Two-Helper RNA System for Production of Recombinant Semliki Forest Virus Particles

Alphavirus expression systems based on suicidal virus particles carrying recombinant replicons have proven to be a very efficient way to deliver genes for heterologous protein expression. However, present strategies for production of such particles have biosafety limitations due to the generation, by RNA recombination, of replication-proficient viruses (RPVs). Here we describe a new packaging system for Semliki Forest virus (SFV) based on a the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 polyprotein are expressed from two independent RNA molecules. The capsid gene contains a translational enhancer and therefore that sequence was also engineered in front of the spike sequence p62-6K-E1. A sequence coding for the foot-and-mouth disease virus 2A autoprotease was inserted in frame between the capsid translational enhancer and the spike genes. This allows production of the spike proteins at high levels with cotranslational removal of the enhancer sequence and normal biosynthesis of the spike complex. The autoprotease activity of the capsid protein was abolished by mutation, further increasing the biosafety of the system. Cotransfection of cells with both helper RNAs and an SFV vector replicon carrying the LacZ gene led to production of recombinant particles with titers of up to 8 × 10⁸ particles per 10⁶ cells. Extensive analysis failed to demonstrate the presence of any RPVs, emphasizing the high biosafety of the system based on two-helper RNAs.     

Inactivation of Semliki Forest Virus in aerosols.

Purified Semliki forest virus in aerosols is inactivated rapidly at 40% and above 70% relative humidity. At all humidities tested the decay of virus infectivity runs parallel with the decrease in hemagglutination activity, whereas the biological integrity of the virus ribonucleic acid is preserved. Also, free infectious ribonucleic acid is stable after spraying at all relative humidities. Evidence is presented for the hypothesis that above 20% relative humidity, virus inactivation in aersols is mainly due to surface-dependent factors, damaging the virus coat.     

Ribonuclease Sensitivity of Semliki Forest Virus Nucleocapsids

Treatment of Semliki Forest virus nucleocapsids with pancreatic ribonuclease (1 mug/ml, 37 C) digests the ribonucleic acid to acid-soluble fragments; the nucleocapsid protein forms a rapidly sedimenting aggregate.     

Semliki Forest Virus in HEp-2 Cell Cultures

The growth and development of Semliki Forest virus (SFV), an arbovirus of serological group A, in HEp-2 cells in tissue culture was examined by various techniques at frequent intervals. Infectivity and fluorescent-antibody studies demonstrated the presence of infective virus and viral antigens within the cells at 8 hr after infection. The antigen was particulate and distributed throughout the cytoplasm. Thereafter, there was rapid progression of virus production and cell destruction. By electron microscopy, tubular structures bounded by a fine membrane were observed in cytoplasm at 12 hr. Rows of small (25 mmu) virus particles were often present on the outer surface of these membranes, and at later times they became progressively more encrusted with the small virus particles. These structures subsequently increased rapidly in number, size, and complexity, and the space between the membrane and the tubules increased, thus forming vacuoles which contained tubules and were covered with the small particles. At later times (24 hr and later) larger (42 to 50 mmu) particles were observed, usually inside of the vacuoles. These larger particles (and occasionally the smaller ones) were also seen at the cell periphery and in the extracellular space. The large SFV particles appear to form by three distinct processes: (i) from the smaller particles, (ii) by development on an intravacuolar membrane, and (iii) at the ends of the tubules. The mode of development of SFV is unique among viruses studied to date, but in some characteristics it resembles that of other group A arboviruses. Its development differs from that of most arboviruses of group B and other serological groups.     

Electroporation-Mediated Delivery of Nucleolar Targeting Sequences from Semliki Forest Virus Nucleocapsid Protein

Abstract

Electroporation was used as a powerful and simple method to probe to the intracellular distribution and trafficking of signal sequences. By coupling synthetic peptides to carrier reporter groups, specific amino acid sequences responsible for nucleolar targeting of Semliki Forest virus (SFV) Core (C) protein were found out. In the N-terminal part of the C protein the sequences ⁶⁶KPKKKKTTKPKPKTQPKK^{83 92}KKKDKQADKKKKP¹⁰⁵ are able to situate BSA or KLH as reporter proteins in the nucleolus, suggesting that SFV C protein contains at least two independent nucleolar targeting sequences.     

