我々は、それぞれ、JFH1とJ6CF分離株のTakaji脇田とイェンス·ブフに感謝しています。さらに我々は、チャールズホ-7.5セルのライスと9E10抗体、E2特異的抗体CBH-23のスティーブンFoung、および実験的ウイルス学、有用な提案や議論のためのTwincore学科のすべてのメンバーに感謝します。

<ol>
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TPはバイオテストから顧問料を受け取った。

我々は、HCVの複製を妨げるドミナントネガティブ制約の分析のために培養細胞におけるヘテロカリオンの形成を誘導する2つのメソッドを紹介します。これらの手順を使用して様々なヒト非肝臓およびマウス肝臓細胞株における支配的な恒常的に発現またはウイルス誘発因子の存在を除外した。制限要因は感染症の子孫のHCVアセンブリと放出を防止する場合、最初のアッセイでは、主に分析し...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 41965-039 </td></tr><tr><td> L-グルタミン酸</td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 25030-024 </td></tr><tr><td>非必須アミノ酸</td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 11140-035 </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 15140-122 </td></tr><tr><td>ウシ胎仔血清</td><td> PAA、コルベ、ドイツ</td><td> A15151 </td></tr><tr><td> α-E2（CBH23） </td><td>親切にスティーブンFoung 10が提供する</td><td></td></tr><tr><td> ATP </td><td>シグマ、シュタインハイム、ドイツ</td><td> A2833-106 </td></tr><tr><td>グルタチオン</td><td>シグマ、シュタインハイム、ドイツ</td><td&gt; G4251-1G </td></tr><tr><td>ブラストサイジン</td><td> Invivoゲン、サンディエゴ、アメリカ合衆国</td><td> ANT-BL-1 </td></tr><tr><td> G418（ゲネチシン） </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 11811-064 </td></tr><tr><td>ポリエチレングリコール-1500 </td><td>ロシュ、マンハイム、ドイツ</td><td> 10783641001 </td></tr><tr><td>パラホルムアルデヒド</td><td>ロス、カールスルーエ、ドイツ</td><td> 0335.3 </td></tr><tr><td>トリトンX-100 </td><td>ロス、カールスルーエ、ドイツ</td><td> 3051.2 </td></tr><tr><td>ヤギ血清</td><td>シグマ、シュタインハイム、ドイツ</td><td> G9023-5mLの</td></tr><tr><td> α-NS5A（9E10） </td><td>親切にチャールズ·ライス7が提供する</td><td></td></tr><tr><td> DAPI（4 &#39;、6&#39;-ジアミジノ-2 - フェニルインドール二塩酸塩） </td><td>インビトロジェン</td><td> D1306 </td></tr><tr><td>アレクサ·弗素546 - ヤギ抗ヒトIgG </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> A21089 </tD&gt; </tr><tr><td>アレクサ·弗素488 - ヤギ抗マウスIgG </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> A10680 </td></tr><tr><td>リポフェクタミン2000 </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td>インビトロジェン社、カールスルーエ、ドイツ</td><td> C2925 </td></tr><tr><td> Fluoromount </td><td>シグマ、シュタインハイム、ドイツ</td><td> F4680-25ML </td></tr><tr><td>他のすべての化学物質</td><td>ロス、カールスルーエ、ドイツ</td><td></td></tr><tr><td>細胞培養材料</td><td>ザルスタット、Nümbrecht、ドイツ</td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

