私たちは、フローサイトメトリーのヘルプのためにUTMBフローサイトメトリーコアファシリティのマウスポリクローナル抗RVFV抗血清とM.グリフィンを提供するためにRBテッシュに感謝します。サポートされていたこの作品は、NIHの助成金R01 AI08764301、優秀西部地域センターを通じて5 U54 AI057156によってサポートされていました、そしてUTMB.BKにおけるワクチン開発のためのシーリーセンターから資金がUTMB.OLでジェームズ·W·マクラフリンフェローシップ基金によってサポートされていましたモーリスR.ハイルマン早期キャリア調査官賞によって。

<ol>
	<li>Fakan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+support+for+RNA+synthesis+in+the+cell+nucleus.">Structural support for RNA synthesis in the cell nucleus.</a> Methods Achiev. Exp. Pathol. 12, 105-140 (1986).</li><li>Jensen, P. O., Larsen, J., Christiansen, J., Larsen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+measurement+of+RNA+synthesis+using+bromouridine+labelling+and+bromodeoxyuridine+antibodies.">Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies.</a> Cytometry. 14, 455-458 (1993).</li><li>Jao, C. Y., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+RNA+transcription+and+turnover+in+vivo+by+using+click+chemistry.">Exploring RNA transcription and turnover in vivo by using click chemistry.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 15779-15784 (2008).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+"ligation"+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.</a> Angew Chem. Int. Ed. Engl. 41, 2596-2599 (2002).</li><li>Tornoe, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+solid+phase:+[1,2,3]-triazoles+by+regiospecific+copper(i)-catalyzed+1,3-dipolar+cycloadditions+of+terminal+alkynes+to+azides.">Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.</a> J. Org. Chem. 67, 3057-3064 (2002).</li><li>Kalveram, B., Lihoradova, O., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSs+protein+of+rift+valley+fever+virus+promotes+posttranslational+downregulation+of+the+TFIIH+subunit+p62.">NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.</a> J. Virol. 85, 6234-6243 (2011).</li><li>Kalveram, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rift+Valley+fever+virus+NSs+inhibits+host+transcription+independently+of+the+degradation+of+dsRNA-dependent+protein+kinase+PKR.">Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.</a> Virology. , (2012).</li><li>Head, J. A., Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Rift+Valley+fever+virus+NSs+encoding+a+partial+truncation.">Functional analysis of Rift Valley fever virus NSs encoding a partial truncation.</a> PLoS One. 7, e45730 (2012).</li><li>Lihoradova, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Rift+Valley+Fever+Virus+MP-12+Strain+Encoding+NSs+of+Punta+Toro+Virus+or+Sandfly+Fever+Sicilian+Virus.">Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus.</a> PLoS Negl Trop Dis. 7, e2181 (2013).</li><li>Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toscana+Virus+NSs+Protein+Promotes+Degradation+of+Double-Stranded+RNA-Dependent+Protein+Kinase.">Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase.</a> J. Virol. , (2013).</li><li>Schmaljohn, C., Nichol, S., Knipe, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Fields Virology. , 1741-1789 (2007).</li><li>Le May, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TFIIH+transcription+factor,+a+target+for+the+Rift+Valley+hemorrhagic+fever+virus.">TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.</a> Cell. 116, 541-550 (2004).</li><li>Caplen, H., Peters, C. J., Bishop, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagen-directed+attenuation+of+Rift+Valley+fever+virus+as+a+method+for+vaccine+development.">Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development.</a> J. Gen. Virol. 66, 2271-2277 (1985).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+infectious+Rift+Valley+fever+virus+entirely+from+cDNA,+analysis+of+virus+lacking+the+NSs+gene,+and+expression+of+a+foreign+gene.">Rescue of infectious Rift Valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.</a> J. Virol. 80, 2933-2940 (2006).</li><li>Vyboh, K., Ajamian, L., Mouland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viral+RNA+by+fluorescence+in+situ+hybridization+(FISH).">Detection of viral RNA by fluorescence in situ hybridization (FISH).</a> J. Vis. Exp. , e4002 (2012).</li><li>Reich, E., Goldberg, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actinomycin+and+nucleic+acid+function.">Actinomycin and nucleic acid function.</a> Prog. Nucleic Acid Res. Mol. Biol. 3, 183-234 (1964).</li><li>Holmes, K. L., Lantz, L. M., Russ, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+of+fluorochromes+to+monoclonal+antibodies.">Conjugation of fluorochromes to monoclonal antibodies.</a> Curr. Protoc. Cytom. Chapter 4 (Unit 4 2), (2001).</li></ol>

