A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

Synthesis of tobacco mosaic virus (TMV)-specific low molecular weight component RNA (LMC) was investigated in relation to that of other TMV-related RNAs and proteins, and formation of progeny virus particles using synchronously infected tobacco mesophyll protoplasts. Timing of LMC synthesis was shown to be almost the same as, but somewhat earlier than that of TMV-RNA synthesis. In contrast, synthesis of TMV-specific double-stranded RNAs (replicative intermediate, RI and replicative form, RF) as well as a high molecular weight virus-induced protein (140 K protein) showed the maximum incorporation rate 4–6 h earlier than LMC synthesis. While, synthesis of coat protein and formation of progeny virus particles lagged behind LMC synthesis for 6–8 h. LMC occurring in polysomes was also investigated during the course of virus replication. The amount of produced coat protein calculated theoretically from the amount of LMC in polysomes of infected protoplasts was shown to be well agreed with the experimental results, indicating that LMC in polysomes is actively functioning as messenger for coat protien synthesis in vivo.     

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

The low molecular weight tobacco mosaic virus (TMV)-specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of ¹⁴C-uridine-labelled RNA from infected protoplasts. Free and membrane-bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV-specific RNA species including full-length viral RNA, its replicative intermediate, and LMC were found in both free and membrane-bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small-sized polysomes (mono- to tetrasomes). Polysomes of this size class produced viral coat protein in a cell-free protein synthesizing system from rabbit reticulocytes. On the other hand, full-length TMV-RNA was associated predominantly with larger polysomes which produced in the cell-free system TMV-specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.     

黄瓜花叶病毒衣壳蛋白存在于被其侵染的烟草叶绿体中

用原生质体法制备出高纯度的完整叶绿体经SDS-PAGE电泳,银染后,发现黄瓜花叶病毒(CMV)侵染的烟草病叶叶绿体蛋白质图谱和健叶叶绿体相比,多出一条染色较弱的迁移率与CMV衣壳蛋白质相同的带,经Western转移,用CMV游离衣壳蛋白亚基抗血清进行斑点酶联(Immunoblot)检测,证明这条带就是CMV衣壳蛋白质。健康叶绿体中加入去掉叶绿体的病叶汁液而制备出的叶绿体中无CMV衣壳蛋白质,说明这不是在叶绿体提纯过程中得到的假象,即衣壳蛋白质存在于被CMV侵染的完整叶片叶绿体中。这个结果否认了以往报道的CMV衣壳蛋白质不存在于烟草叶绿体中的结论。另外还发现,叶绿体中的衣壳蛋白质浓度与叶片症状严重程度呈正相关。     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Properties of the Coat Protein of a New Tobacco Mosaic Virus Coat Protein ts-Mutant

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 Å from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21 T and D66 G, both in the 70-Å region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21–66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-Å region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.     

Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.     

烟草花叶病毒对烟草叶片光合特征和POD表达的影响

以烤烟(Nicotiana tabacum L.)品种'中烟5号'为实验材料,对烟草健康株与感染烟草花叶病毒(TMV)株的叶绿素、光合速率、光合速率对光强的响应曲线、光暗反应荧光特征、POD活性及其表达等进行研究,以探讨TMV感染对烟草植株生理生态特征的影响.结果显示:病株的叶绿素a(Chl a)和叶绿素b(Chl b)含量显著低于健康株,但Chl a/Chl b值基本相同;病株暗中初始荧光(F0)、暗中最大荧光(Fm)、暗中可变荧光(Fv)、光下初始荧光(F0′)、光下最大荧光(Fm′)、光下可变荧光(Fv′)、非光化学猝灭系数(NPQ)、PSⅡ捕光效率(Fv′/Fm′)、PSⅡ实际光化学效率(ФPSⅡ)及光饱和点显著低于健康株;净光合速率在光强较大(＞1 500 μmol·m-2·s-1)时病株比健康株低,光强适中(1 500 μmol·m-2·s-1左右)时两者相差不大,光强较弱(＜1 500 μmol·m-2·s-1左右)时病株比健康株高;病株叶片的过氧化物酶(POD)活性显著升高,POD同工酶中一些大分子量蛋白分子表达量加大.研究表明,感染TMV使烟草植株对光抑制更为敏感,叶片的荧光激发能力和热耗散能力下降,PSⅡ反应中心捕光效率和光化学反应效率降低,光合电子传递能力和碳同化能力受到抑制;POD活性提高和表达量增加可能是诱导烟草抗病性的一个关键生理过程.     

Cytochrome Oxidase and Mitochondrial Protein in Extracts of Leaves of Nicotiana glutinosa L. Infected with Tobacco Mosaic Virus

Leaves of N. glutinosa were inoculated with tobacco mosaic virus,and infection allowed to develop in situ or in discs cut fromthe leaves and floated on water or culture solution. Extracts,made from the leaves and discs when their respiration was greaterthan that of comparable uninfected tissue, contained amountsof protein N, chlorophyll, glycollic oxidase, and mitochondrialprotein N, similar to those in extracts from uninfected tissue,but slightly less cytochrome oxidase and considerably more polyphenoloxidase.No evidence was obtained for the view that the virus-inducedrespiration reflects an increase in the amount of mitochondrialmaterial.     

