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.     

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.     

Amino Acid Composition of Cucumber Green Mottle Mosaic Virus Coat Protein and Sequence Determination of Its Tryptic Peptides

Amino acid composition of the CGMMV* coat protein was determined to be as follows: Asp₂₀, Thr₁₀, Ser₂₄, Glu₁₀. Pro₆, Gly₉, Ala₂₁, Val₇, Ile₇, Leu₁₈, Tyr₄, Phe₉, Lys₄, His₁, Arg₈, Trp₂. No terminal α-amino group was detected by dinitrophenylation method. The carboxyl-terminus was found to be serine by hydrazinolysis of the protein and digestion with carboxypeptidase A.

For sequence analysis of the coat protein, tryptic digestion was accomplished at pH 8.0 resulting in ten soluble and several insoluble peptides at pH 4.5. The amino acids contained in soluble peptides accounted for 91 out of 160 residues in the whole protein. The amino acid sequences of ten soluble peptides were determined.

From the similarities of amino acid sequence of the peptides to those of TMV* protein, CGMMV was assumed to be a strain of TMV group.     

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。     

大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。     

Nuclear Targeting of the Cauliflower Mosaic Virus Coat Protein

The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.     

A Point Mutation in the Coat Protein Gene Affects Long-Distance Transport of the Tobacco Mosaic Virus

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic spread of the mutant virus was delayed by 5–6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.     

Soybean Mosaic Virus: Vector Specificity and Coat Protein Properties

Aphid transmission studies of two soybean mosaic virus isolates have shown that both isolates are transmitted by Myzus persicae. Only one of the isolates is transmitted by Rhopalosiphum maidis. The R. madis non-transmissible isolate could be transmitted from plants co-infected with the R. maidis transmissible isolate; aphid acquisition factor did not seem to mediate this transmission. The two isolates could be differentiated by enzyme-linked immunosorbent assay experiments, but peptide mapping experiments revealed few differences between the isolates.     

侵染落葵的黄瓜花叶病毒分离物CP基因序列分析

通过间接酶联免疫法(ID-ELISA)检测到染病落葵病样中存在黄瓜花叶病毒（Cucumber Mosaic Virus,CMV）。从病叶中提取总RNA,用RT-PCR方法扩增得到657bp的CMV CP基因片断,将扩增产物与T载体连接并进行测序。用DNA MAN将得到的CP基因序列与GenBank收录的黄瓜花叶病毒两亚组部分株系或分离物的CP基因序列进行比较,结果表明该CP基因与CMV亚组Ⅰ、亚组Ⅱ之间的核苷酸序列同源性分别为91.17~95.43%和75.30~75.76%,推导氨基酸序列同源性分别为95.41~97.71%和81.28~81.74%,表明CMV-Ba与亚组Ⅰ同源关系密切。     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

States of Aggregation of Tobacco Mosaic Virus Protein

Self-assembly of the protein subunits of tobacco mosaic virus takes place by a series of steps. The natural state of aggregation of TMV protein is not the helix but the 20S disk, which consists of two paired rings of seventeen sub-units each. Movement of 5 Å is involved in the pairing. Control of the aggregation and its consequences for virus assembly are described in the next two articles.     

Polymerization of Tobacco Mosaic Virus Protein and its Control

Near neutral pH₉ TMV protein sub-units polymerize into two-layer aggregates culminating in the 20S disk, whereas the helical rod forms cooperatively at acid pH. The mode of aggregation is controlled by the state of ionization of two abnormally titrating carboxyl groups.