Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation. During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities.     

Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity,cell survival,and mitochondria number in CHO-DG44 grown in suspension and serum-free media

We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.     

Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild-type Bcl-2 was compared to a Bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA "ladder" and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl-2 mutant, cell death due to Sindbis virus was inhibited in a concentration-dependent manner. Furthermore, the Bcl-2 mutant provided increased protection as compared to wild-type Bcl-2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl-2 variants compared to the parental cell line. In order to understand the reasons for the improved anti-apoptosis properties of the mutant, wild-type Bcl-2 and mutant Bcl-2 were examined by Western blot following each model insult. Wild-type Bcl-2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl-2 protein was not degraded during the same period. The processing of Bcl-2 was found to correlate with reduced cell viabilities following the two external insults to suggest that Bcl-2 degradation may limit its ability to inhibit apoptosis. These studies indicate that the cells regulate anti-apoptosis protein levels and these processing events can limit the effectiveness of cell death inhibition strategies in mammalian cell culture systems.     

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.     

Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells

Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.     

Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Improved Semliki Forest virus vectors for receptor research and gene therapy.

We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in beta-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.     

Novel mutant Semliki Forest virus vectors: gene expression and localization studies in neuronal cells

Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology.     

IMPROVED SEMLIKI FOREST VIRUS VECTORS FOR RECEPTOR RESEARCH AND GENE THERAPY

We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in β-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.     

Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells

Alphavirus glycoproteins have broad host ranges. Human immunodeficiency virus type 1 (HIV-1) vectors pseudotyped with their glycoproteins could extend the range of tissues that can be transduced in both humans and animal models. Here, we established stable producer cell lines for HIV vectors pseudotyped with alphavirus Ross River virus (RRV) and Semliki Forest virus (SFV) glycoproteins E2E1. RRV E2E1-stable clones could routinely produce high-titer pseudotyped vectors for at least 5 months. SFV E2E1-stable clones, however, produced relatively low titers. We examined the properties of RRV E2E1-pseudotyped vectors [HIV-1(RRV)] and compared them with amphotropic murine leukemia virus Env- and vesicular stomatitis virus glycoprotein G-pseudotyped vectors. HIV-1(RRV) displayed a number of characteristics which would be advantageous in ex vivo and in vivo experiments, including resistance to inactivation by heat-labile components in fresh human sera and thermostability at 37 degrees C. Upon single-step concentration by ultracentrifugation of HIV-1(RRV), we could achieve vector stocks with titers up to 6 x 10(7) IU/ml. HIV-1(RRV) efficiently transduced cells from several different species, including murine primary dendritic cells, but failed to transduce human and murine T cells as well as human hematopoietic stem cells (HSC). These results indicate that HIV-1(RRV) could be used in a number of applications including animal model experiments and suggest that expression of RRV cellular receptors is limited or absent in certain cell types such as T cells and human HSC.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%. Total viable cell densities were similar and specific productivities were enhanced by almost two-fold on scale-up to bioreactors. Comparison in a chemically defined media demonstrated an even greater sustained viability in bcl-x(L) expressing cells by 50% and up to 90% increase in titer with no impact on product quality. Caspase 3 activities were monitored as a marker for apoptotic cell death. In the presence of Bcl-x(L), caspase activities were reduced to background levels. The role of Bcl-x(L) in protecting cells from premature death was further examined in studies performed in the presence of NaBu, at concentrations known to trigger cell death. Results demonstrated that cells expressing bcl-x(L) retained 88% cell viability with >2 fold increase in titer. Bcl-x(L) was similarly overexpressed in a different CHO cell line producing a humanized mAb against the chemokine MCP1. Once again, production titer was increased by 80% and viability by 75%. Together the studies have shown that overexpression of bcl-x(L) in production cell lines was able to significantly increase the titer by enhancing both the specific activity and total cell viability while maintaining product quality.     

Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures

Apoptosis is now recognized as a significant problem in mammalian cell culture. Therefore, in this study, a single gene and multigene approach to inhibit apoptosis has been examined. Stable Chinese hamster ovary (CHO) cell lines were generated to overexpress different single, dual, and triple combinations of three apoptosis inhibitor genes. Two upstream inhibitors involved in the mitochondrial pathway, Bcl-X(L) and Aven, were expressed in addition to a downstream inhibitor of caspases. The caspase inhibitor, a variant of XIAP containing only the caspase inhibitory BIR domains (XIAP-BIRs), has been shown previously to enhance viabilities in mammalian cultures. Stable clonal cell lines were generated and tested for three apoptotic insults: Sindbis virus infection, the chemical reagent etoposide, and spent medium. For all single gene experiments, the Bcl-X(L)-containing cell lines provided superior protection to either the Aven- or XIAP-BIRs-containing cell lines following initial exposure to the insults. However, the cell lines expressing two or more anti-apoptosis proteins were more effective at inhibiting cell death than those expressing just one anti-apoptosis gene. The cell lines overexpressing Bcl-X(L) in combination with XIAP-BIRs were especially effective in delaying cell death for all three apoptotic insults. Expression of all three anti-apoptosis genes in concert was only slightly more effective than using Bcl-X(L) and XIAP-BIRs for some insults. During exposure to spent medium, CHO-BIRS + Aven + BclX(L) was the best inhibitor of apoptosis (IAP) initially, whereas CHO-BIRs + BclX(L) was particularly effective at later times of the experiment. In conclusion, the utilization of a mitochondrial dysfunction inhibitor used in combination with a caspase inhibitor was more effective in thwarting the progression of apoptosis than either inhibitor expressed individually. Thus, the concurrent expression of multiple apoptosis inhibitors may be the most effective strategy to increase survival of mammalian cells in culture.     

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.     

Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19K and Aven in the production of a monoclonal antibody with Chinese hamster ovary cells

Mammalian cells are used for the production of numerous biologics including monoclonal antibodies. Unfortunately, mammalian cells can lose viability at later stages in the cell culture process. In this study, the effects of expressing the anti-apoptosis genes, E1B-19K and Aven, separately and in combination on cell growth, survival, and monoclonal antibody (MAb) production were investigated for a commercial Chinese Hamster Ovary (CHO) mammalian cell line. CHO cells were observed to undergo apoptosis following a model insult, glucose deprivation, and at later stages of batch cell culture. The CHO cell line was then genetically modified to express the anti-apoptotic proteins E1B-19K and/or Aven using an ecdysone-inducible expression system. Stable transfected pools induced to express Aven or E1B-19K alone were found to survive 1-2 days longer than the parent cell line following glucose deprivation while the expression of both genes in concert increased cell survival by 3 days. In spinner flask batch studies, a clonal isolate engineered to express both anti-apoptosis genes exhibited a longer operating lifetime and higher final MAb titer as a result of higher viable cell densities and viabilities. Interestingly, survival was increased in the absence of an inducer, most likely as a result of leaky expression of the anti-apoptosis genes confirmed in subsequent PCR studies. In fed-batch bioreactors, the expression of both anti-apoptosis genes resulted in higher growth rates and cell densities in the exponential phase and significantly higher viable cell densities, viabilities, and extended survival during the post-exponential phase. As a result, the integral of viable cells (IVC) was between 40 and 100% higher for cell lines engineered to express both Aven and E1B-19K in concert, and the operational lifetime of the fed-batch bioreactors was increased from 2 to 5 days. The maximum titers of MAb were also increased by 40-55% for bioreactors containing cells expressing Aven and E1B-19K. These increases in volumetric productivity arose primarily from enhancements in viable cell density over the course of the fed-batch culture period since the specific productivities for the cells expressing anti-apoptosis genes were comparable or slightly lower than the parental hosts. These results demonstrate that expression of anti-apoptosis genes can enhance culture performance and increase MAb titers for mammalian CHO cell cultures especially under conditions such as extended fed-batch bioreactor operation.     

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.     

我国D2-43病毒株PrM-E基因的重组SFV的制备及生物学鉴定

 为构建含PrM E基因的重组SFV病毒 ,将含我国登革 2型病毒PrM E基因的SFV表达载体和辅助载体DNA线性化后 ,进行体外转录 .然后将获得的 2种RNA转录物共转染BHK2 1细胞 ,以RT PCR法证明转染后的细胞培养上清中含有重组SFV .进一步将该重组病毒经蛋白酶激活后可使BHK2 1细胞产生细胞病变 ,并且以间接免疫荧光法在感染细胞内可检测到登革 2型病毒所特有的蛋白 .含我国登革 2型病毒PrM E基因的重组SFV的获得 ,为进一步观察该重组病毒的免疫原性奠定了基础 ,从而为登革新型疫苗的研制提供新途径     

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.