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zmfzj至尊木蟲 (著名寫手)
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[交流]
玉米褪綠斑駁病毒(Maize chlorotic mottle virus，MCMV)
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The genome organization of the single viral RNA of MCMV is most similar to panicum mosaic virus (PMV). Key taxonomic features of the genus Machlomovirus are a unique open reading frame(ORF) at the 5′end of the genome that largely overlaps the pre-readthrough portion of the viral replicase gene, and a readthrough ORF preceding and overlapping the CP gene near the 3′end of the genome. The virion consists of a 4437 nt single-stranded RNA surrounded by 25.1 kDa CP subunits that lack the protruding domain found on CPs of viruses from many genera in the family Tombusviridae. Sequence similarity to the CPs of PMV, tobacco necrosis virus genus Necrovirus, family Tombusviridae, and southern bean mosaic virus genus Sobemovirus suggest that MCMV is a T = 3 icosahedral virion with 180 copies of its CP in the viral shell. The plus-sense RNA is 4437 nt long and contains seven overlapping ORFs that encode proteins of 7 kDa or larger. The RNA was reported to have no poly(A) tail and an m7 G cap at the 5′end. However, in common with other viruses in the family, the encoded MCMV replicase does not have any motifs characteristic of the methyltransferase domain found in viral replicases of capped RNA viruses. None of the other MCMV-encoded proteins contain a methyltransferase domain, so it is likely that the RNA in MCMV, like in other members of the family, is in fact uncapped. The 5′UTR is 117 nt long, and ORF1 encodes a 32 kDa highly acidic protein. ORF2 begins 19 nt downstream and encodes a 50 kDa highly basic protein so the migration of these two proteins in sodium dodecyl sulfate (SDS) polyacrylamide gels is likely to be anomalous. Suppression of the UAG stop codon of ORF2 would produce a 111 kDa protein. A cluster of four ORFs is encoded in the 30 third of the viral RNA downstream of the transcription start site for the 1467-nt-long subgenomic RNA1 (sgRNA1). ORF4 encodes a 7.5 kDa protein (p7a), and suppression of its UGA stop codon would produce a 31 kDa protein. The second AUG of sgRNA1 begins ORF7 which encodes the viral CP. ORF6 was identified by similarity of its gene product p7b to small peptides encoded in similar locations on carmoviruses, necroviruses, and PMV, and it begins with a noncanonical start codon. In vitro translation of MCMV virion RNA in rabbit reticulocyte lysate produces