without direct exposure of cells to air bubbles. Nutrients are supplied convectively to the attached cells on support material as the fluid flows through the downcomer. The split-flow, air-lift bioreactor appears to be suitable for insect cell culture and is potentially scalable. It can provide a high surface-to-volume ratio and can be operated in batch or continuous mode. A lab-scale prototype bioreactor has been constructed and tested for the production of a secreted, glycosylated recombinant protein (human alkaline phosphatase) or seAP using an Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) vector. With a ratio of riser cross-sectional area to downcomer cross-sectional area of 1, an aspect ratio of 4.4, an air-flow rate of 54 mL/min in the riser, and a bed of 2400 3-min nonporous glass beads, 10.7 micrograms/mL of seAP was produced using an MOI (multiplicity of infection or the ratio of plaque-forming units to cells) of 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Replication of Autographa californica baculovirus (AcMNPV) in a coleopteran cell line.

A coleopteran cell line (AGE) derived from the cotton boll weevil Anthonomus grandis supported replication of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). The titer of extracellular virus (ECV) and the number of occlusion bodies (OB) produced in AGE cells were approximately equal to those produced by a Trichoplusia ni cell line (TN-CL1), and the OB produced by both cell lines were equally infectious for T. ni larvae. The identity of the AGE cell line was established by chromosome and isoenzyme analyses.

Expression of an eclosion hormone gene in insect cells using baculovirus vectors

The ability of a baculovirus vector system to produce insect eclosion hormone (EH) was investigated by insertion of a Manduca sexta cDNA encoding EH into the genome of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) such that transcription was under the control of a strong, modified polyhedrin promoter. Two polypeptides, approx. 6.0–6.5 kDa, were synthesized in and secreted from recombinant virus-infected cells but not wild-type (wt) virus-infected cells. Both polypeptides were immunoprecipitated by polyclonal antiserum directed againnst natural M. sexta EH. Immunoblotting revealed only a single polypeptide, suggesting that only one form is stable in the expression system. The size of this polypeptide and its elution from a reverse phase HPLC column indicate that this polypeptide is similar, if not identical, in structure to natural EH. Bioassay data revealed that biologically active EH was synthesized and secreted at high levels (e.g. 10 μg of active polypeptide per 10 6 cells). Thus, the baculovirus expression system is an excellent source of EH for further studies of this unique insect neurohormone.

Replication of Autographa californica nuclear polyhedrosis virus in five lepidopteran cell lines

The doubling time, virus yield, and susceptibility of five lepidopteran cell lines, Trichoplusia ni, Spodoptera frugiperda, Heliothis virescens, Plutella xylostella, and Anticarsia gemmatalis to the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) were compared. Isoelectric focusing provided isoenzyme patterns that specifically characterized each cell line. Cell doubling times ranged from 24 hr ( A. gemmatalis) to 30 hr ( H. virescens). Although the H. virescens cell line produced the highest viral titer (statistically equivalent to the T. ni cell line), the P. xylostella line produced the greatest number of polyhedral inclusion bodies (PIB). PIB from all five cell lines were virulent for T. ni larvae; PIB from the T. ni and H. virescens lines had the highest and lowest virulence, respectively.