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At least 775 records · Page 43Linked to original sources
PubMed2006

Assay and characterization of a strong promoter element from B. subtilis.

A new strong promoter fragment isolated from Bacillus subtilis was identified and characterized. Using the heat stable beta-galactosidase as reporter, the promoter fragment exhibited high expression strength both in Escherichia coli and B. subtilis. The typical prokaryotic promoter conservation regions were found in the promoter fragment and the putative promoter was identified as the control element of yxiE gene via sequencing assay and predication of promoter. To further verify and characterize the cloned strong promoter, the putative promoter was sub-cloned and the beta-Gal directed by the promoters was high-level expressed both in E. coli and B. subtilis. By means of the isolated promoter, an efficient expression system was developed in B. subtilis and the benefit and usefulness was demonstrated through expression of three heterologous and homogenous proteins. Thus, we identified a newly strong promoter of B. subtilis and provided a robust expression system for genetic engineering of B. subtilis.

Bacillus subtilis↗
PubMed2004

Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system.

Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin. Potential functions of the BARF1 early gene in EBV oncogenesis emerged from our observations showing expression of BARF1-encoded protein in nasopharyngeal carcinoma biopsies, and induction of either malignant transformation (in rodent fibroblast and human B cell lines) or immortalization (in monkey primary epithelial cells) following BARF1 transfection. We previously reported expression of the BARF1 product as a cytoplasm/membrane-associated protein from 293-tTA cells infected with a BARF1-recombinant adenovirus. Since constitutive expression of BARF1 from this heterologous system became inefficient, we developed a tetracycline-regulatable recombinant vector expressing BARF1 and green fluorescent protein from a dicistronic message. As here reported, stable and efficient expression of BARF1 from this vector in either permissive or non-permissive cell lines, allowed the first sequencing identification and further molecular characterization of BARF1-encoded protein.

Adenoviridae↗
PubMed2004

A mathematical model of baculovirus infection on insect cells at low multiplicity of infection.

The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI described. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.

Animals↗
PubMed1990

Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte.

The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.

Alkaline Phosphatase↗
PubMed1990

Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27.

Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 alpha, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of gamma-1 genes, (ii) induction of expression of gamma-2 genes, (iii) down regulation of expression of alpha and beta genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of gamma-1 proteins but appeared to be unable to efficiently express gamma-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates gamma-1 gene expression and a second one required for gamma-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral beta genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.

Animals↗
PubMed1986

Molecular analysis of 21-hydroxylase gene expression in mouse adrenal cells.

Two homologs of the gene encoding the adrenocortical 21-hydroxylase (21-OHase) are located within the S region of the mouse major histocompatibility complex. Only one of these homologs, however, encoded the full-length sequence of 21-OHase, directed the synthesis of 21-OHase RNA in the mouse adrenal gland, and was capable of restoring 21-OHase activity when transfected into 21-OHase-deficient Y1 adrenocortical tumor cells. Y1 cells transfected with the 21-OHase gene, when stimulated with ACTH, increased the number of 21-OHase transcripts up to 10-fold. The 21-OHase gene was not expressed when transfected into mouse fibroblast L cells, and was poorly expressed and poorly regulated by ACTH when transfected into a Y1 mutant harboring a defective cAMP-dependent protein kinase. Marked decreases in expression of the 21-OHase gene were noted when DNA constructs that contained fewer than 230 base pairs in the 5' flanking region of the gene were transfected into Y1 cells. These results indicate that the 21-OHase gene encodes information required for the tissue-specific expression and hormonal regulation of 21-OHase. The cAMP-dependent protein kinase is important for both aspects of gene expression. At most, 230 base pairs of 5' non-coding information are required for efficient expression of the 21-OHase gene in Y1 cells.

Adrenal Cortex↗
PubMed2005

Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester on milk production and composition by Holstein cows.