利害の衝突は宣言されていない。

提供されたプロトコールは、新生RNAへウリジンアナログEUの取り込みを介して宿主細胞の転写に対するウイルス感染の影響を測定するための方法が記載されている。それは、高速で敏感であり、それは、放射性同位元素の使用に依存しない：この方法は、従来の方法に比べていくつかの利点を有する。さらに、この方法は、蛍光顕微鏡又は本質的に同じ試薬を用いてフローサイトメトリーを?...

<html>伝統的に、転写活性は、放射性（3 H-ウリジン）1のいずれかの取り込みを介して測定または新生RNAに（BRU）2ヌクレオシドハロゲン化されています。これらウリジン類似体は、細胞に取り込まヌクレオシドサルベージ経路を介しウリジン三リン酸（UTP）に変換され、続いて新たに合成されたRNAに組み込ま。3 H-ウリジンは、時間がかかることがオートラジオグラフィー、またはシンチレーションによって検出することができているカウント、特殊な装置を必要とします。さらに、放射性同位元素の使用が高い封じ込めバイオ研究所を含め、実験室の設定の数には実用的ではないかもしれない。 BRUは、RNA二次構造は、抗体結合のための立体障害かもしれないとして、RNAの核または部分変性に対する抗体のアクセスを確保するため厳しい透過処理が必要になる場合があります抗BRU抗体による免疫染色で検出することができます。 _content &quot;&gt;ここで記述されたプロトコルは、最近RVFV株MP-12に感染した細胞内で転写活性を測定する化学3をクリックして開発した。クリックケミストリーは非常に選択的かつ効率的な銅（I）触媒によるアルキンとの間の付加環化反応に依存して利用4,5アジド部分は、このプロトコルでは、感染細胞における新生RNAは、第ウリジンアナログEUの取り込みを介して標識される。第二 ​​工程では、取り込まれたEU、蛍光アジドとの反応により検出される。この方法の主な利点はそれは放射性同位元素の使用を必要とせず、それはRNAの過酷な透過又は変性を必要としないこと。50μm未満Daの分子量と（2）、蛍光アジドのアクセスが不十分なことによって妨げられない（1）ということである透過またはRNAの二次構造。クラスにRNAの検出を組み合わせた場合また、研究者は一次抗体の彼らの選択に限定されるものではないウイルスタンパク質の免疫染色のiCal。 蛍光顕微鏡（ステップ1-4）を介して転写を可視化するための一つであり、フローサイトメトリー（ステップ5-8）を介して転写を定量化するためのいずれかの2つの異なる方法は、このプロトコルに記載されている。どちらの方法でも、セル単位で転写活性とウイルス感染との間の相関を可能にするウイルスタンパク質に対する免疫細胞内で新生RNAの標識を結合する。 著者らは、正常プロトコルがRVFV株MP-12 6、トスカーナウイルス（TOSV）10に感染した別のMP-12変異体7,8、9、及び細胞に感染した細胞に感染した細胞において転写活性を決定するために、ここで説明に使用した。ここで説明するプロトコルは、簡単に異なるウイルスの使用に適合し、特定のウイルス又は細胞タンパク質の免疫蛍光染色を含むように修正される可能性を有することができる。

