Genome of Sindbis Virus

³²P-labeled ribonucleic acid (RNA) from purified Sindbis virus was examined for the presence of hidden breaks. Viral RNA was treated with acid at pH 2.9 or with formaldehyde and was analyzed on sucrose gradients or by polyacrylamide gel electrophoresis. The sedimentation pattern and mobility on polyacrylamide gels of the 42S RNA was unaffected by heating and quick cooling and was not altered by denaturing agents such as dimethyl sulfoxide and urea. No evidence that Sindbis RNA is a polyaggregate of fragments was obtained. It is concluded that the genome consists of a continuous length of single-stranded polynucleotide.     

Agglutination of Sindbis Virus and of Cells Infected with Sindbis Virus by Plant Lectins

We have examined the agglutination of Sindbis virus and of chick and hamster cells infected with Sindbis virus by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. Both lectins agglutinate the virus by binding to the polysaccharide chains of the envelope glycoproteins. Both chick and hamster cells exhibit increased agglutination by the lectins after infection by Sindbis virus. In the case of chick cells infected with Sindbis virus, this increase in agglutinability occurs between 3 and 5 h after infection. Infected and mock-infected cells bind the same amount of (3)H-labeled concanavalin A, which suggests that the increase in agglutination after infection is due to rearrangements at the cell surface rather than to insertion of new lectin binding sites per se.     

Phosphorylated Proteins of Sindbis Virus

The capsid and two membrane proteins of Sindbis virus, grown in chicken cells, contain 0.03 to 0.1 mol of phosphate per mol of protein.     

Large-Molecular-Weight Precursors of Sindbis Virus Proteins

Infection of chicken embryo fibroblasts with a temperature-sensitive mutant of Sindbis virus at the nonpermissive temperature leads to the accumulation of a large-molecular-weight protein. We have shown that this protein contains (14)C-arginine tryptic peptides present in the three virion proteins. We have also found that a slightly smaller protein which is detected in Sindbis-infected BHK cells contains the (14)C-arginine tryptic peptides of the two envelope proteins but not those of the capsid protein. Pulse-chase experiments indicate that the Sindbis virus protein in BHK cells is cleaved to the envelope proteins.     

Enzymatic Iodination of Sindbis Virus Proteins

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.     

Homologous Interference Induced by Sindbis Virus

Homologous interference during Sindbis virus infection has been investigated. Prior infection of either chicken embryo fibroblast or BHK(21) cell cultures results in reduced yields of progeny virions of the superinfecting genotype. This reduction in yield results from a reduction in the number of cells in the cultures capable of producing the superinfecting genotype. The development of interference parallels the attachment kinetics of Sindbis virus. Interference requires an active viral genome since the activity is sensitive to inactivation by ultraviolet light, and an RNA(-) mutant, ts-24, fails to induce interference under nonpermissive conditions. However, ts-6, an RNA(-) mutant belonging to a different complementation group, and the RNA(+) mutants, ts-2 and ts-20, interfere at both permissive and nonpermissive temperatures.     

Location of the Glycoprotein in the Membrane of Sindbis Virus

SINDBIS virus, which is transmitted by arthropods, consists of a nucleoprotein core within a lipid-containing envelope. Its components assemble at a cellular membrane and virus particles form by an outfolding of this membrane. Thus, such viruses provide useful systems for studies of the structure and synthesis of membranes. The Sindbis virus particle contains only two proteins, one associated with the viral envelope and the other with the viral RNA in the core, or nucleocapsid¹. The protein associated with the membrane is a glycoprotein, whereas the core protein contains no carbohydrate². The exact location of the glycoprotein within the viral envelope has not been determined, nor has information been obtained about the function of the carbohydrate in the virion. The results described here indicate that the spikes which cover the surface of the virion are glycoprotein in nature.     

Glycoproteins of Sindbis Virus: Preliminary Characterization of the Oligosaccharides

The carbohydrate content of Sindbis virus was determined by gas chromatographic analysis. The two viral glycoproteins were found to be approximately 8% carbohydrate by weight. Mannose is the sugar present in the largest amount. Smaller amounts of glucosamine, galactose, sialic acid, and fucose were also detected. Each of the two viral glycoproteins appears to contain two structurally unrelated oligosaccharides. Two of the three Sindbis-specific glycoproteins found in infected chick cells were shown to contain short, unfinished oligosaccharides.     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Comparison of Membrane Protein Glycopeptides of Sindbis Virus and Vesicular Stomatitis Virus

A comparison has been made of the membrane glycoproteins and glycopeptides from two enveloped viruses, Sindbis virus and vesicular stomatitis virus (VSV). Glycopeptides isolated from Sindbis virus and VSV grown in the same host appear to differ principally in the number of sialic acid residues per glycopeptide; when sialic acid is removed by mild acid treatment, the glycopeptides of the two viral proteins are indistinguishable by exclusion chromatography. Preliminary evidence argues that the carbohydrate moiety covalently bound to different virus-specified membrane proteins may be specified principally by the host.     