The esterification of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) to isopropanol (HMBi) decreases the rate and extent of its ruminal breakdown. The modes of action of HMB and HMBi appear to be different. The quantification of the production response to HMBi has not been done. The objectives of this study were (1) to determine the lactation response to HMB, (2) to determine the lactation response to HMBi, and (3) to evaluate whether the response to HMBi is affected by HMB in the diet. Sixty-one Holstein cows (24 primiparous, 37 multiparous) were assigned to 1 of 4 dietary treatments 21 to 28 d after calving. The base diet consisted of [on a dry matter (DM) basis] 32.5% corn silage, 17.5% alfalfa hay, 10% whole cottonseed, and 40% of a pelleted concentrate made primarily of ground corn, soybean meal, and blood meal, and was fed for 16 wk as a control diet. To prepare the dietary treatments, the base diet was supplemented with 0.1% of diet DM with HMB (treatment 2), with 0.15% HMBi (treatment 3), or with 0.045% HMB and 0.15% HMBi (treatment 4). Results showed a significant increase in milk yield (2.9 kg/d), protein content (0.15%), protein yield (115 g/d), fat yield (165 g/d), and lactose yield (182 g/d) from HMBi. Supplementation of HMB had small and nonsignificant effects on milk yield and composition. There were no significant interaction effects of HMB with HMBi on any of the production traits measured in this experiment. Plasma free Met as a proportion of essential amino acids was increased by HMBi, but not by HMB. Dietary supplementation of HMBi increased gross N efficiency expressed as the proportion of ingested N secreted in milk. Consequently, HMBi significantly improved N efficiency.

2-Propanol↗
PubMed2006

[Inhibitory effect of N-terminal of p55PIK--regulatory subunit of phosphoinositide-3 kinase--on proliferation of gastric cancer cell line MGC803 and its mechanism].

BACKGROUND & OBJECTIVE: p55PIK is one of the regulatory subunits of phosphoinositide-3 kinase (PI3K). The unique 24 amino acids at N-terminal of p55PIK can bind Rb, and their ectopic expression may inhibit cell cycle progression. This study was to observe the effects of ectopic expression of the 24 amino acids at N-terminal of p55PIK (N24p55PIK) on cell proliferation and tumor growth of gastric cancer, and explore possible mechanism. METHODS: Plasmid pEGFPN24 was transfected into gastric cancer cell line MGC803 (MGC803/GFP-N24); pEGFPC1 was transfected into MGC803 cells as control (MGC803/pEGFPC1). Transient expression of GFP-N24 fusion protein was confirmed by Western blot. The growth of cell clones was determined by MTT assay. Effect of N24p55PIK overexpression on cell clonogenic ability was detected by colony formation assay. Tumorigenic capacity of MGC803/GFR-N24 cells was tested by tumorigenicity assay in nude mice. Influence of N24p55PIK on the expression of cell cycle protein Cyclin D1 was analyzed by Western blot. RESULTS: N24p55PIK was efficiently expressed in MGC803 cells, but the level of GFP-N24 fusion protein in MGC803/GFP-N24 cells was much lower than that of GFP in MGC803/pEGFPC1 cells. Compared with MGC803/pEGFPC1 cells, the growth of MGC803/GFP-N24 cells was suppressed and the cell doubling time was prolonged. The volume of MGC803/GFP-N24 cell colonies was smaller than that of MGC803/pEGFPC1 cell colonies. The tumorigenic capacity of MGC803 cells was decreased after transfection of pEGFPN24 in nude mice. The tumor weight and volume were (0.398+/-0.244) g and (408+/-268) mm(3) in MGC803/pEGFPN24 group, and were (0.763+/-0.193) g and (829+/-271) mm(3) in MGC803/pEGFPC1 group (P<0.05). The expression of Cyclin D1 was down-regulated in MGC803/GFP-N24 cells. CONCLUSIONS: Ectopic expression of N24p55PIK might inhibit tumor cell growth both in vitro and in vivo through decreasing the expression of Cyclin D1. The N24 peptide, derived from PI3K regulatory subunit p55PIK, may be a potential drug in antitumor treatment.

Adenocarcinoma, Mucinous↗
PubMed1989

Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation.

An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.

Actinobacillus↗
PubMed1989

Regulation of chicken alpha and beta actin genes and their hybrids inserted into myogenic mouse cells.

We have investigated the regulation of intact non-muscle (beta) and muscle-specific (skeletal alpha) chicken actin genes and of hybrids of these two genes (alpha 5'-beta 3' and beta 5'-alpha 3') transferred into the mouse myogenic non-fusing cell line BC3H1. BC3H1 cells express members of the actin multigene family in a differentiation-dependent manner. When proliferating, the cells accumulate large amounts of non-muscle actin mRNA; when the cells are induced to differentiate, the amount of non-muscle actin mRNA decreases and the amount of muscle-specific actin mRNA increases. The transferred beta-actin gene is efficiently expressed in undifferentiated cells and appropriately down-regulated upon differentiation. In contrast, the transferred alpha-actin gene is inefficiently expressed and not consistently up-regulated. Results with the intact and hybrid genes, taken together, are consistent with the hypothesis that both 5' and 3' halves of these genes contain sequences important in regulating the efficiency and/or developmental timing of their expression in BC3H1 cells. By nuclear run-on experiments we found no evidence for gene-specific changes in the rate of transcription of the transferred actin genes during myogenesis. We conclude that the differentiation-dependent changes in expression of the intact beta-actin gene in BC3H1 cells must be regulated at the post-transcriptional level.