p32, p50, p111, and p25. The two 7 kDa peptides and p31 were not detected in rabbit reticulocyte lysate translations. The 3′UTR is 343 nt long and encodes a 337-nt-long sgRNA2. The replication strategy of MCMV has not been completely determined, but inoculation of maize protoplasts with transcripts from wild type and mutant versions of an infectious cDNA has provided some information. Transcripts with mutations in the 30third of the genome that stop expression of one or more of the proteins encoded on sgRNA1 are capable of replication. Additionally, mutations just upstream of the sgRNA1 transcription start site that stop expression of sgRNA1 but do not alter the sequence of p111 are capable of replication, indicating that none of the proteins encoded on sgRNA1 are necessary for replication. Based on the replication mechanisms of other tombusvirus family members it is likely that after virion disassembly, MCMV viral RNA is translated to produce the viral replicase which then synthesizes the negative strand of genomic RNA after recognizing sequences and structures located at the viral 3′terminus that have sequence and structural similarities to the promoters of carmoviruses. The com- plementary strand is then used as template for synthesis of progeny viral RNA strands. sgRNA synthesis mechanisms differ between genera in the family Tombusviridae, and it is not known which mechanism is used by MCMV. sgRNA1 synthesis may initiate by replicase binding internally to the sgRNA promoter on the genomic complementary strand. Alternatively, occasional premature termination of viral complementary strand synthesis at a specific location may produce separate complementary strand copies of sgRNA1 that are used as templates to synthesize many copies of sgRNA1. Although sgRNA2 accumulates in infected maize plants and inoculated protoplasts, its function and method of transcription are not known.  |
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至尊木蟲 (著名寫手)
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7樓2010-09-30 08:26:48
wxy20052002
木蟲 (著名寫手)
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翻譯
zmfzj(金幣+85, 翻譯EPI+1):謝謝 2010-09-30 08:21:43
