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PubMed2001

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.

Animals↗
PubMed1999

The nongastric H+-K+-ATPases: molecular and functional properties.

The Na-K/H-K-ATPase gene family is divided in three subgroups including the Na-K-ATPases, mainly involved in whole body and cellular ion homeostasis, the gastric H-K-ATPase involved in gastric fluid acidification, and the newly described nongastric H-K-ATPases for which the identification of physiological roles is still in its infancy. The first member of this last subfamily was first identified in 1992, rapidly followed by the molecular cloning of several other members. The relationship between each member remains unclear. The functional properties of these H-K-ATPases have been studied after their ex vivo expression in various functional expression systems, including the Xenopus laevis oocyte, the insect Sf9 cell line, and the human HEK 293 cells. All these H-K-ATPase alpha-subunits appear to encode H-K-ATPases when exogenously expressed in such expression systems. Recent data suggest that these H-K-ATPases could also transport Na+ in exchange for K+, revealing a complex cation transport selectivity. Moreover, they display a unique pharmacological profile compared with the canonical Na-K-ATPases or the gastric H-K-ATPase. In addition to their molecular and functional characterizations, a major goal is to correlate the molecular expression of these cloned H-K-ATPases with the native K-ATPases activities described in vivo. This appears to be more complex than anticipated. The discrepancies between the functional data obtained by exogenous expression of the nongastric H-K-ATPases and the physiological data obtained in native organs could have several explanations as discussed in the present review. Extensive studies will be required in the future to better understand the physiological role of these H-K-ATPases, especially in disease processes including ionic or acid-base disorders.

Amino Acid Sequence↗
PubMed1985

In vitro host range of five baculoviruses in lepidopteran cell lines.

The in vitro host range of five nuclear polyhedrosis viruses (NPV) was assessed in five lepidopteran cell lines from three genera. Multiple-enveloped baculoviruses of Autographa californica (ACMNPV), Trichoplusia ni (TNMNPV), and Galleria mellonella (GMMNPV) replicated in cells of T. ni, Spodoptera frugiperda, and Heliothis virescens to a titer of approximately 10(7) TCID50/ml. The multiple-enveloped baculovirus of S. frugiperda (SFMNPV) replicated only in S. frugiperda cells. The single-enveloped baculovirus of H. zea (HZSNPV) replicated in cells of H. zea and H. virescens but not in cells of H. armigera, T. ni, or S. frugiperda. Low levels of replication of ACMNPV, TNMNPV, and SFMNPV in cultures of H.zea, H. virescens, and T. ni, respectively, could not be detected by using a sensitive tritiated thymidine technique. However, two characteristically labelled peaks at densities of 1.145 and 1.245 g/ml were obtained in H. virescens cells inoculated with ACMNPV.60 min postinoculation ACMNPV particles were observed both entering and inside S. frugiperda cells but were not observed in H. zea or H. armigera cells. None of the five baculoviruses replicated in H. armigera cells.

Animals↗
PubMed1992

Heat resistance and thermotolerance in a radiation-resistant cell line.

Exponentially growing TN-368 lepidopteran insect cells have a normal growth temperature of 28 degrees C. These cells were heated in water baths at various temperatures between 33 and 44 degrees C under conditions of constant or fractionated heating. Determinations of cell survival using colony formation as well as measurements of DNA and protein synthesis were performed to assess relative heat resistance and development of thermotolerance. The results demonstrate a marked heat resistance over previously reported findings from the same laboratory for dipteran Drosophila cells in culture. The degree of heat resistance is remarkable, especially when compared to the heat resistance of mammalian cells, i.e. TN-368 cell survival at 41.5 and 44 degrees C was somewhat similar to mammalian cell survival, even though these temperatures are 13.5 and 16 degrees C above the normal growth temperature for TN-368 cells and 4.5 and 7 degrees C above the growth temperature of mammalian cells. Furthermore, the lepidopteran cells maintain the ability to develop a notable amount of thermotolerance in addition to this heat resistance. Thermotolerance development alone is capable of enhancing survival by an additional 10,000-fold. Thermotolerance could also be detected at the level of protein synthesis as a more rapid recovery following heat treatment. In contrast, DNA synthesis inhibition was prolonged even further in cells receiving a prior heat treatment to induce thermotolerance. In summary, it appears that, in addition to their pronounced radiation resistance, the TN-368 cells are also quite resistant to heat. It remains to be seen whether a single mechanism could be responsible for resistance to these agents which act very differently.

Acclimatization↗
PubMed1994

Cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from Bacillus thuringiensis toward cultured insect cells.

