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。     

细胞穿透肽Tat-LK15介导siRNA的转染效率和细胞毒性研究

通过与Lipofectamine TM RNAi MAX(Lipo)比较,探讨细胞穿透肽Tat-LK15对人肾上皮细胞(human embryonic kidney 293T cells,293T)和大鼠视神经节细胞(retinal ganglion cell line,RGC-5)转染小干扰RNA(small interference RNA,si RNA)的效率和细胞毒性,为后期实验提供依据。以羧基荧光素(carboxyfluorescein,FAM)标记的si RNA为报告基因,不同剂量的Tat-LK15(Tat-LK15组)和Lipo(Lipo组)为转染试剂,分别转染293T和RGC-5细胞。转染24 h后荧光显微镜观察FAM荧光强度,计算转染效率,并以CCK-8法检测细胞毒性。随着Tat-LK15和Lipo剂量的增加,si RNA转染效率逐渐提高。当Tat-LK15与si RNA配比为2∶1(μg/μg)时,RGC-5细胞的转染效率达到最高((87.3±4.5)%),低于Lipo与si RNA配比为5∶1(μL/μg)时的最高转染效率((96.2±3.2)%(P0.05));作用于293T细胞时,两转染试剂的最高转染效率没有统计学差异(P0.05)。转染试剂剂量增加,Lipo细胞毒性显著增加,而Tat-LK15毒性无明显变化。转染效率最高时,Lipo(5μL)组293T和RGC-5的细胞存活率仅为((77.8±4.1)%,(73.4±7.7)%)显著低于Tat-LK15(2∶1)组同种细胞的存活率((91.0±3.7)%,(90.0±6.1)%)(P0.05)。Tat-LK15可有效地转染si RNA,细胞毒性低,安全性突出,有一定的应用前景。     

Template RNA Length Determines the Size of Replication Complex Spherules for Semliki Forest Virus

The replication complexes of positive-strand RNA viruses are always associated with cellular membranes. The morphology of the replication-associated membranes is altered in different ways in different viral systems, but many viruses induce small membrane invaginations known as spherules as their replication sites. We show here that for Semliki Forest virus (SFV), an alphavirus, the size of the spherules is tightly connected with the length of the replicating RNA template. Cells with different model templates, expressed in trans and copied by the viral replicase, were analyzed with correlative light and electron microscopy. It was demonstrated that the viral-genome-sized template of 11.5 kb induced spherules that were ∼58 nm in diameter, whereas a template of 6 kb yielded ∼39-nm spherules. Different sizes of viral templates were replicated efficiently in trans, as assessed by radioactive labeling and Northern blotting. The replication of two different templates, in cis and trans, yielded two size classes of spherules in the same cell. These results indicate that RNA plays a crucial determining role in spherule assembly for SFV, in direct contrast with results from other positive-strand RNA viruses, in which either the presence of viral RNA or the RNA size do not contribute to spherule formation.     

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。     

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.     

Comparison of 6/94 Virus and Sendai Virus RNA by RNA-RNA Hybridization

The genomic RNA of 6/94 virus, an agent isolated from the brains of multiple sclerosis patients, was studied for sequence homology by RNA-RNA hybridization with closely related Sendai virus and another paramyxovirus virus, Newcastle disease virus. It was found that the genomic RNA of 6/94 virus hybridizes equally as well to the virus-specific 18S RNA found in Sendai-infected cells as that of Sendai virus.     

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。     

可磷酸化短肽偶联壳聚糖促进CS/DNA转染效率

合成基序为LLLRRRDNEY*FY*VRRLL的短肽(pSP),其中含有两个可被JaK2蛋白激酶磷酸化的酪氨酸残基.将此短肽与壳聚糖(CS)相偶联,体外磷酸化及DNA释放实验检测哺乳动物细胞裂解液对短肽的磷酸化及pSP-CS/DNA复合物中DNA释放的影响.放射性标记DNA转移实验验证pSP-CS/DNA复合物的入胞能力后,将荷荧光素酶或GFP报告基因的质粒与pSP-CS制成pSP-CS/DNA复合物,转染体外培养的C2C12小鼠成肌细胞,观察GFP的分布及细胞裂解液中的荧光素酶活性以表征转染效率.继而进行多种细胞系的转染,衡量pSP偶联的壳聚糖对不同种属细胞的转染效率.结果表明,哺乳动物细胞裂解液可有效地使短肽发生磷酸化,并藉此促进DNA与壳聚糖载体的解离.以pSP修饰的壳聚糖进行转染时,细胞裂解液的荧光素酶活性可达普通壳聚糖转染的两倍,细胞中GFP的含量也明显增加.据此推论,短肽被磷酸化后产生电荷属性的改变,促进DNA与壳聚糖载体的解离从而显著提高壳聚糖的转染效率.     

