Human RNA silences viral DNA
MicroRNA plays an unexpected role in the process, researchers report in Science | By Charles Q Choi
RNA silencing can defend against viruses in humans, French scientists report in this week's Science. Surprisingly, say the scientists, microRNA (miRNA) appears to form the basis of this system.
"MiRNAs were thought to be involved in the regulation of endogenous genes, whereas exogenous RNAs, in particular viral RNAs, were thought to be regulated by siRNA [small interfering RNA]," lead author Charles-Henri Lecellier at the Institute of Plant Molecular Biology in Strasbourg, France, told The Scientist.
Prior studies have revealed that RNA interference can destroy viruses in plants and insects, but a similar role in vertebrates has not been demonstrated. Since RNA silencing can suppress endogenous retroviruses from mobilizing in plants, yeast, worms, and flies, Lecellier and colleagues reasoned that retrotransposition of mammalian exogenous viruses might also prove vulnerable. They chose as their model system the primate foamy virus type 1, a retrovirus akin to HIV.
PFV-1 accumulation in cultured human embryonic kidney cells was strongly enhanced by the expression of the P19 silencing suppressor, suggesting that a siRNA or miRNA pathway limited PFV-1 replication in human cells, because P19 specifically binds to and inactivates both.
To identify the target and means of human RNA silencing, the investigators fused viral sequences spanning the PFV-1 genome to a green fluorescent protein (GFP)–tagged reporter gene into constructs cotransfected with PFV-1 into baby hamster kidney cells. Northern and Western analysis revealed GFP levels from construct F11 were disproportionately reduced compared to F11 mRNA accumulation, which reminded researchers of miRNA translational inhibition. The DIANA-microT algorithm revealed a high probability match between the F11 sequence and the human miR-32.
Further studies demonstrated miR-32 silencing was suppressed in P19-expressing cells. Also, anti-miR-32 locked nucleic acid oligonucleotide almost doubled progeny virus production, unlike anti-miR-23, suggesting miR-32 has a direct, sequence-specific antiviral effect.
In plants and insects, all viruses targeted by RNA interference encode proteins that suppress RNA silencing. Further studies found that in PFV-1, Tas, a viral transactivator, was that protein.
In Arabidopsis, transgenic Tas expression strongly decreased siRNAs and led to developmental anomalies reminiscent of those elicited by suppressors interfering with miRNA functions, such as leaf elongation and serration, suggesting Tas suppresses a fundamental step conserved from plants to mammals shared between the miRNA and siRNA pathways.
Like Tas, another protein, AC2, encoded by the DNA plant viruses, geminiviruses, is a viral transactivator that can suppress RNA silencing. "We want to investigate whether transactivation and suppression are linked or completely separate," Lecellier said.
Researchers currently think each cell type harbors its own specific miRNA repertoire, Lecellier said. "This idea could partially explain some of the differences in viral permissivity observed between specific tissues," he said, with viruses preferentially replicating in cell types where antiviral miRNAs are not expressed or are only weakly expressed.
A miRNA response could also lead to the emergence of viral quasispecies, as viruses that can rapidly introduce synonymous mutations into their genomes, such as HIV or influenza, do so to evade silencing by miRNA. "The emergence of quasispecies is important for resistance to antiviral strategies, so studying this miRNA response could be important for studying resistance," Lecellier said.
The team saw no evidence that human cells used siRNAs to disable viruses. "So it'd be interesting to investigate whether or not mammals have lost the ability to respond to viruses using siRNAs because they have a more advanced immune system than plants and flies and worms," said Phillip Zamore at the University of Massachusetts, who did not participate in this study. It was uncertain whether the miRNAs were part of a dedicated antiviral response or whether they accidentally silenced viral RNA, he said. "It'd be interesting to see whether expression of miRNAs are vastly upregulated in viral infection," he told The Scientist.
Future directions should involve testing any of the several hundred human microRNAs against viruses such as HIV and influenza, said Shou-Wei Ding at the University of California at Riverside, who did not participate in this study. "Also, they studied this in cell culture, and it'd be interesting to look at this at the whole animal level," Ding told The Scientist.