B cells and antibodies in the pathogenesis of myelin injury in Semliki Forest Virus encephalomyelitis

To determine the contribution of B cells to brain myelin injury in Semliki Forest Virus (SFV) encephalomyelitis, normal C57BL/6 (B6) and B-cell-deficient (C57BL/6-tm1Cgn) B6 mice were infected with SFV. The peak of clinical disease, i.e., the time at which the greatest proportions of mice had moderate to severe clinical signs, appeared earlier in B6 mice [day 7 postinfection (pi)] than in B-cell-deficient mice (day 21 pi). By flow cytometry, no clear differences were found in the percentages of CD3(+)CD4(+) T cells in the brains of B6 and B-cell-deficient mice. However, by day 21 pi, percentages of CD3(+)CD8(+) T cells were greater in brains of B-cell-deficient than in those of B6 mice. On day 21 pi, percentages of CD19(+) B cells were maximal in B6 mice, but B cells were absent in B-cell-deficient mice at all time points. Sera obtained from B6 mice showed antibody responses to SFV, to SFV E2 peptides p137-151 and p115-133, and to peptides of myelin oligodendrocyte glycoprotein p18-32 and myelin basic protein (MBP) p64-75. Sera obtained from B-cell-deficient mice showed minimal or no reactivity to SFV, E2, or myelin peptides. CNS inflammatory and PAS-positive macrophage foci were maximal on days 7-14 pi in all mice. Additionally, B6 mice had brain white matter vacuolation, whereas B-cell-deficient mice did not. These data suggest that brain infiltrating B cells and anti-myelin antibodies contribute to myelin injury in SFV encephalomyelitis.     

Inhibition of phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells infected with Semliki Forest virus.

Semliki Forest virus inhibits phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells 6 h after infection. Viral infection reduced the incorporation of [1,2-14C]-ethanolamine into intact cells by approximately 50%. A similar reduction in the activity of the ethanolaminephosphotransferase (EC 2.7.8.1) was also observed. The apparent Km for CDPethanolamine was 60 muM for the microsomal enzymes from infected or mock-infected cells. In addition, exogenous diglyceride only stimulated by 1.5-fold the ethanolaminephosphotransferase from virus- or mock-infected cells, whereas the same diglyceride preparations stimulated the cholinephosphotransferase (EC 2.7.8.2) from baby hamster kidney cells by sixfold. Generation of endogenous diglyceride by pretreatment of the microsomes with phospholipase C (EC 3.1.4.3) stimulated the activity of the cholinephosphotransferase but not the ethanolaminephosphotranferase. Semliki Forest virus does not inhibit all microsomal enzymes, since the activities of NADH- K3Fe(CN)6 reductase and NADH dehydrogenase (EC 1.6.99.3) were not affected. The ethanolaminephosphotransferase from virus- and mock-infected cells showed similar profiles of activity as a function of temperature; this result and other studies suggest that that membranous environment of the ethanolaminephosphotransferase was not significantly modified by the virus.     

一种基于塞姆利基森林病毒复制子的新型复制子载体

RNA复制子是能自主复制的病毒RNA。基于RNA复制子的表达载体和基因疫苗比常规真核表达载体和DNA疫苗具有更大的优越性。以塞姆利基森林病毒RNA复制子衍生的真核表达载体pSFV1为骨架 ,插入CMV立即早期启动子和SV40晚期Poly(A)信号 ,构建了一种完全基于DNA的复制型表达载体pSFV1CS ,将增强型绿色荧光蛋白基因EGFP插入其中 ,构建了重组质粒pSFV1CS EGFP ,通过转染 2 93T细胞 ,证实外源基因能在其中高效表达。该载体可用于表达真核蛋白和构建复制子载体疫苗。     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Recombinant protein production using the Semliki Forest Virus expression system

We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial β-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 10⁹ cells⁻¹) with an average specific activity of 3942 ± 765 pg PGE₂ μg⁻¹ microsomal protein 5 min⁻¹. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protein Synthesis in BHK-21 Cells Infected with Semliki Forest Virus