Actins↗
PubMed2003

Rat mammary carcinogenesis induced by in situ expression of constitutive Raf kinase activity is prevented by tethering Raf to the plasma membrane.

Mammary carcinogenesis induced through expression of activated Raf was investigated using a model in which retroviral vectors were infused into the central ducts of rat mammary glands. This model allows efficient expression of experimental proteins in a small fraction of endogenous mammary epithelial cells in situ. We previously reported that Raf is the dominant oncogenic signaling pathway from activated Ras in rat mammary glands. We show here that mammary gland carcinogenesis is rapidly induced by the expression of c-Raf-1 kinase that is activated by N-terminal truncation (Delta-Raf). Interestingly, targeting Raf to the plasma membrane via C-terminal fusion with Ras membrane localization signals (Raf-Caax) induces Raf kinase activity that transforms 3T3 cells more frequently than Delta-Raf, yet in situ expression of Raf-Caax does not induce mammary carcinomas. To investigate these contrasting results and begin elucidating the mechanisms of Raf-induced mammary carcinogenesis, we combined both activating mutations (N-terminal truncation and C-terminal membrane localization motifs) in one Raf construct (Delta-Raf-Caax). While Delta-Raf-Caax transforms 3T3 cells more efficiently than Delta-Raf or Raf-Caax, in situ expression of Delta-Raf-Caax does not induce carcinomas in vivo, demonstrating that lipid modification on the C-terminus of Delta-Raf negates its oncogenic potential in rat mammary gland.

3T3 Cells↗
PubMed2003

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

Adenoviridae↗
PubMed2005

Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.

This is the first report of an insect esterase efficiently expressed in the methylotrophic yeast Pichia pastoris (so far insect esterases have been produced only in the baculovirus system). Having isolated a Tribolium castaneum carboxylesterase cDNA (TCE), we were initially unable to express it in Escherichia coli or P. pastoris despite significant transcription levels. As codon usage bias is different in T. castaneum and P. pastoris, we assumed this was a possible explanation for the translational barrier observed in yeast. Accordingly, we designed and constructed by recursive PCR a synthetic TCE gene (synTCE) optimized for heterologous expression in P. pastoris, i.e., a gene in which certain TCE codons are replaced with synonymous codons 'preferred' in P. pastoris. When the altered gene was placed under the control of either the P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase (AOX1) promoter and introduced on an expression vector into P. pastoris, its product was produced intracellularly. We also successfully explored the possibility of obtaining a secreted product: P. pastoris cells expressing an in-frame fusion of synTCE with the alpha-factor secretion signal under the control of the GAP promoter were found to secrete the recombinant esterase into the external medium (to a concentration of 7 mg/L). In addition to this demonstration of TCE production in yeast, our results suggest that the GAP promoter could advantageously replace the AOX1 promoter as a driver of synTCE expression. TCE specific activity was approximately 5 U/mg when p-nitrophenyl acetate was used as substrate.

Animals↗
PubMed2006

Stable gammaretroviral vector expression during embryonic stem cell-derived in vitro hematopoietic development.

Unlike conventional gammaretroviral vectors, the murine stem cell virus (MSCV) can efficiently express transgenes in undifferentiated embryonic stem cells (ESCs). However, a dramatic extinction of expression is observed when ESCs are subjected to in vitro hematopoietic differentiation. Here we report the construction of a self-inactivating vector from MSCV, MSinSB, which transmits an intron embedded within the internal transgene cassette to transduced cells. The internal transgene transcriptional unit in MSinSB comprises the composite cytomegalovirus immediate early enhancer-chicken beta-actin promoter and associated 5' splice site positioned upstream of the natural 3' splice site of the gammaretroviral envelope gene, and is configured such that the transgene translational initiation sequence is coincident with the envelope ATG. MSinSB could be produced at titers approaching 10(6) transducing units/ml and directed higher levels of transgene expression in ESCs than a splicing-optimized MSCV-derived vector, MSGV1. Moreover, when transduced ESCs were differentiated into hematopoietic cells in vitro, MSinSB remained transcriptionally active in greater than 90% of the cells, whereas MSGV1 expression was almost completely shut off. Persistent high-level expression of the MSinSB gammaretroviral vector was also demonstrated in murine bone marrow transplant recipients and following in vitro myelomonocytic differentiation of human CD34(+) cord blood stem/progenitor cells.