Links for this article
C. Lecellier et al., "A cellular microRNA mediates antiviral defense in human cells," Science, 308:557-60. April 22, 2005.
http://www.sciencemag.org
Charles-Henri Lecellier
http://www.sigu7.jussieu.fr/B2M/pages/doc/2K4/virord11va.html
A.J. Hamilton, D.C. Baulcombe, "A species of small antisense RNA in posttranscriptional gene silencing in plants," Science, 286:950-2, October 29, 1999.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10542148/]
H. Li et al. "Induction and suppression of RNA silencing by an animal virus," Science, 296:1319-21, May 17, 2002.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/12016316http://www.biomedcentral.com/pubmed/12016316]
C. Lecellier, A. Saib. "Foamy viruses: between retroviruses and pararetroviruses," Virology, 271:1-8, May 25, 2000.
[PubMed Abstract:http://www.biomedcentral.com/pubmed/10814564http://www.biomedcentral.com/pubmed/10814564]
Phillip Zamore
http://www.umassmed.edu/bmp/faculty/zamore.cfm
Shou-Wei Ding
http://www.cepceb.ucr.edu/members/ding.htm
據(jù)《科學(xué)》雜志4月23日報道,，MicroRNA在人體發(fā)育過程中起著意想不到的作用,。
法國科學(xué)家稱核糖核酸沉默機(jī)制（RNA silencing）能夠抵抗人體中的病毒，不可思議的是,，小RNA (miRNA)竟然是組成這一機(jī)制的基礎(chǔ),。
“科學(xué)家一直認(rèn)為miRNA與調(diào)節(jié)內(nèi)源基因有關(guān)，而小分子干擾核糖核酸（iRNA）能夠控制外源基因,，尤其是病毒RNA基因,，”該文的主要作者、來自法國斯特拉斯堡的植物分子生物學(xué)院的查利斯·亨利·萊西利亞在《科學(xué)》雜志上說道,。
科學(xué)家們先前的研究顯示,，核糖核酸干擾（RNAi）能夠消滅植物以及昆蟲體內(nèi)的病毒，但目前還沒有研究表明它在脊椎動物也擁有類似功能,。自從科學(xué)家們發(fā)現(xiàn)RNA沉默機(jī)制能夠壓抑內(nèi)生反轉(zhuǎn)錄病毒,，使其無法在植物、酵母,、蚯蚓以及蒼蠅等等體內(nèi)游動,，萊西利亞和他的同事就推斷哺乳動物體內(nèi)的外源病毒基因的反轉(zhuǎn)錄移位功能應(yīng)當(dāng)也是同樣脆弱。因此他們選擇了一種原始的1型泡沫病毒（PFV-1）——一種與人體免疫缺損病毒（HIV）同類的逆轉(zhuǎn)錄酶病毒——作為實驗的模擬系統(tǒng),。
由于P19沉默干擾基因的表達(dá),，PFV-1病毒在人工“人體胚胎腎臟細(xì)胞株”（human embryonic kidney cells）中聚集并且變得十分明顯。這意味著，由于P19細(xì)胞控制著siRNA和miRNA,，而且這兩種核糖核酸的路徑也控制著PFV-1在人體細(xì)胞中的繁殖,。