<table><tbody><tr><td>5-ethynyl-uridine</td><td>Berry Associates</td><td>PY 7563</td><td>dissolve in DMSO for 100 mM stock</td></tr><tr><td>12-mm round coverslips</td><td>Fisherbrand</td><td>12-545-82</td><td></td></tr><tr><td>5 ml polystyrene tubes</td><td>BD Biosciences</td><td>352058</td><td></td></tr><tr><td>Actinomycin D (ActD)</td><td>Sigma</td><td>9415</td><td>dissolve in DMSO for 10 mM stock</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG</td><td>Invitrogen</td><td>A-11029</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) </td><td>Santa Cruz</td><td>sc-2323</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>C-8027</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 100 mM stock</td></tr><tr><td>DAPI </td><td>Sigma</td><td>D-9542</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 5 mg/ml stock</td></tr><tr><td>DMEM </td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000044</td><td></td></tr><tr><td>fluorescent azide (Alexa Fluor 594-coupled)</td><td>Invitrogen</td><td>A10270</td><td></td></tr><tr><td>fluorescent azide (Alexa Fluor 657-coupled)</td><td>Invitrogen</td><td>A10277</td><td></td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A-5960</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 500 mM </td></tr><tr><td>paper filter</td><td>VWRbrand</td><td>28333-087</td><td></td></tr><tr><td>paraformaldehyde </td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>poly-L-lysine (MW &amp;#8805; 70000)</td><td>Sigma</td><td>P1274</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T-1503</td><td>dissolve in H&lt;sub&gt;2&lt;/sub&gt;O for 1.5 M stock, pH 8.5</td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25200056</td><td></td></tr><tr><td>Fluorescence Microscope</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Olympus IX71</td></tr><tr><td>Flow Cytometer</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; BD Biosciences LSRII Fortessa</td></tr></tbody></table>

using click chemistry to measure the effect of viral infection on host-cell rna synthesis

多くのRNAウイルスは、細胞の防御を回避する手段として、宿主細胞の転写を阻害する能力を進化させてきた。これらのウイルスの研究のために、感染細胞において転写活性を測定する迅速かつ信頼できる方法を有することが重要である。伝統的に、転写はH-ウリジンは、このような免疫を介して検出が続き、5 -ブロモウリジン（BRU）などのハロゲン化ウリジン類似体のオートラジオグラフィーまたはシンチレーション計数、または組み込みを介した検出が続く3などの放射性ヌクレオシドの取り込みのいずれかにより測定された。 BRUの検出が煩雑になることができ、低感度に苦しむかもしれない放射性同位体の使用は、しかしながら、特殊な装置を必要とし、実験室の設定の数には不可能である。 アジドとアルキンから銅触媒トリアゾール形成を含む最近開発されたクリックケミストリーは、今では迅速かつhighlを提供していますこれら2つの方法にyを敏感代わる。クリックケミストリーは、新生RNAは、第1の蛍光アジドとの標識の検出に続いてアナログウリジン-5 - ethynyluridine（EU）の取り込みにより標識された2段階プロセスである。これらのアジドは可視化のためのオプションの広い範囲を可能にする、いくつかの異なる蛍光体として利用可能です。 このプロトコルは、クリックケミストリーを用いたリフトバレー熱ウイルス（RVFV）株MP-12に感染した細胞内で転写抑制を測定する方法を説明しています。同時に、これらの細胞におけるウイルスタンパク質の発現は、古典的な細胞内の免疫染色によって決定される。 4詳細手順1〜8詳細を通して、フローサイトメトリーを介した転写抑制を定量化する方法を、手順5ながら、蛍光顕微鏡を介した転写抑制を可視化する方法。このプロトコルは、他のウイルスとの使用に容易に適応可能である。

P62のプロテアソームによる分解を促進することにより隔離することでは、（i）基本転写因子TFIIH 11と、（ii）のP44サブユニット：RVFVのNSsのタンパク質は、（ ブニヤウイルス科 、属フレボ ）11は、2つの異なるメカニズムを通じて宿主細胞一般的な転写を阻害TFIIH 6のサブユニット。 MP-12株は、生物学的安全性レベル2実験室で処理することができ、他の野?...

Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis at JoVE.com

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

この方法は、リフトバレー熱ウイルス（RVFV）MP-12株による感染の後、宿主細胞転写の変化を測定するためにクリックケミストリーの使用を記載している。結果は、蛍光顕微鏡を介して、定性的に可視化またはフローサイトメトリーを介して定量的に得ることができる。この方法は、他のウイルスとの使用に適合可能である。

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

using-click-chemistry-to-measure-effect-viral-infection-on-host-cell

Research

JoVE Journal

Immunology and Infection

11.6K ビュー.  University of Texas Medical Branch. この方法は、リフトバレー熱ウイルス（RVFV）MP-12株による感染の後、宿主細胞転写の変化を測定するためにクリックケミストリーの使用を記載している。結果は、蛍光顕微鏡を介して、定性的に可視化またはフローサイトメトリーを介して定量的に得ることができる。この方法は、他のウイルスとの使用に適合可能である。

ビデオ: Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

Bioorthogonalクリックケミストリーを介したウイルス表面の化学選択的修飾

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

免疫・感染学

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

(CARIC) 戦略をキャプチャするキャプチャとクリック化学支援 RNA インタラクトームを用いた RNA 結合タンパク質の同定

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

生化学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

The protocol describes a method for introducing controllable genetic diversity in the hepatitis C virus genome by combining full-length mutant RNA synthesis using error-prone PCR and reverse genetics. The method provides a model for phenotype selection and can be used for 10 kb long positive-sense RNA virus genomes.

ポジティブセンスRNAウイルス変異体を改変するためのリバースジェネティクス

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA ...

遺伝学

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C型肝炎ウイルスの複製を分析するためのプロトコル

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸器間性ウイルスのウイルス特異的ヌクレオカプシドの生成と組み立て

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

細菌人工染色体：プラス鎖RNAウイルスの研究のための機能ゲノミクスツール

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

アルボ ウイルス感染症ウイルス免疫調節剤およびホスト抗ウイルス因子の同定のためのスクリーニング ツールとして

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

世代、増幅、および組換え呼吸器合胞体ウイルスの滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

要約

さらに動画を探す

この動画の章

Bioorthogonalクリックケミストリーを介したウイルス表面の化学選択的修飾

(CARIC) 戦略をキャプチャするキャプチャとクリック化学支援 RNA インタラクトームを用いた RNA 結合タンパク質の同定

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

ポジティブセンスRNAウイルス変異体を改変するためのリバースジェネティクス

C型肝炎ウイルスの複製を分析するためのプロトコル

呼吸器間性ウイルスのウイルス特異的ヌクレオカプシドの生成と組み立て

細菌人工染色体：プラス鎖RNAウイルスの研究のための機能ゲノミクスツール

アルボウイルス感染症ウイルス免疫調節剤およびホスト抗ウイルス因子の同定のためのスクリーニングツールとして

世代、増幅、および組換え呼吸器合胞体ウイルスの滴定

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

要約

さらに動画を探す

この動画の章

Bioorthogonalクリックケミストリーを介したウイルス表面の化学選択的修飾

(CARIC) 戦略をキャプチャするキャプチャとクリック化学支援 RNA インタラクトームを用いた RNA 結合タンパク質の同定

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

ポジティブセンスRNAウイルス変異体を改変するためのリバースジェネティクス

C型肝炎ウイルスの複製を分析するためのプロトコル

呼吸器間性ウイルスのウイルス特異的ヌクレオカプシドの生成と組み立て

細菌人工染色体：プラス鎖RNAウイルスの研究のための機能ゲノミクスツール

アルボ ウイルス感染症ウイルス免疫調節剤およびホスト抗ウイルス因子の同定のためのスクリーニング ツールとして

世代、増幅、および組換え呼吸器合胞体ウイルスの滴定

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

アルボウイルス感染症ウイルス免疫調節剤およびホスト抗ウイルス因子の同定のためのスクリーニングツールとして