The pattern of in vitro toxicity of activated toxins from several classes of entomocidal inclusion genes from Bacillus thuringiensis was measured using eight established cell lines from lepidopteran insects. Protoxins representing CryIA(b), CryIA(c), and a mixture of all three CryIA toxins (subtypes a, b, and c; from B. thuringiensis subsp. kurstaki HD-1) were compared with the protoxin representing CryIC in a bioassay which measured the viability of cultured insect cells upon exposure to entomocidal toxin proteins. The responses of the various cell lines were very specific toward the individual toxin proteins. CryIC activated protoxin was toxic for cells of Manduca sexta, Plodia interpunctella, and to a lesser extent Spodoptera frugiperda. CryIA(b) and CryIA(c) proteins were toxic toward M. sexta but relatively non-toxic for P. interpunctella or S. frugiperda. The toxicity of CryIA(b), CryIA(c), and the composite CryIA activated toxins toward cells of Choristoneura fumiferana varied substantially, with the CryIA mixture being slightly more toxic than CryIA(c) alone. CryIC toxin had no effect toward C. fumiferana cells. Probit regression analysis of dose-response relationships between insect species and crystal protein composition demonstrated specific patterns of toxicity which may be related to membrane-receptor site binding by specific toxins. Membrane binding analysis of 125I-labeled CryIA(b), CryIA(c), and CryIC toxins to insect cells from three of the cell lines yielded high specific binding only with M. sexta cells toward CryIA(c) toxin. Lower levels of binding were observed with CryIA(b) and CryIC toward cells of C. fumiferana and P. interpunctella. Although relatively low binding levels for CryIC were observed with P. interpunctella cells, toxicity was high for these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Gypsy moth cell lines divergent in viral susceptibility. I. Culture and identification.

A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus.

Cell Division↗
PubMed1999

Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line.

The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2,and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glyco-proteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to GlcNAcMan5(Fuc)GlcNAc2 intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located.

Animals↗
PubMed2005

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.

Animals↗
PubMed2005

Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus.

Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.

Animals↗
PubMed2003

Characterization of cell lines developed from field populations of Culicoides sonorensis (Diptera: Ceratopogonidae).

Two cell lines, ABADRL-Cs-W3 (W3) and ABADRL-Cs-W8A (W8), were developed from a field population of Culicoides sonorensis Wirth & Jones. The cell lines were characterized by isozyme phenotyping and the ability to support the replication of bluetongue virus (BLU) and epizootic hemorrhagic disease virus (EHDV) (Orbivirus, Reoviridae). Comparison of isozymes found in the cell lines with those found in adult C. sonorensis colony insects confirmed that the cell lines were of C. sonorensis origin. There was, however, sufficient isozyme variation present in the cell lines to construct a unique isozyme profile for each cell line. Although both cell lines supported BLU and EHDV replication to the same level, one-step growth curves for BLU indicated that virus replication was faster and attained a peak titer earlier in the W3 cell line than in the W8 cell line. Viral proteins and RNA were detected earlier in the W3 cell line as well. The accelerated virus growth kinetics observed in the W3 cell line and the adherent nature of the cells makes it more suitable for certain Orbivirus studies.

Alleles↗
PubMed2006

Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus.

Members of the baculovirus p35 gene family encode proteins that specifically inhibit caspases, cysteine proteases that are involved in apoptosis. To date, p35 homologs have only been found in baculoviruses. We have identified AMVp33, a gene from Amsacta moorei entomopoxvirus with low but significant homology to baculovirus p35 genes. Expression of AMVp33 blocked apoptosis in several different insect and human cell lines. Purified recombinant P33 protein was an efficient inhibitor of insect and human effector caspases, but not initiator caspases. P33 was cleaved by effector caspases, and the resulting cleavage fragments stably associated with the caspases. Mutation of the predicted caspase cleavage site in P33 eliminated cleavage, caspase inhibition and anti-apoptotic function. Thus, AMVp33 encodes a caspase inhibitor similar to baculovirus P35 with a preference for effector caspases. This is the first report of a p35 homolog from any viral or cellular genome outside of the baculovirus family.

Amino Acid Sequence↗
PubMed2002

Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson).

Microplitis croceipes wasps overcome host immunity by inducing changes in host physiology using factors derived from the embryo and/or larva. Teratocytes of some parasitic wasps circulate in the host hemolymph after egg hatch and synthesize proteins (TSPs), some of which are secreted to alter host physiology in support of endoparasitoid development. TSPs appear to alter host physiology, at least in part, by inhibiting synthesis of certain proteins. M. croceipes teratocytes synthesize a 13.9 kDa protein (TSP14), which inhibits synthesis of host proteins that are linked to larval growth and development. A cDNA encoding TSP14 was generated by RT-PCR from teratocyte RNA, and cloned into yeast expression vectors to produce sufficient recombinant protein for functional analyses. RecTSP14 was produced using the yeast expression system at a concentration of more than 300 micrograms/L. The recTSP14 inhibited in vitro translation of larval Heliothis virescens RNA, with the activity sensitive to boiling, protein concentration, incubation time, and storage temperatures. Although recTSP14 inhibited translation of some cellular RNAs in vitro, the in vivo incorporation of [35S]-methionine into proteins of selected insect and mammalian cell lines was not inhibited. These findings suggest that recTSP14 entry is cell type-specific and required to inhibit synthesis of target protein(s).