[³H]leucine-labeled proteins synthesized in BHK-21 cells infected with Semliki Forest virus were fractionated by polyacrylamide gel electrophoresis (PAGE). Cellular and virus-specific proteins were identified by difference analysis of the PAGE profiles. The specific activity of intracellular [³H]leucine was determined. Two alterations of protein synthesis, which develop with different time courses, were discerned. (i) In infected cultures an inhibition of overall protein synthesis to about 25% of the protein synthesis in mock-infected cultures develops between about 1 and 4 h postinfection (p.i.). (ii) The relative amount of virus-specific polypeptides versus cellular polypeptides increases after infection. About 80% of the proteins synthesized at 4 h p.i. are cellular proteins. Since significant amounts of nontranslocating ribosomes in polyribosomes were not detected up to 7 h p.i., the inhibition of protein synthesis is not caused by inactivation of about 75% of all polyribosomes but by a decreased protein synthetic activity of the majority of polyribosomes. Indirect evidence indicates that an inhibition of elongation and/or release of protein synthesis develops in infected cells, which is sufficient to account for the observed inhibition of protein synthesis. Inhibition of over-all protein synthesis developed when virus-specific RNA began to accumulate at the maximal rate. This relationship was observed during virus multiplication at 37, 30, and 25 C. A possible mechanism by which synthesis of virus-specific RNA in the cytoplasm could inhibit cellular protein synthesis is discussed. Indirect evidence and analysis of polyribosomal RNA show that the increased synthesis of virus-specific protein is brought about by a substitution of cellular by viral mRNA in the polyribosomes.     

基于Semliki森林病毒复制子的新型RNAi质粒载体的构建

目的:以semliki森林病毒复制子为基础,构建一类可迅速高效表达shRNA的新型RNAi载体。方法:以Semliki森林病毒衍生的复制子载体pSFV1为骨架,用CMV IE启动子替换SP6启动子并在3′-UTR下游插入SV40 polyA转录终止子,在原26S亚基因组启动子后插入带有相应改良多克隆位点的shRNA表达元件,同时加入抗新霉素选择复合体,并去掉3′-UTR的重复序列。所获载体用于沉默EGFP基因,通过体外细胞转染、病毒颗粒制备、荧光显微镜观察、RT-PCR分析等初步验证、评估其效果。结果:构建了基于Semliki森林病毒复制子的新型RNAi质粒载体pSFV-RNAi Ready。经体外实验初步证实,该载体直接转染细胞,或与辅助载体共转染,制备成具有感染能力的重组病毒颗粒后使用,均可高水平表达shRNA,沉默目的基因。其中使用病毒颗粒抑抑效率可高达90%以上。结论:该载体的成功构建,可望显著拓宽SFV载体的应用范围,丰富RNAi实施手段,并用于相关科学研究及基因药物技术开发。     

Cytoplasmic Fractions Associated with Semliki Forest Virus Ribonucleic Acid Replication

When actinomycin D-treated chick fibroblasts were labeled with (3)H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26S form, and a ribonuclease-resistant 20S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65S cytoplasmic fraction which contained only the 26S RNA form. Finally, after a 1-hr labeling period, a 140S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42S viral RNA. Negative-contrast electron micrographs of the 140S particle and the virion demonstrated structural differences between them. The base compositions of the 42S and 26S viral RNA forms were not significantly different. The base composition of the 20S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20S form differed, and depended on the period during the virus growth cycle in which (32)P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20S RNA appeared to be a structure engaged in viral RNA replication and the 140S particle appeared to be a virus precursor.     

A model of the binding, entry, uncoating, and RNA synthesis of Semliki Forest virus in baby hamster kidney (BHK-21) cells

A quantitative understanding of viral trafficking would be useful in treating viral-mediated diseases, designing protocols for viral gene therapy, and optimizing heterologous protein production. In this article, a model for the trafficking of Semliki Forest virus and its RNA synthesis in baby hamster kidney (BHK-21) cells is presented. This model includes the various steps leading to infection such as attachment, endocytosis, and viral fusion in the endosome. The model estimates a mean fusion time of 4 to 6 min for the wild-type virus, and 38 min for Fus-1, an SFV mutant which requires a lower pH for fusion. These mean fusion times are consistent with the time-scale of endosomal acidification, suggesting viruses fuse almost instantaneously with the endosomal membrane as soon as the pH of the endosome drops below the pH threshold of the virus. Infection is most likely controlled at the level of viral uncoating, as shown by the close agreement between the efficiency of uncoating and the experimentally determined fraction of viruses that is infectious. The viral RNA synthesized per cell is best described by assuming that it depends on the number of uncoated viruses prior to the onset of replication according to a saturation-type expression. A Poisson distribution is used to determine the distribution of uncoated viruses among the cells. Because attachment is the rate-limiting step in the uncoating of the virus, increasing the attachment rate can lead to enhanced RNA synthesis and, hence, new virion production. Such an increase in the attachment rate may be obtained by lowering the medium pH or the addition of a polycation. (c) 1995 John Wiley & Sons, Inc.     

