Biz reaktif bize sağlamış Yohei Yamauchi ve Roberta Mancini teşekkür ederiz. AH laboratuvar Marie Curie ilk Eğitim Ağları (ITN) tarafından desteklenmiştir, ve İsviçre Ulusal Bilim Vakfı tarafından Avrupa Araştırma Konseyi (ERC) (Sinergia).

Tamponlar ve Stok Çözümleri hazırlanması 1. <ol><li> 80 mi GKD 2 O 470 mg TRIS-hidroklorid, 390 mg MES hidrat ve 580 mg NaCI çözülmesiyle (20 mM MES, 30 mM Tris ve 100 mM NaCl) MNT tamponu hazırlayın PH 7.4 tampon ayarlamak ve GKD 2 O 100 ml&#39;lik bir nihai hacme getirmek </li><li> 80 ml ​​dH 2 O her 9.76 gr MES hidrat çözerek 500 mM MES tamponu üç özdeş çözümler hazırlayın. konsantre NaOH ile titre edilerek, sırasıyla pH 5.8, 5.4 ve 5.0, tamponları ayarlayın. DH 2 O 100 ml&#39;lik nihai hacme kadar her bir tampon getirmek </li><li> DH 2 O 80 ml, her 7.88 gr TRIS-hidroklorid çözülmesiyle 500 mM Tris tamponu, iki özdeş çözüm hazırlanması ve konsantre HCI ile titre edilerek, sırası ile 7.4 ve 6.4, pH ayarlamak. DH 2 O 100 ml&#39;lik nihai hacme kadar tampon getirmek </li><li> DH 2 O% 50 gliserol çözeltisi hazırlayın ve glisin kadar iyice karıştırınerol berrak bir karışım. Bir cam şişe içine Steritop filtre birimi (0.22 um gözenek boyutu) ile çözelti filtre. </li><li> 10% NP-40 çözeltisi ve DH 2 O 1 M NaCl hazırlanması </li><li> 4 ml GKD 2 O. iki tablet çözerek 25x konsantre proteaz inhibitörü hazırlayın </li></ol> Gliserol Gradyanların 2. Hazırlık <ol><li> 5 mi deterjan tamponu ana karışımı hazırlanması (pH 7.4, 6.4, 5.8, 5.4 ve 5.0) test etmek için, her bir pH durumu için çözeltisi (300 mM NaCl,% 2 NP-40, 2 x proteaz inhibitörü konsantre edildi). Bu amaçla, 1 M NaCI 9 mi, 10% NP-40, 6 ml 25x proteaz inhibitörü stokunun 2.4 mi, 9 mi GKD 2 0 karıştırın. </li><li> Her pH koşulu pipet için 50 ml konik tüp içine ana karışımı 4.4 ml. </li><li> pH değerleri 5.8 üzerinde tüp için ilgili pH&#39;ı ayarlanmış 500 mM Tris stok çözeltisi 0.6 ml ekleyin. pH 5.8 ve daha düşük için ilgili pH&#39;ı ayarlanmış 500 mM MES, stok çözeltisi 0.6 ml ekleyin. </li><li> 5 yapmak300 mM NaCl ihtiva eden tampon çözeltisi mi, 2x proteaz inhibitörü, 60 mM Tris pH 7.4, GKD 2 O ayarlandı Bu deterjan içermeyen kontrol degrade tampon olarak görev yapacak. </li><li> Gerekirse, sırasıyla konsantre edildi HCI ve NaOH çözeltileri eklenerek pH 7.4, 6.4, 5.8, 5.4 ve 5.0 için çözeltilerin pH ince ayarlar. </li><li> altı farklı% 25 gliserol Çözeltilerin elde edilen deterjan içeren deterjan içermeyen tampon karışımları, 5 ml% 50 gliserol stokunun 5 ml. pH indikatör şeritleri kullanarak pH değerlerini kontrol edin. Not: ölçülü bir pH istenen değerden önemli ölçüde farklıdır durumda, ilgili çözümleri tekrar hazırlanmalıdır. </li><li> 7 oranında a 3 dH 2 O ile% 50 gliserol