Apoptosis induced by Semliki Forest virus is RNA replication dependent and mediated via Bak

The RNA alphavirus Semliki Forest (SFV) triggers apoptosis in various mammalian cells, but it has remained controversial at what infection stage and by which signalling pathways host cells are killed. Both RNA synthesis-dependent and -independent initiation processes and mitochondrial as well as death receptor signalling pathways have been implicated. Here, we show that SFV-induced apoptosis is initiated at the level of RNA replication or thereafter. Moreover, by expressing antiapoptotic genes from recombinant SFV (replicons) and by using neutralizing reagents and gene-knockout cells, we provide clear evidence that SFV does not require CD95L-, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)- or tumor necrosis factor-mediated signalling but mitochondrial Bak to trigger cytochrome c release, the fall in the mitochondrial membrane potential, apoptotic protease-activating factor-1/caspase-9 apoptosome formation and caspase-3/-7 activation. Of seven BH3-only proteins tested, only Bid contributed to effective SFV-induced apoptosis. However, caspase-8 activation and Bid cleavage occurred downstream of Bax/Bak, indicating that truncated Bid formation serves to amplify rather than trigger SFV-induced apoptosis. Our data show that SFV sequentially activates a mitochondrial, Bak-mediated, caspase-8-dependent and Bid-mediated death signalling pathway that can be accurately dissected with gene-knockout cells and SFV replicons carrying antiapoptotic genes.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Human MxA Protein Confers Resistance to Semliki Forest Virus and Inhibits the Amplification of a Semliki Forest Virus-Based Replicon in the Absence of Viral Structural Proteins

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the β-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

靶向山羊痘病毒DNA聚合酶基因shRNA质粒表达载体的构建及筛选

本研究针对山羊痘病毒基因组高度保守的DNA聚合酶区段,选取了5个干扰靶位点。根据RNAi技术原理,构建了包括对照在内的6个shRNA重组表达质粒,转染细胞后接种病毒,通过细胞毒液的TCID_(50)测定和用实时荧光定量PCR检测siRNA重组质粒对羊痘病毒DNA聚合酶基因表达的抑制作用。结果显示,重组表达质粒组的抑制率在63%以上,其中以pSI-W2最为明显,抑制率为93.8%。该研究应用RNAi技术在细胞水平筛选出了能够高效抑制山羊痘病毒增殖的干扰片段,为抗山羊痘病毒转基因羊的研究奠定了基础。     

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.     

传统DNA疫苗载体与Semliki森林病毒复制子对HIV-1 Pr55gag表达与体液免疫原性的比较性研究

为探索Semliki森林病毒(SFV)衍生的复制型DNA载体可否用于HIV疫苗的候选载体,对该载体与传统DNA疫苗载体对HIV-1Pr55gag的表达与体液免疫原性进行了系统比较研究.将野生型(wtgag)及密码子改造(syngag)的HIV-1 ⅢB gag基因分别克隆于SFV DNA载体及传统DNA疫苗载体[pCDNA3.1(+)],对其Pr55gag细胞内表达水平、Pr55gag病毒样颗粒释放、以及在BALB/c鼠的体液免疫原性进行了比较.在293T、H1299、C2C12和BHK细胞系中,SFV-wtgag可以Rev非依赖方式有效表达Pr55gag,而pC-wtgag转染的细胞不能有效表达Pr55gag,从而不能诱导小鼠产生免疫反应.虽然SFV质粒的细胞转化效率明显低于pCDNA载体,SFV-wtgag和SFV-syngag在细胞内Pr55gag的表达量与pC-syngag相似,而Pr55gag病毒样颗粒的释放明显低于pC-syngag.在肌内注射免疫的小鼠中,低剂量(0.1和1.0μg)的SFV及pCDNA gag表达质粒均未诱导出GAG特异性免疫反应.在高剂量(10,30,100μg)免疫组中,与SFV gag表达质粒相比,pC-syngag可诱导出较高水平的TH1型GAG特异性抗体.SFV-syngag较SFV-wtgag可诱导出高水平的体液免疫反应.结果提示,SFV衍生的复制子单独使用不能在小鼠诱导出优于传统DNA疫苗载体的HIV-1 GAG特异性体液免疫反应,其原因可能与病毒样颗粒的释放有关.密码子改造的gag基因的优势在SFV载体系统中得到了进一步证实.     

Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.     

The Lipidomes of Vesicular Stomatitis Virus,Semliki Forest Virus,and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.Enveloped viruses acquire their lipid envelope from the membranes of host cells (43). In this process, the nucleocapsid or the nucleocapsid-matrix complex of the viruses buds out of the cell and becomes enveloped by a segment of the host membrane. This membrane segment is modified during the budding process, such that virally encoded membrane proteins are included in the viral envelope, while most host proteins are excluded. Since viruses usually do not carry lipid-synthesizing enzymes, the lipids in the viral envelope are derived from the host membrane. The lipid compositions of enveloped viruses have been studied for years (2, 15, 17, 18, 23, 25, 34, 36, 38, 40). One question that remains to be answered is whether the lipids are included passively, and thus the lipid composition of the envelope reflects the lipid composition of the host membrane, or whether lipid sorting occurs, leading to selective inclusion of some lipids and exclusion of others. This issue has been complicated by the fact that the lipid bilayer is no longer considered a homogenous liquid but contains fluctuating nanoscale assemblies of sphingolipids, saturated phospholipids, cholesterol (Chol), and proteins, called lipid rafts (13, 44). Lipid rafts can be induced to coalesce—usually by protein-protein interactions—into larger, dynamic platforms that function in signal transduction, intracellular membrane transport, and other membrane functions (45). It was also proposed that viruses make use of these membrane domains during their exit from cells (29, 32).A major complication in comparing viral envelopes with host cell membranes is the difficulty in obtaining host cell membranes of purity similar to that of the easily purified viruses. Many studies are faulted by the impurity of the cell membranes analyzed. Moreover, the early work in this field employed conventional analytical methods (such as thin-layer chromatography) that provide only semiquantitative estimates of the total abundance of the major lipid classes. Most importantly, lipid species diversity could not be analyzed. Recent developments in mass spectrometry (MS) have enabled comprehensive and quantitative analyses of lipidomes at the level of individual molecular species. The lipidomes of human immunodeficiency virus (HIV), murine leukemia virus (6, 7), and several bacteriophages (20, 21) were recently analyzed by these new methods.This paper focuses on two well-characterized enveloped viruses, Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV). SFV is an RNA virus belonging to the Togaviridae family of the Alphaviridae that acquires its envelope by budding from the host cell plasma membrane (PM) (46). Early studies analyzed the lipid composition of the viral envelope and also that of the host cell PM (39, 40). These studies revealed strong similarity between the envelope of SFV and the host PM, but one important discrepancy was the higher Chol-to-phospholipid ratio in the virus.VSV is an RNA virus belonging to the Rhabdoviridae family and also hijacks its envelope from the host cell PM (35), but the lipid specificity of the budding process remains controversial. The most recent studies claim that VSV buds from localized regions that do not reflect the average composition of the PM (23, 36). It has also been claimed that lipid rafts are involved in VSV envelope assembly during budding (37).We used BHK-21 cells as host cells to purify SFV and VSV. The purposes of this study were (i) to establish a robust, comprehensive, and quantitative method to analyze lipidomes, including the full complement of glycerolipid, glycerophospholipid, and sphingolipid species as well as Chol; (ii) to establish a protocol for purification of PM suitable for MS analysis; and (iii) to analyze and compare the lipidomes of SFV, VSV, and the BHK-21 PM.We found that the lipidomes of SFV and VSV are similar in molecular composition and are closely related to that of the BHK-21 PM. The small differences observed could be explained by the high degrees of curvature generated during the viral budding process.     

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.     

A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.