基于DNA和RNA的双功能Semliki森林病毒复制子载体的构建

以semliki森林病毒衍生的复制子载体pSFV1和辅助载体pSFV-helper2为骨架, 用CMV IE和T7启动子替换SP6启动子并在3′ UTR下游插入BGH转录终止子,构建了基于DNA和RNA的复制子表达载体pSMCTA和辅助载体pSHCTA。在DNA和RNA二种递送方式上证实该表达载体可高水平表达外源基因,与辅助载体共转染可制备具有感染能力并能表达外源基因的重组病毒颗粒。构建的基于DNA和RNA的双功能复制子载体显著地提高SFV载体应用范围,在体外可用于高水平表达外源基因及大规模制备重组病毒颗粒,在体内也可用于研制复制子疫苗和基因治疗载体。     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

Expression of Ligand-Gated Ion Channels with the Semliki Forest Virus Expression System

Abstract

Two types of ligand-gated ion channels were expressed with the Semliki Forest virus (SFV) expression system.The cDNAs for mouse serotonin 5-HT₃ receptor and rat and human purinoreceptor P_2x subtypes were introduced into the pSFV1 vector. In vitro transcribed RNAs were coelectroporated with pSFV-Helper2 RNA into BHK cells, where in vivo packaging resulted in high titer SFV-5-HT₃ and SFV-P_2x virus stocks. Infection of BHK, CHO and RJN cells resulted in high-level expression of recombinant receptors. Saturation binding analysis indicated the presence of more than 3 × 10⁶ 5-HT3 receptors per cell. Binding studies on isolated membranes yielded from 10 to 60 pmol of either 5-HT₃ or P_2x receptor per mg protein. Functional responses to the P_2x receptors were demonstrated in SFV-infected CHO cells by Ca²⁺ mobilization or by ⁴⁵Ca²⁺ influx. High amplitude electrophysiological responses were also detected for both SFV-5-HT₃ and SFV-P_2x infected CHO cells in whole-cell patch clamp recordings. To facilitate the purification procedure of SFV-expressed recombinant receptors a histidine tag was introduced at the C-terminus of the 5-HT₃ receptor. This 5-HT₃His receptor showed high levels of expression, specific binding and high amplitude electrophysiological responses. For large scale expression the BHK cells were adapted to suspension culture and were efficiently infected in a 11.5 liter fermentor culture with SFV-5-HT3His resulting in high-level expression, 52 pmol receptor per mg protein corresponding to 3.2 × 10⁶ receptors per cell.     

Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus

Baby hamster kidney (BHK) cells were infected with Semliki Forest virus (SFV) and, 2 h later, were treated for 4 h with 10 microM monensin. Each of the four to six flattened cisternae in the Golgi stack became swollen and separated from the others. Intracellular transport of the viral membrane proteins was almost completely inhibited, but their synthesis continued and they accumulated in the swollen Golgi cisternae before the monensin block. In consequence, these cisternae bound large numbers of viral nucleocapsids and were easily distinguished from other swollen cisternae such as those after the block. These intracellular capsid-binding membranes (ICBMs) were not stained by cytochemical markers for endoplasmic reticulum (ER) (glucose-6-phosphatase) or trans Golgi cisternae (thiamine pyrophosphatase, acid phosphatase) but were labeled by Ricinus communis agglutinin I (RCA) in thin, frozen sections. Since this lectin labels only Golgi cisternae in the middle and on the trans side of the stack (Griffiths, G., R. Brands, B. Burke, D. Louvard, and G. Warren, 1982, J. Cell Biol., 95:781-792), we conclude that ICBMs are derived from Golgi cisternae in the middle of the stack, which we term medial cisternae. The overall movement of viral membrane proteins appears to be from cis to trans Golgi cisternae (see reference above), so monensin would block movement from medial to the trans cisternae. It also blocked the trimming of the high-mannose oligosaccharides bound to the viral membrane proteins and their conversion to complex oligosaccharides. These functions presumably reside in trans Golgi cisternae. This is supported by data in the accompanying paper, in which we also show that fatty acids are covalently attached to the viral membrane proteins in the cis or medial cisternae. We suggest that the Golgi stack can be divided into three functionally distinct compartments, each comprising one or two cisternae. The viral membrane proteins, after leaving the ER, would all pass in sequence from the cis to the medial to the trans compartment.