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玉米褪綠斑駁病毒 玉米褪綠斑駁病毒單一RNA的基因組組成方式與黍花葉病毒屬（PMV）非常相似。玉米褪綠斑駁病毒屬的關(guān)鍵分類學(xué)特征是在基因組5’端具有一個獨一無二、與病毒復(fù)制酶基因復(fù)制前通讀部分有較大重疊的開放閱讀框；此外，在基因組3’端附近，具有一個通讀的開放閱讀框，該開放閱讀框位于病毒外殼蛋白CP基因上游，并于CP基因重疊。 玉米褪綠斑駁病毒顆粒由一個4437nt的單鏈RNA組成，周圍有分子量為25.1KDa的CP蛋白亞基環(huán)繞。在番茄叢矮病毒家族的許多屬中，其病毒顆粒的CP蛋白都具有一個凸出的結(jié)構(gòu)域，但玉米褪綠斑駁病毒顆粒的CP蛋白不具有這樣的凸出結(jié)構(gòu)。分析玉米褪綠斑駁病毒與黍花葉病毒屬、煙草壞死病毒屬、番茄叢矮病毒科以及南方菜豆花葉病毒屬的序列相似性，研究表明，玉米褪綠斑駁病毒衣殼亞基的組裝方式遵循二十面體立體對稱T=3的規(guī)則，病毒顆粒表面由180個拷貝的CP蛋白組成。正鏈RNA長4437nt，包含7個重疊的開放閱讀框，這些開放閱讀框編碼的蛋白質(zhì)分子量為7kDa或者更大。具帽狀結(jié)構(gòu)RNA病毒的重組酶序列中，常具有甲基轉(zhuǎn)移酶結(jié)構(gòu)域，但玉米褪綠斑駁病毒重組酶與家族中的其它病毒一樣，不具有該結(jié)構(gòu)域的任何特征基序。玉米褪綠斑駁病毒編碼的其它蛋白也都不具有甲基轉(zhuǎn)移酶的結(jié)構(gòu)域，因此，與家族中其它成員一樣，玉米褪綠斑駁病毒的RNA的帽狀結(jié)構(gòu)很可能已經(jīng)被去除。5’端非翻譯區(qū)長117nt，開放閱讀框1編碼一個分子量為32kDa的蛋白，該蛋白具有高度的酸性。開放閱讀框2的第一個密碼子位于下游19nt處，編碼一個分子量為50kDa的蛋白，該蛋白具有高度的堿性。因此這兩個蛋白質(zhì)分子在SDS-PAGE膠中的遷移率很可能是不一致的。若抑制開放閱讀框中的UAG終止密碼子，使其作用不能發(fā)揮，則可以產(chǎn)生一個111 kDa的蛋白質(zhì)分子。玉米褪綠斑駁病毒的1／33序列處編碼一個由4個開放閱讀框組成的基因簇，位于轉(zhuǎn)錄起始點下游，該基因簇編碼一個長1467nt的亞基因組RNA1(sgRNA1)。 開放閱讀框4編碼一個7.5 kDa的蛋白質(zhì)分子（p7a），若抑制其開放閱讀框中的UGA終止密碼子，使其作用不能發(fā)揮，則可以產(chǎn)生一個31kDa的蛋白質(zhì)分子。開放閱讀框7由亞基因組sgRNA1的第二個AUG處開始，編碼病毒外殼蛋白。開放閱讀框6具有一個非典型的起始密碼子，編碼產(chǎn)物是p7b，p7b是一個小肽。在香石竹斑駁病毒屬、壞死病毒屬和黍花葉病毒屬病毒基因組的相同位點處，都編碼與p7b相似的小肽。體外翻譯玉米褪綠斑駁病毒顆粒的RNA，在兔網(wǎng)織紅細(xì)胞裂解物中獲得四個產(chǎn)物p32，p50，p111和p25。未發(fā)現(xiàn)兩個7kDa的肽和p31。3′非翻譯區(qū)長343nt，編碼一個337 nt的亞基因組sgRNA2。 玉米褪綠斑駁病毒顆粒的復(fù)制機(jī)制尚未徹底闡明，然而，通過給玉米原生質(zhì)體接種一個致病性玉米褪綠斑駁病毒cDNA的野生型和突變型轉(zhuǎn)錄產(chǎn)物，人們已經(jīng)獲得了一些數(shù)據(jù)。終止病毒cDNA亞基因組sgRNA1序列中的一個或多個蛋白質(zhì)表達(dá)，同時在基因組1／33序列處引入突變，病毒基因組轉(zhuǎn)錄產(chǎn)物仍然能夠復(fù)制。另外，在亞基因組sgRNA1轉(zhuǎn)錄起始點上游引入突變，同時終止亞基因組sgRNA1的表達(dá)，但未改變序列p111，病毒仍然能夠復(fù)制；表明亞基因組sgRNA1不編碼病毒復(fù)制的必需蛋白。綜合考慮番茄叢矮病毒家族中其它成員的復(fù)制機(jī)制，很可能在病毒粒子解體后，玉米褪綠斑駁病毒RNA被翻譯產(chǎn)生了病毒復(fù)制酶，復(fù)制酶識別3’末端與香石竹斑駁病毒啟動子類似的序列和結(jié)構(gòu)，進(jìn)而合成了基因組RNA的負(fù)鏈。其互補(bǔ)鏈再被用作模板以合成子代RNA鏈。番茄叢矮病毒家族中，不同屬有不同的病毒亞基因組RNA合成機(jī)制，玉米褪綠斑駁病毒亞采用何種亞基因組RNA合成機(jī)制尚不明確。重組酶與亞基因組互補(bǔ)鏈上的啟動子內(nèi)部結(jié)合后，起始亞基因組sgRNA1的合成。另外，病毒互補(bǔ)鏈的合成過程偶爾會在一個特定位點處發(fā)生成熟前終止現(xiàn)象，由此導(dǎo)致產(chǎn)生不同的亞基因組sgRNA1的互補(bǔ)鏈，這些亞基因組sgRNA1的互補(bǔ)鏈可以用作合成多種亞基因組sgRNA1的模板。盡管亞基因組sgRNA2可以在感病玉米植株及接種了病毒的玉米原生質(zhì)體中累積，但其轉(zhuǎn)錄機(jī)制和功能仍然未知。 圖2 玉米褪綠斑駁病毒基因組組成及蛋白質(zhì)產(chǎn)物。用方框標(biāo)出了基因組RNA和亞基因組RNA（sgRNA1）上每個閱讀框中的7個開放閱讀框。同時標(biāo)出了兩個被抑制掉的終止密碼子（UAG和UGA），開放閱讀框6的非典型的起始密碼子用短劃線表示。蛋白質(zhì)用深黑色條形表示，位于其所在的mRNA下方。深藍(lán)色方框代表編碼復(fù)制酶的區(qū)域，該復(fù)制酶的蛋白質(zhì)組成與番茄叢矮病毒家族中具有單分體基因組的其它成員具有高度相似性；淡藍(lán)色方框區(qū)域也代表編碼復(fù)制酶的區(qū)域，該復(fù)制酶的序列組成僅與黍花葉病毒復(fù)制酶相似。菱形表示編碼“GDD”基序的區(qū)域。開放閱讀框4中的綠色方框表示編碼保守小肽的序列，黍花葉病毒、香石竹斑駁病毒、壞死病毒中都具有該小肽。彎曲的紅色箭頭表示sgRNA轉(zhuǎn)錄起始點。sgRNA2未包含任何重要的開放閱讀框。 |
3樓2010-09-29 12:12:59
wxy20052002
木蟲 (著名寫手)
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4樓2010-09-29 13:05:20
wxy20052002
木蟲 (著名寫手)
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5樓2010-09-29 15:23:05
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