Animals↗
PubMed1993

Human papillomavirus type 16 E6, E7 and L1 and type 18 E7 proteins produced by recombinant baculoviruses.

Proteins derived from the E6, E7 and L1 ORFs of HPV16 and the E7 ORF of HPV18 were produced in insect cells using a baculovirus expression system. HPV ORFs were inserted into baculovirus transfer vectors pAcYM1 or pVL1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. Using HPV-specific antisera and monoclonal antibodies HPV proteins were identified in lysates of Spodoptera frugiperda (Sf-21) cells infected with HPV-recombinant baculovirus. Immunoreactive HPV16 E7 protein produced in Sf-21 cells had an apparent M(r) of 19 kDa, larger than that predicted from the amino acid sequence, and similar to that of native HPV16 E7 protein in HeLa and CaSki cells. The apparent M(r) of recombinant HPV18-E7, HPV16-L1 and HPV16-E6 proteins was equivalent to the M(r) values predicted from the amino acid sequence. Thermostability studies revealed that the half-life of HPV16-E7 protein in Sf-21 cell lysate was approx. 20 h at 4 degrees C, 2 h at 22 degrees C, and less than 30 min at 37 degrees C. HPV16 L1, HPV16 E7 and HPV18 E7 proteins were predominantly localised in the nucleus of recombinant baculovirus-infected Sf-21 cells, whereas recombinant HPV 16 E6 protein was localised in both the cytoplasm and nucleus of infected insect cells. Northern blot analysis of RNA derived from insect cells infected with vAc16E6E7, a recombinant baculovirus containing both HPV16 E6 and E7 ORF's, revealed the presence of only E6 ORF transcripts, suggesting that the splicing of RNA products derived from the E6 and E7 ORF's, as observed in cervical cancer-derived cell lines, is not performed in insect cells. Baculovirus-derived HPV proteins have similar biological properties to the native proteins and should be suitable for studies on the immunology of HPV.

Animals↗
PubMed2003

Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells.

Successive viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did not show the accumulation of such concatemers. When introduced into SeUCR1 and SeIZD2109 cells, the non-hr ori concatemers generated in Se301 cells were maintained but did not increase. This suggests that the non-hr ori confers a strong selective advantage in Se301 cells, but not or to a lesser extent in the other cell lines. The cell line-specific accumulation of non-hr ori concatemers might be due to a higher intrinsic recombination frequency in Se301 cells and may reflect tissue related differences involving some host cell factor(s). Since non-hr ori concatemers in Se301 cells were more abundant in intracellular than in extracellular viral DNA preparations, episomal replication and the requirement of a minimal DNA size for packaging into nucleocapsids is hypothesized.

Animals↗
PubMed1998

Cell growth activity of growth-blocking peptide.

Growth-blocking peptide (GBP) is an insect biogenic peptide that retards the development of lepidopteran larvae. cDNAs encoding GBP were isolated from three lepidopteran species: Pseudaletia separata, Mamestra brassicae and Spodoptera litura. Comparison of these molecules revealed that the GBP coding regions were 70% homologous. In contrast, the upstream regions of the deduced propeptides were only 33% identical. Sequence analysis further suggested that GBP shares some structural similarities with human epidermal growth factors. Bioassay data revealed that several pmol/ml of GBP stimulated DNA synthesis of a human keratinocyte cell line and of SF-9 insect cells. However, several nmol/ml of GBP did not stimulate cell proliferation at all. In vivo studies similarly indicated that low concentrations of GBP stimulated larval growth while high concentrations of GBP retarded growth. These data suggest that GBP acts as a growth factor that regulates insect larval development.

Amino Acid Sequence↗
PubMed1994

Regulation of the cryptic sequence-specific DNA-binding function of p53 by protein kinases.

p53 is an allosterically regulated protein with a latent DNA-binding activity. Posttranslational modification of a carboxy-terminal regulatory site in vitro, by casein kinase II and protein kinase C, can activate the sequence-specific DNA-binding function of the wild-type protein. The latent form of p53 is produced in a variety of eukaryotic and prokaryotic cell lines, including E. coli, Sf9 insect cells, and C6 cells, indicating that the activation of p53 in vivo is rate-limiting. In addition, phosphorylation of p53 at the protein kinase C site and activation in vivo correlate with the loss of reactivity of active p53 protein to the carboxy-terminal antibody, PAb421. These results suggest that two highly conserved protein kinases modify polypeptide structure through a common biochemical mechanism and that different enzymatic pathways may channel information into the carboxy-terminal regulatory site of p53, allosterically activating its function as a tumor suppressor.

Allosteric Site↗