Effects of Cucumber Mosaic Virus Infection on Photosynthetic Activities of Tobacco Leaves and Chloroplasts

Changes in photosynthetic activities were studied with tobacco (Nicotiana tabacum L.) leaves and chloroplasts infected by cucumber mosaic virus (CMV) at the top, middle and bottom located leaves. Net photosynthetic rate was reduced at all three positioned leaves, with the maximum reduction occurring at the top leaves (31.9% of control). The infected chloroplasts showed a reduction in electron transport rates of the whole chain electron transport, photosystem Ⅱ (PSⅡ) and photosystem Ⅰ (PSⅠ). Since the decline in the whole chain electron transport (15.6% of control, H2O→MV) closely paralleled the decline in PSⅡ activity (20.9% of control, H2O→PBQ), the inhibition of the latter was probably responsible for the overall decrease. Chlorophyll a fluorescence measurements showed a variable reduced fluorescence yield (Fv/Fo) which indicated that PSⅡ was impaired and the CO2 assimilation was disturbed by CMV infection. Fluorescence emission spectra at 77 K indicated that energy distribution between PSⅡ and PSⅠ was affected. F686/F734 of infected leaves and chloroplasts increased and the greatest increase (331.1% of control ) was found in the top leaves. These data may conclude that the infection inhibited mainly the PSⅡ activity.     

嘧肽霉素影响烟草花叶病毒RNA蓄积量的研究

嘧肽霉素是新报道的由土壤中分离筛选出来的不吸水链霉菌辽宁变种(Streptomyces achygroscopicus var.liaoningensis)产生的,其有效成分是胞嘧啶核苷肽类化合物,已获得农药临时登记,该药剂对烟草花叶病毒(Tobacco mosaic virus,TMV)等多种病毒病害具有很好的防治效果。定量竞争PCR(Quantitative competitive PCR,QCPCR)是一种定量检测细胞因子较为精确的方法,其实质是待测核酸(DNA或RNA)与内参模板一起竞争参与PCR反应,并通过电泳将扩增产物在凝胶上明显的分开,从而进一步进行定性或定量分析。     

黄瓜花叶病毒对烟草叶片和叶绿体光合活性的影响

研究了烟草（Ｎｉｃｏｔａｎａ ｔａｂａｃｕｍ Ｌ．）感染黄瓜花叶病毒后烟草植株不同部位（顶层、中层、下层）中片的光合速率和叶绿体光化学反应的变化。感病的叶片净光合速率减少,顶层叶砬少最多。病毒侵染抑制ＰＳⅡ活性比ＰＳⅠ活性更明显,特别是顶层叶。叶绿素ａ荧光测定表明还原态Ｑ库减少,ＣＯ２同化减少。从低温荧光发射光谱图变化中发现能量分配受干扰。     

Spatial Distribution of Lectin Activity in Perilesional Areas of Tobacco Leaves Inoculated with Tobacco Mosaic Virus

The activities of lectins and peroxidase and lignin content were studied in the perilesional area of leaves in two tobacco species (Nicotiana tabacumL., cultivar Samsun NN, and N. glutinosa L.) inoculated with tobacco mosaic virus. The development of hypersensitivity response proved to be accompanied by a complex spatial and temporal distribution of lectin activity. The area 5–9 mm away from the lesion center was characterized by the highest activity of loosely bound membrane lectins eluted with 0.05% Triton X-100. In the fraction of tightly bound membrane lectins (eluted with 0.5% detergent), lectin activity decreased during the first two days but increased on day 4 after inoculation. The activity of loosely bound membrane lectins increased in the leaf areas distant from the lesion. Two-phase dynamics in the interlesional area were also observed for lectin activity in the tightly bound membrane fraction (decrease on day 2 days and increase on day 4 after inoculation) and for peroxidase activity (increase on day 2 days and decrease on day 4). The relationship between the dynamics and spatial distribution of lectins in the perilesional area and the possible involvement of these proteins in pathogen-induced changes in photosynthesis are discussed.     

Isolation of Polyribosome from Tobacco Plants Infected with Tobacco Mosaic Virus

Polyribosomes have been isolated from tobacco leaves. Upon infection with TMV, a new polyribosome has appeared which is specific for infection, and TMV-antigenic protein is formed on this polyribosome. This new polyribosome has an S-value of 360–380. From these results, it is suggested that this TMV specific polyribosome is an aggregate of 60–80 ribosomes which is bound by TMV-RNA as messenger, and that TMV-RNA is translated polycistronically.     

Effect of Tobacco Mosaic Virus on Phospholipid Content and Phospholipase D Activity in Tobacco Leaves

The phospholipid content and phospholipase D activity in the leaves of two tobacco (Nicotiana tabacum L.) cultivars were investigated. These cultivars are characterized by different response to the infection with tobacco mosaic virus (TMV). In the infected leaves of a susceptible cv. Samsun, phospholipid content and phospholipase D activity did not change within seven days after TMV infection. The development of a hypersensitive response in the leaves of a resistant cv. Xanthy necrotic was not accompanied by a change in the total phospholipid content as compared to the noninfected leaves. However, the appearance of necrotic lesions and their subsequent expansion resulted in a steady decrease in the level of phosphatidylglycerol in infected leaves. At the same time, phosphatidic acid and diphosphatidylglycerol contents increased. Leaf zones remote from the regions of necrosis development were also characterized by an increased level of phosphatidic acid. There was a tendency for an increase in phospholipase D activity in both the sites of necrosis development and in the leaf regions remote from these sites. The changes in phosphatidic acid content were of similar nature, and therefore a relative increase in phosphatidic acid could result from the phospholipase D activity. This fact suggests a possible involvement of phospholipase D in the development of the hypersensitive response, and this suggestion is supported by a higher enzyme activity in the leaves of healthy plants of the resistant cultivar as compared to the susceptible one. Causes for the changes in the content of some phospholipids, as well as the physiological role of phospholipase D in the hypersensitive response are discussed.