RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.     

Semliki Forest virus vectors expressing transforming growth factor beta inhibit experimental autoimmune encephalomyelitis in Balb/c mice

Cytokine immunomodulation of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, has remained a formidable treatment option, but access into the CNS is hampered due to the impermeability of the blood-brain barrier. In this report, we describe the construction and characterization of CNS-homing gene delivery/therapy vectors based on avirulent Semliki Forest virus (SFV) expressing either native or mutant transforming growth factor beta 1 (TGF-beta1). Biological activity of the expressed inserts was demonstrated by PAI-1 promoter driven luciferase production in mink cells and TGF-beta1 mRNA was demonstrated in the CNS of virus treated mice by in situ hybridization and RT-PCR. Both vectors, when given intraperitoneally to EAE mice significantly reduced disease severity compared to untreated mice. Our results imply that immunomodulation by neurotropic viral vectors may offer a promising treatment strategy for autoimmune CNS disorders.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.     

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.     

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.     

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient tor low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1–2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a co-factor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.     

Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.     

猪2型圆环病毒ORF2基因在塞姆利基森林病毒RNA复制子衍生的新型真核表达载体中的表达

猪 2型圆环病毒 (porcinecircovirus 2 ,PCV2 )是断乳仔猪多系统衰竭综合征 (postweaningmultisystemicwastingsyndrome,PMWS)的原发性病原。PCV2的ORF2编码病毒唯一的结构蛋白Cap。根据GenBank中公布的PCV2JXL株的序列设计一对引物 ,应用PCR方法从该毒株感染的PK 15细胞中扩增出完整的ORF2基因 ,将此基因克隆于本实验室此前构建的塞姆利基森林病毒 (SemlikiForestvirus,SFV)RNA复制子衍生的新型真核表达载体Psfv1cs中的BamHⅠ位点 ,获得重组质粒pSFV1CS Cap。用pSFV1CS-Cap分别转染BHK-21细胞和293T细胞 ,经间接免疫荧光试验检测表明 ,PCV2 ORF2基因在转染细胞中得到表达。小鼠接种试验表明 ,该重组质粒能诱导小鼠产生特异性抗体.     

Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties

BACKGROUND: Sendai virus (SeV) is a new type of cytoplasmic RNA vector, which infects and replicates in most mammalian cells, directs high-level expression of the genes on its genome and is free from genotoxicity. In order to improve this vector, both the matrix (M) and fusion (F) genes were deleted from its genome. METHODS: For the recovery of the M and F genes-deleted SeV (SeV/DeltaMDeltaF), the packaging cell line was established by using a Cre/loxP induction system. SeV/DeltaMDeltaF was characterized and compared with wild-type and F or M gene-deleted SeV vectors in terms of transduction ability, particle formation, transmissible property and cytotoxicity. RESULTS: SeV/DeltaMDeltaF was propagated in high titers from the packaging cell line. When this vector was administered into the lateral ventricle and the respiratory tissue, many of the ependymal and epithelial cells were transduced, respectively, as in the case of wild-type SeV. F gene-deletion made the SeV vector non-transmissible, and M gene-deletion worked well to inhibit formation of the particles from infected cells. Simultaneous deletions of these two genes in the same genome resulted in combining both advantages. That is, both virus maturation into particles and transmissible property were almost completely abolished in cells infected with SeV/DeltaMDeltaF. Further, the cytopathic effect of SeV/DeltaMDeltaF was significantly attenuated rather than that of wild type in vitro and in vivo. CONCLUSIONS: SeV/DeltaMDeltaF is an advanced type of cytoplasmic RNA vector, which retains efficient gene transfer, gains non-transmissible properties and loses particle formation with less cytopathic effect.     

A型肉毒毒素受体结合区Hc在重组SFV复制子载体中的表达

为了在哺乳动物细胞中表达A型肉毒毒素Hc抗原, 构建了含A型肉毒毒素受体结合区Hc基因的基于RNA和DNA的重组Semliki森林病毒(Semliki forest virus, SFV)复制子表达载体。RNA和DNA复制子载体转染BHK21细胞后, 经间接免疫荧光、Western印迹和ELISA检测, 结果表明非分泌型和分泌型的Hc抗原在细胞中都得到了有效地表达; 而且复制子表达载体与辅助病毒载体共转染均可制备高滴度的重组病毒颗粒, 该重组病毒颗粒感染细胞后, 也都能表达Hc抗原。以上结果表明, 基于RNA和DNA的重组SFV复制子表达载体在细胞中均可有效地表达Hc抗原和制备具有感染能力并能表达Hc抗原的重组病毒颗粒。基于RNA和DNA的重组SFV复制子表达载体的构建和含A型肉毒毒素受体结合区Hc基因的重组病毒颗粒的获得, 为进一步观察SFV复制子疫苗的免疫原性奠定了基础, 从而为A型肉毒毒素新型疫苗的研制提供了新途经。     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

The icosahedral RNA virus as a grotto: organizing the genome into stalagmites and stalactites

There are two important problems in the assembly of small, icosahedral RNA viruses. First, how does the capsid protein select the viral RNA for packaging, when there are so many other candidate RNA molecules available? Second, what is the mechanism of assembly? With regard to the first question, there are a number of cases where a particular RNA sequence or structure—often one or more stem-loops—either promotes assembly or is required for assembly, but there are others where specific packaging signals are apparently not required. With regard to the assembly pathway, in those cases where stem-loops are involved, the first step is generally believed to be binding of the capsid proteins to these “fingers” of the RNA secondary structure. In the mature virus, the core of the RNA would then occupy the center of the viral particle, and the stem-loops would reach outward, towards the capsid, like stalagmites reaching up from the floor of a grotto towards the ceiling. Those viruses whose assembly does not depend on protein binding to stem-loops could have a different structure, with the core of the RNA lying just under the capsid, and the fingers reaching down into the interior of the virus, like stalactites. We review the literature on these alternative structures, focusing on RNA selectivity and the assembly mechanism, and we propose experiments aimed at determining, in a given virus, which of the two structures actually occurs.     

Flavors of Flaviviral RNA Structure: towards an Integrated View of RNA Function from Translation through Encapsidation

For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision‐making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA‐centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro‐derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self‐regulation of viral replication is revealed.     

Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.     

Sequence comparisons for a hepatitis C virus genome RNA isolated from a patient with liver cirrhosis

The nucleotide (nt) sequence was determined for an isolate of hepatitis C virus (HCV) derived from cirrhotic tissue of a patient with hepatocellular carcinoma. The 9408-nt sequence (EMBL Acq. No. X61596) showed homology of 90.7-91.4% on the nt level, as compared to two Japanese isolates from patients with a high titer of serum transaminase, 78.4-78.8% to those obtained in the United States, and 65.0% to that from an asymptomatic Japanese carrier. The phylogenetic tree of the six isolates classified them into three groups.