為了確定人體RNA沉默機(jī)制的具體作用，研究者們將PFV-1病毒所含有的病毒基因序列與一種附有報告基因綠色熒光蛋白（GFP）鏈接在一起,，然后使它們與人體胚胎腎臟細(xì)胞一起染上病毒,。報告顯示，F(xiàn)11架構(gòu)中的綠色熒光蛋白與F11mRNA聚集中所含有的綠色熒光蛋白比例完全不同,。這一點使研究者們想到了miRNA轉(zhuǎn)錄抑制,。利用DIANA-microT運(yùn)算法則計算后發(fā)現(xiàn)F11序列與人體miR-32具有高度的相似性。
進(jìn)一步的研究表明,，miR-32沉默受到P19表達(dá)細(xì)胞的壓抑,。同樣，受抗miR-32細(xì)胞控制的核酸低聚核苷酸使子代病毒的繁殖量增加了一倍,，并不是像抗miR-32細(xì)胞所起的作用一樣,。這意味著miR-32起著直接反病毒作用。
植物與昆蟲中,，所有的核糖核酸干擾（RNAi）能夠消滅的病毒都含有可以抑制核糖核酸沉默的蛋白質(zhì),。而在后來的研究結(jié)果顯示，PFV-1中含有一種叫做Tas的病毒反激活蛋白,。
一種叫做“阿拉伯芥”的植物中,，轉(zhuǎn)基因Tas能夠大量減少siRNAs，并且使干擾基因引發(fā)的細(xì)胞產(chǎn)生發(fā)育變異現(xiàn)象,，同時干擾miRNA的功能,，例如植物葉子變長、長出鋸齒等,。這意味著Tas具有抑制miRNA 和siRNA某種基本功能的作用,，而這種功能是植物與哺乳動物共同擁有的。
類似Tas的另一種蛋白質(zhì),，AC2,，其中含有植物基因病毒，也是一種病毒反激活蛋白,，同樣能夠壓抑RNA沉默機(jī)制,。
目前，研究者們認(rèn)為每種類型的細(xì)胞都有自己獨(dú)有的miRNA細(xì)胞庫,。萊西利亞說：“這樣的概念能夠部分解答一些有關(guān)病毒反應(yīng)方面的問題,。”他認(rèn)為，在細(xì)胞種類中,，優(yōu)先繁殖的病毒是處在抗病毒miRNAs沒有表現(xiàn)的地方,，或者表現(xiàn)并不明顯的地方,。
MiRNA的反應(yīng)也會導(dǎo)致基因準(zhǔn)種群出現(xiàn)，如果病毒能夠快速使其基因組發(fā)生變異,，例如HIV與流感病毒,，它就能避免受到miRNA沉默機(jī)制的影響。“基因準(zhǔn)種群的出現(xiàn)對于躲避抗病毒功能的影響是十分重要的,，因此研究這種miRNA反應(yīng)也十分重要,，” 萊西利亞說。
這一研究小組并沒有發(fā)現(xiàn)人類細(xì)胞能夠利用siRNA來消滅病毒,。“所以研究哺乳動物是用還具有利用siRNA來對付病毒的能力是一個十分有趣的課題,，因為與蚯蚓、蒼蠅這些昆蟲相比,，哺乳動物擁有更加先進(jìn)的免疫系統(tǒng),，”麻省理工大學(xué)的飛利浦·賽摩說。他并沒有參加這次研究活動,。雖然miRNAs是否是抗病毒反應(yīng)的原因之一，或者只是偶然對病毒核糖核酸起到了沉默作用,，但是他認(rèn)為：“不論miRNAs的表達(dá)是否會抑制病毒傳染,，這項研究都會引起科學(xué)界的廣泛關(guān)注。”
美國加州大學(xué)的丁守維（音譯）稱,，未來的研究方向可能會包括測試幾百個人體microRNAs對諸如HIV病毒和流感病毒等病毒的反應(yīng),，他也未參加此次研究。他說：“他們已經(jīng)在細(xì)胞培養(yǎng)室做了試驗,，如果在動物身上做相關(guān)實驗的話會引起科學(xué)界更大的關(guān)注,。”
法國研究人員發(fā)現(xiàn)，哺乳動物細(xì)胞能關(guān)閉入侵的病毒,。當(dāng)病毒感染細(xì)胞后,，它把自己的基因插入細(xì)胞的基因組，這樣在細(xì)胞復(fù)制時也產(chǎn)生許多的病毒拷貝,。研究人員已經(jīng)知道植物和昆蟲用RNA干擾使病毒基因沉默,，在這個過程中，小的RNA分子將自己插入到基因表達(dá)機(jī)器中使某個基因沉默,。動物也將RNA干擾用在一個調(diào)節(jié)功能上：它們在發(fā)育過程中通過RNA干擾改變自己基因的表達(dá),。Charles Henri Lecellier和同事現(xiàn)在發(fā)現(xiàn)，人類細(xì)胞也用RNA干擾來阻礙一個侵襲哺乳類的病毒的積累,。