stok karıştırılarak% 15 gliserol çözeltisi hazırlayın. </li></ol> IAV 3. Ultrasantrifüj <ol><li> 5 ml&#39;lik bir şırınga ve bir öncesine kullanılarak ultra net santrifüj tüpleri içine (13.2 mi, 14 mm x 89 mm), 3 ml% 15 gliserol solüsyonu ilavedle (9 cm uzunluğunda 21 G). Bu eğim bütünlüğünü bozacağı olarak borunun iç duvarında damla bırakmayın. Altı santrifüj tüpleri test beş pH koşulları her biri için, kontrol örneği için bir toplam için tekrarlayın. </li><li> Dikkatli bir şekilde 5 ml şırınga ve uzun bir iğne kullanılarak% 15 gliserol tabakasının altında 3.4 mL% 25 gliserol çözeltisi yerleştirin. iki kat karıştırmak değil özen gösterin. ilgili pH ayarlı gliserol çözeltileri test etmek altı koşulları için tekrarlayın. NOT: Aşağıdaki adımlar için sınıf II biyogüvenlik kabini içinde çalışmak. </li><li> Yavaşça 1 mi MNT tamponu içinde seyreltilmiş IAV (X31, H3N2) içeren alantoik akışkan açıklık 30 ul gliserol gradyanlar Bindirme her gradient (20-30 ug toplam viral proteinin karşılık gelir). </li><li> Denge tüpleri karşı ve SW41 Ultrasantrifügasyon rotor içine koyun. 55.000 x g&#39;de 150 dakika, ve 12 ° C santrifüj. </li><li> Santrifüj işleminden sonra, dikkatli bir şekildeTemiz bir Pasteur pipeti ve bir aspiratör kullanarak süpernatant, yani hem gliserol katmanlarını kaldırmak. 40 ul (1 x), LDS numune tamponu indirgeyici olmayan pelletini. Tamamen pelet çözülür aşağı birkaç kez pipetleyin ve önemlidir. 1.5 ml mikrosantrifüj tüpü içine örnek aktarmak. </li></ol> Topak bölümleri ve Coomassie boyama 4. SDS-PAGE <ol><li> 10 dakika boyunca 95 ° C &#39;de, tüm numuneler ısıtın. bu, SDS-PAGE ile analiz edilinceye kadar, bu noktada numune -20 ° C&#39;de muhafaza edilebilir. </li><li> Yük bir prekast gradyanı (% 4-12) üzerinde eritildi pellet 20 ul Bis-Tris Mini jeli ve çalışan tampon 1x MOPS SDS içinde 200 V de 1 saat boyunca çalışır. </li><li> GKD 2 O&#39;da% 40 metanol ve% 10 buzlu asetik asit ile sabitleme çözeltisi yapmak </li><li> 1 saat için tespit çözeltisi içinde jel inkübe ve Coomassie solüsyonu yeterli bir hacmi ile 15 cm&#39;lik bir hücre kültür kabı içinde gece boyunca hafifçe lekeOda sıcaklığında çalkalayarak. NOT: boyama çözeltisi buharlaşmasını önlemek için yemek kapatmak için önemlidir. </li><li> GKD 2 O. jel destain Jel arka plan berrak hale gelinceye kadar GKD 2 O her 15-20 dakikada bir değiştirin. Bu bant ölçümü için taranır kadar 4 ° C &#39;de GKD 2 O jel saklayın. </li><li> yüksek çözünürlükte jel tarayın ve protein bandı yoğunluklarının ölçümü için piyasada mevcut veya ısmarlama yazılımı kullanın. İlgili bantları yakın bir bölgeden arka plan sinyalini çıkartın ve (pH 7.4&#39;te) deterjan içermeyen kontrol numunelerine bu değerleri normalize. </li></ol>