基孔肯雅病毒和辛德毕斯病毒可视化基因芯片与荧光基因芯片的建立

根据GenBank中收录的基孔肯雅病毒和辛德毕斯病毒E蛋白基因序列,设计及筛选针对2种病毒的寡核苷酸探针及引物,制备基孔肯雅病毒与辛德毕斯病毒可视化基因芯片与荧光基因芯片,对芯片的灵敏性、特异性进行了验证,并将可视化基因芯片、荧光基因芯片进行灵敏性比较.结果显示,制备的两种基因芯片都能检测到基孔肯雅病毒和辛德毕斯病毒特异性杂交信号.可视化基因芯片、荧光基因芯片检测两种病毒质粒的灵敏度达到9.1×103 copies/mL, 6.8×101 copies/mL和9.1×104 copies/mL, 6.8×103 copies/mL,与普通PCR比较差异显著. 荧光基因芯片灵敏度是PCR方法的10倍,可视化基因芯片是荧光基因芯片灵敏度的100倍. 模拟病毒检测过程特异性检验证明,可视化基因芯片都具有良好的特异性.本试验建立了基孔肯雅病毒与辛德毕斯病毒两种特异的可视化和荧光基因芯片检测方法,两种方法灵敏度高、特异性强,适用于基孔肯雅病毒与辛德毕斯病毒的流行病学调查和种特异性鉴定.     

Temperature-Sensitive Virus from Aedes albopictus Cells Chronically Infected with Sindbis Virus

Cultures of Aedes albopictus cells persistently infected with wild-type Sindbis virus (SV-W) give rise to small plaque-forming mutants which are also temperature sensitive. These mutants, designated SV-C, are neutralized by antiserum produced against SV-W. Mutant ts clones were isolated from SV-C by plaque purification. After serial undiluted passage in BHK or mosquito cells, each of the clones gave rise to ts(+) revertants which, however, remained mutant with respect to plaque morphology. Nineteen of 20 clones derived from SV-C were RNA(+), and one was RNA(-) (SV-C-2). The RNA synthesizing activity, once induced in infected cells by SV-C-2, was stable at the nonpermissive temperature (39.5 C). All clones derived from SV-C were inactivated at 60 C much more quickly than was SV-W. It was not possible to demonstrate complementation between any of the SV-C clones.     

Proteins Specified by Sindbis Virus in HeLa Cells

HeLa cells infected with Sindbis virus were found to contain five species of nonvirion proteins besides the structural proteins of the virus. Some of the nonvirion proteins were found to serve as precursors to the viral structural proteins in a pulse-chase experiment.     

Modification of Sindbis Virus Glycoprotein by Host-Specified Glycosyl Transferases

The amino acid sequence of the membrane glycoprotein of Sindbis virus is specified by the viral genome, but it has not been determined whether the carbohydrate portion of this molecule is specified by the cell or by the virus. We have examined two of the enzyme activities which catalyze transfer of monosaccharides to glycoprotein (sialyl and fucosyl transferases). Comparison of particulate enzyme preparations from infected and uninfected cells showed no difference in either the specific activity or acceptor specificity of these enzymes. This is impressive in view of the fact that the Sindbis membrane glycoprotein is the only glycoprotein synthesized in the infected cell. It was also determined that sialyl transferase from uninfected cells is capable of transferring ((3)H) sialic acid to acceptor prepared from Sindbis membrane glycoprotein. These results imply that at least some of the carbohydrate of the virus glycoprotein can arise by host modification.     

Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123_C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23_C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123_C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.     

Binding of Sindbis Virus to Cell Surface Heparan Sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

云南辛德毕斯病毒的生物学性状研究

本文对云南首次分离到的Sindbis病毒进行了滤过试验,耐酸耐醚试验、致细胞病变、动物敏感性试验、血压 凝特性、空斑和毒力等试验研究,结果符合披膜病毒科的病毒特性。交 因抑试验和免疫荧光试验,以及空斑减小中和试验进一步证实为甲病毒属的Sindbis病毒,其空斑纯化株的生物学特性也与原株相符,纯化株的制备为该株病毒分子生物学研究的准确性和一致性提供了条件。云南Sindbis病毒的首次分离具有重要的流行病学意义,其生物学特性研究结果对我省该病的诊断和防治具有重要的指导意义。     

云南辛德毕斯病毒的生物学性状研究

本文对云南首次分离到的Sindbis病毒进行了滤过试验、耐酸耐醚试验、致细胞病变、动物敏感性试验、血凝特性、空斑和毒力等试验研究,结果符合披膜病毒科的病毒特性。交叉血抑试验和免疫荧光试验,以及空斑减少中和试验进一步证实为甲病毒属的Sindbis病毒。其空斑纯化株的生物学特性也与原株相符,纯化株的制备为该株病毒分子生物学研究的准确性和一致性提供了条件。云南Sindbis病毒的首次分离具有重要的流行病学意义,其生物学特性研究结果对我省该病的诊断和防治具有重要的指导意义。     

辛德毕斯病毒YN87448株与宿主细胞凋亡的研究

为了了解云南辛德毕斯病毒在BHK2 1细胞上的生长特性 ,并观察该病毒在BHK2 1细胞、3d龄和 2周龄小白鼠中是否有凋亡反应出现。在接种后不同时间取样 ,检测病毒的滴度 ,电镜检测凋亡细胞 ,检测细胞的DNA阶梯。一步生长曲线结果表明病毒在出现CPE之前就有大量繁殖。秋水仙素对该病毒的成熟释放没有抑制作用。该病毒对BHK2 1、小白鼠均可产生细胞凋亡