Animals↗
PubMed2003

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris.

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.

Animals↗
PubMed1994

Identification of a human immunodeficiency virus type 1 TAR binding protein in human hepatoblastoma HepG2 cells that trans-activates HIV-1 LTR-directed gene expression.

Recently, we have shown that the human immunodeficiency virus (HIV-1) long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) gene is efficiently expressed in human hepatoblastoma HepG2 cells and these cells can support productive HIV-1 replication. In this study we show that HepG2 cells contain a nuclear factor that binds to the HIV-1 trans-activating region (TAR), which we named HepG2-derived TAR binding protein (HTBP). Gel retardation assays using synthetic oligonucleotide probes carrying different mutations in the TAR region and competition DNA mobility-shift experiments using these oligonucleotides revealed the binding site encompassing between +7 and +13 nucleotides (5'-TCTGGTT-3') in the HIV-1 LTR. An in vivo CAT competition assay using -65HIV-1 LTR CAT as a reporter plasmid and various competitor plasmids containing these mutated oligonucleotides also demonstrated that HTBP can influence the HIV-1 LTR-directed CAT gene expression in HepG2 cells by interaction with a specific sequence in the TAR region.

Base Sequence↗
PubMed2001

High level expression of human factor VIII in mammalian cells after retroviral-mediated gene transfer.

OBJECTIVE: To develop a retroviral-mediated high efficient expression system of human coagulation factor VIII. METHODS: The LNC-FVIIIBD retroviral vector was generated by cloning a human B-domain-deleted (760aa-1639aa) Factor VIII (FVIII) cDNA (FVIII cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FVIII in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FVIIIBD mRNA. RESULTS: Human FVIII was expressed in all four target cells, with the highest FVIII expression observed in NIH3T3. The coagulant activity of secreted FVIII was up to 1.6 U/10(6) cells.24 hrs-1, and the FVIII antigen was 500 ng/10(6) cells.24 hrs-1. FVIII coagulant activity and antigen expressed by transduced CHO cells were 0.12 U/10(6) cells.24 hrs-1 and 62.4 ng/10(6) cells.24 hrs-1, respectively. Human FVIII expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FVIII cDNA BD in the target cells. CONCLUSIONS: The constructed retroviral vector was able to direct high level expression of human FVIII in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.

Animals↗
PubMed1995

Sindbis vectors suppress secretion of subviral particles of Japanese encephalitis virus from mammalian cells infected with SIN-JEV recombinants.

Double-subgenomic Sindbis virus (dsSIN) recombinants that express cassettes encoding prM-E or a C-terminally truncated form of E of Japanese encephalitis virus (JEV) were constructed. The products were efficiently expressed in both mammalian and mosquito cell lines infected with the dsSIN recombinants. However, suppression of prM-E secretion from mammalian cells infected with dsSIN-prM-E recombinants was observed. This suppression was more pronounced late in infection (< 5% of total product was secreted during an 8-hr chase) than early in infection (15% secretion during a 6-hr chase). In comparison, a vaccinia virus-prM-E recombinant (vP829) described previously (E. Konishi et al. (1991) Virology 185, 401-410) was shown to secrete 35-50% of total product during a 6- to 8-hr chase both early and late in infection. In contrast, secretion of prM-E from dsSIN-prM-E-infected mosquito (C6/36) cells was found to be efficient (> 50% during an 8-hr chase). The prM-E secreted from both mammalian and mosquito cells was in the form of subviral particles as determined by velocity gradient centrifugation, sensitivity to nonionic detergent, and analysis of processing of N-linked glycans. The truncated E protein expressed by the dsSIN recombinants was secreted efficiently from both mammalian and mosquito cells. Coinfection experiments with the dsSIN-JEV recombinants + wild-type vaccinia virus and vP829 + SIN demonstrated that the reduced level of secretion of subviral particles exhibited by the dsSIN-JEV recombinants was due to an inhibitory effect of the dsSIN vectors. Furthermore, this inhibitory effect was accounted for by the SIN nonstructural proteins since SIN replicons that express prM-E cassette in place of the SIN structural protein open reading frame exhibited a low level of subviral particle secretion. No self-propagating infectious particles were produced in cells transfected with SIN replicons that encode the JEV prM-E cassette. The suppression of subviral particle secretion was apparently correlated with the inhibition of cell protein synthesis which is mediated in SIN-infected vertebrate cells by expression of the SIN nonstructural proteins.

Aedes↗