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The authors have nothing to disclose.

Viral kapsidler metastabil makromoleküler kompleksler bulunmaktadır. virionlar montaj virüs genomunun encapsidation ve yoğunlaşmayı gerektirir, enfeksiyon sonraki turda başlanması bu kompakt kapsid yapısının sökülmesi bağlıdır. Virüsler hücresel reseptörler, şaperonlar, proteolitik enzim, motor protein ve helikazlar yanı sıra pH ve iyonik anahtarları 26,27 tarafından sağlanan fiziksel kuvvetler de dahil olmak üzere kılıf kılıfının döngüsü kontrol etmek için çeşitli hücre...

İnfluenza A virüsü (IAV) zarflı bir virüstür ve Ortomiksoviridae ailesine aittir. Bu genler, parça parça, negatif sens, tek iplikçikli bir RNA genomu üzerindeki kodlanır. İnsanlarda, IAV mevsimsel salgın salgınlar meydana solunum yolu enfeksiyonları, nedenleri ve küresel salgın hastalıklar 1 potansiyelini taşımaktadır. Ev sahibi hücre yüzeyi 2 sialik asit artıkları bağlanma üzerine, IAV klatrin bağlı endositoz ve klatrin bağımsız yollar 3-8 ile yerleştirilebilir. Endositik vakuollerde asidik ortam (pH &lt;5.5), viral füzyonu ve geç endosomal zarı 9 sonuçlanır IAV başak glikoprotein hemaglutinin (HA), büyük bir yapısal değişim tetikler. Site o - IAV kapsid kez geç endosomes (LES), bu çekirdeğin içine viral ribonükleoproteinlerin (vRNPs) taşınması ve ardından sitozolde kaplanmamış olan kaçan (burada da viral &quot;çekirdek&quot; olarak anılacaktır)f virüs replikasyonu ve transkripsiyonu 10-13. HA asit aktivasyonu öncesinde virüs ileride bunun demontajı 14-16 çekirdek hazırlar endositik sistemi, pH tedrici bir azalma yaşar. Bu &quot;priming&quot; proton ve K +10,14,16 akını aracılık viral membran M2 iyon kanallarını adım. Virüs iç iyonik konsantrasyon değişim etkileşimleri, viral matriks protein M1 ve sekiz vRNP demet tarafından inşa rahatsız ve membran füzyon 10,14-17 aşağıdaki sitoplazmada IAV olmama kolaylaştırır. hazırlama aşamasında doğrudan ve kantitatif analiz endozomlar deneysel ulaşılması zor olduğu gerçeği engel olmuştur. giriş süreci, dahası, son derece olmayan eşzamanlı olduğunu. Buna ek olarak, bu tür serbest viral RNA veya enfekte ölçümlerinin QRT-PCR gibi son nokta tahlilleri, viral capsi biyokimyasal durumu hakkında detaylı bir resmini sağlamazgiriş herhangi bir aşamasında, d. SiRNA veya ilaç tedavisi ile endozomlardan pertürbasyon anlamlı IAV girişi 18-20 anlayışına katkıda bulunurken, ince ayar sıkı kontrol endosome olgunlaşma programında belirsiz yan etkileri zor ve eğilimli. Bu sorunları önlemek için, hız gradyanı ile santrifüjleme 17 kullanımına dayanmaktadır, daha önce geliştirilmiş in vitro protokol adapte edilmiştir. Çoğunlukla proteolitik bölünme EM analizi ile izlenmiştir deterjan muamelesi kombinasyonları dayanmaktadır diğer girişimlerde 21,22, 23,24, aksine, bu yaklaşım kolayca ölçülebilir bir sonuç sağladı. Farklı gradyan katmanları üzerinden Santrifüj çöktürülmesi parçacıklar maruz bırakılmış ve kontrollü bir şekilde değişen koşullara reaksiyona sağlar. Sunulan protokol olarak, IAV açıklık allantoik akışkan ya da saflaştınlmış viral parçacıkların elde edilen iki tabakalı bir gliserine çöktürülürgradyan olup, buradaki alt tabaka, iyonik olmayan bir deterjan NP-40 (Şekil 1) içerir. Virüs, ikinci deterjan içeren katmanı girdiğinde, viral lipid zarfı ve zarf glikoproteinleri yavaşça çözülmüş ve geride bırakılır. çekirdek, sekiz vRNP demet oluşan ve bir matris tabakasıyla çevrili, pelet fraksiyonu içine kararlı bir yapı olarak çökelleri. M1 ve vRNP ilişkili nükleoprotein (NP) ve viral çekirdek proteinleri, SDS-PAGE ve Coomassie boyama ile pelet içinde tespit edilebilir. Özel olarak, ticari olarak temin edilebilen gradyan jelleri yararlanarak ve son derece hassas Coomassie 25 ile boyama, yüksek hassasiyet ve viral çekirdek ilişkili proteinlerin küçük miktarlarda bile tespitini sağlar. Bu pH, tuz konsantrasyonu ve varsayılan kılıfının faktörler temel bileşenleri ve çekirdek stabil ve sedimantasyon davranışları üzerindeki etkileri gibi farklı koşullar olup olmadığını test etmek için temel ayarlar<html> Sığ. Bu amaç için, gliserol geçişi olarak sadece düşük, deterjan içeren katman faktörü ya da ilgi duyulan durum girerek modifiye edilir. Teknik IAV çekirdek 16 bütünlüğüne pH değerleri ve tuz konsantrasyonları farklı etkisini araştıran özellikle değerli olmuştur. IAV olmama arasında ara adımlar pH 5.5 ve daha düşük 16,17 (Şekil 1) vRNP ayrışma ardından hafif asidik pH&#39;ta (&lt;6.5) matris tabakasının dahil olmak üzere ayrılmasını takip edilebilir. Ikinci adım geç endositik ortamı 16 yansıtan gliserol tabakasında yüksek K + konsantrasyonu varlığında tarafından geliştirilmiştir. Virüs ve çekirdek degrade ile tortulaşmış Böylece, onlar bir değişen ortam endosemelerde taklit koşullar yaşadı. Sonuç hücre biyolojisi deneyleri türetilen sonuçları tamamlayan, in vitro viral çekirdek kademeli sökme oldu. t &quot;&gt; Burada sunulan yöntem, hızlı etkin ve IAV son derece tekrarlanabilir analiz etti (X31 ve A / KAA / 33) ve IBV (B Lee / 40 /) asidik pH ile tetiklenir ve demontaj yanı sıra K +16,17 artan olmama Bu düşünülebilir alkali pH maruz 17 üzerine paramiksovirüs çekirdeklerin. yaklaşım hücre girişi sırasında viral kapsid yapısı ve kapsid sökme biyokimyasal özellikleri içgörü kazanmak için diğer zarflı virüsler adapte edilebilir.

<table><tbody><tr><td>cOmplete&amp;trade;, EDTA-free protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>11873580001</td><td>The stock solution can be stored at 2 to 8 &amp;deg;C for 1 to 2 weeks</td></tr><tr><td>Glacial acetic acid</td><td>Merck Millipore</td><td>100063</td><td></td></tr><tr><td>Glycerol anhydrous BioChemica</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>100317</td><td></td></tr><tr><td>Long injection needle (21&amp;nbsp;G, 9 cm, bevel or blunt-end)</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>I8896</td><td>Now commercially available as IGEPAL&amp;reg; CA-630</td></tr><tr><td>NuPAGE 4-12% Bis-Tris mini gels, 10&amp;nbsp;wells, 1.0 mm</td><td>Life Technologies</td><td>NP0321</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Technologies</td><td>NP0008</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer (20x)</td><td>Life Technologies</td><td>NP0001</td><td></td></tr><tr><td>pH indicator strips, pH 4.0-7.0</td><td>Merck Millipore</td><td>109542</td><td></td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>BIO RAD</td><td>1610803</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>106406</td><td></td></tr><tr><td>Sodiumhydroxide</td><td>Merck Millipore</td><td>106498</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>SW41 Ti, ultracentrifuge rotor set</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Thinwall, Ultra-clear centrifuge tubes, 13.2&amp;nbsp;ml, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Tris hydrochloride&amp;nbsp;</td><td>AppliChem</td><td>A1087</td><td></td></tr><tr><td>X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td><td>Virapur</td><td></td><td>Freshly thawed on 4 &amp;deg;C</td></tr></tbody></table>

in vitro disassembly of influenza a virus capsids by gradient centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

Daha önce tartışıldığı gibi, endosemelerde priming yetkili olmama IAV çekirdeğini oluşturmak için gereklidir. çekirdeğin protonlanma M1 ve vRNPs etkileşimini zayıflatır (viral RNA, NP oluşan ve polimeraz kompleksi PB1 / PB2 / PA). Gelen virüsü erken endozomlarda (AİS) 6.5 (veya daha düşük) bir pH değerine maruz kalmış ve LES yaklaşık pH 5.0&#39;a virüs sigorta kadar devam eder, bu işlem başlatılır. <p class="jove_content" fo:keep-together.within-page=...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Gradient Santrifüj tarafından İnfluenza A Virüsü Kapsidlerin Vitro Demontaj içinde

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K Görüntüleme.  ETH Zurich. Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Video: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV-1 Çekirdek in vitro Uncoating

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

İmmünoloji ve Enfeksiyon

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Basit ve Sağlam In vivo Ve In vitro</emVirüs Meclisi Öğrenmenin> Yaklaşım

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Enfekte Hücre Kromatin gelen Gribi ribonükleoprotein Komplekslerinin Affinity Arıtma

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

İnfluenza A Viral ribonükleoprotein Kompleksleri saflaştırılması ve Görselleştirme

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Biyoloji

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Viral kapsid yapısal bütünlük belirlenmesi için alternatif Vitro yöntemleri

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

Derleme ve prototip köpüklü virüs Intasomes arıtma

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenovirüs Üretimi İçin Verimli Bir Yöntem

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Biyomühendislik

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Bir hücre fabrikası platformu kullanarak yüksek titre, yüksek kaliteli adeno-ilişkili virüs vektörleri oluşturmak için kolaylaştırılmış ve standartlaştırılmış bir prosedür

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Dev Virüs Karışım Ayırma için Floresan-Aktive Hücre Sıralama alternatif bir strateji olarak Tek Hücreli Mikro-aspirasyon

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Çift İodiksanol Yoğunluk Gradyan Santrifüjleme ile AAV İzolasyonu

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Yazar Spotlight: Klinik Öncesi Uygulamalar için Verimli Adeno İlişkili Virüs İzolasyonu

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

Gradient Santrifüj tarafından İnfluenza A Virüsü Kapsidlerin Vitro Demontaj içinde

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