C型肝炎ウイルス（HCV）は、ヒトとチンパンジーに制限宿主域を持つ肝炎ウイルスです。 HCV RNAの複製は、人間の非肝臓およびマウス細胞株で観察されているが、効率が非常に低く、HCVレプリコンを使用して、必要な長期的な選定方法については支配的な抗生物質選択マーカー1-5を発現して構築します。 in vitroでの研究で HCVしたがって、ウイルスのライフサイクルのウイルスのエントリと終了を許容するヒト肝癌細胞株に限定されています。狭い種の指向性をHCVsのため、完全なHCV複製サイクル6-8を維持使用可能な免疫小動物モデルは存在しません。マウス由来の例えば、非ヒト細胞中のHCVの非効率的な複製は、おそらく本質的なホストの依存因子および/または制限因子の発現の遺伝的不和合性の欠如によるものである。 我々はHCVの伝播が支配的な制限FAによって抑制されるかどうかを調べた非肝組織から、またはマウスの肝臓細胞株の由来のヒト細胞株のいずれかでctors。この目的のために、我々は、体細胞融合に頼るつの独立した条件付きのトランス相補方法を開発した。両方のケースでは、ウイルス複製サイクルの完了は、ヘテロカリオンでのみ可能です。その結果、感染性のウイルス子孫の de novoの生産を測定することによって決定される成功したトランス相補性は、支配的な制限がないことを示します。 具体的には、ルシフェラーゼ遺伝子を運ぶサブゲノムHCVレプリコンは非常に寛容なヒト肝癌細胞（ホ-7.5細胞）にトランスフェクトした。その後、これらの細胞は共培養し、HCV構造タンパク質コア、エンベロープ1および2（E1、E2）とアクセサリータンパク質P7とNS2を表現する種々のヒトおよびマウス細胞に融合させた。細胞融合は、ポリエチレングリコール（PEG）で処理することにより開始されている限り、文化は、感染性ウイルスのPAを発表受容体に依存する形でナイーブ細胞に感染rticles。 セルのエントリ、RNAの翻訳、複製およびウイルスのアセンブリを含む完全なウイルスのライフサイクル上の支配的な制限の影響を評価するために、我々は、CD81の内因性発現を欠いているヒト肝細胞株（ホ- 7 Lunet N細胞9）を利用したHCVの本質的な侵入因子。異所的に発現CD81の欠如では、これらの細胞は、HCV感染症の10〜本質的に難治性である。重要なのは、共培養し、少なくとも別の重要な細胞侵入因子（すなわち、SR-BI、CLDN1、OCLN）ヒトCD81が不足して発現する細胞と融合させたときにのみ、結果としてヘテロカリオンは、感染症のために必要なHCVエントリー因子の完全なセットを表示します。したがって、主要な制限因子はHCVの複製サイクルの完了を抑制するかどうかを分析するために、我々は、上記のcritを満たすヒトとマウスの原点からの様々な細胞でLunet N個の細胞を融合ERIA。共培養細胞は、プロトタイプの泡沫状ウイルスの変異性の高い融合ウイルスエンベロープタンパク質（PFV 11）でトランスフェクトし、その後、感染性HCV粒子（HCVcc）でチャレンジしたときに、感染性ウイルスの de novo生産が観察された。これは、HCVが成功し、これらの細胞株における主要な制限因子の発現を除外ヘテロカリオンでのレプリケーションサイクルを完了したことを示します。これらの新しい条件付きトランス相補方法は、HCV特有の主要な制限因子の発現のための細胞株および初代培養細胞の大型パネルをスクリーニングに有用であろう。

Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

我々は、条件は、次の2つの方法について説明します。<em&gt;トランス</em&gt;-相補性C型肝炎ウイルス（HCV）は、アセンブリとヘテロカリオンの形成に依存している完全なウイルスのライフサイクルの完了。これらの技術は、感染性HCV子孫の生産を妨げる主要な制限因子を発現する細胞系の画面に適しています。

HCVの制限要因を発見するヘテロカリオンの形成の2つの方法

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

two-methods-heterokaryon-formation-to-discover-hcv-restriction

Research

JoVE Journal

Immunology and Infection

11.5K ビュー.  Twincore, Centre for Experimental and Clinical Infection Research. 我々は、条件は、次の2つの方法について説明します。トランス-相補性C型肝炎ウイルス（HCV）は、アセンブリとヘテロカリオンの形成に依存している完全なウイルスのライフサイクルの完了。これらの技術は、感染性HCV子孫の生産を妨げる主要な制限因子を発現する細胞系の画面に適しています。

ビデオ: Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

定量的質量分析のための脂肪滴の分離

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

生化学

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 ...

免疫・感染学

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

評価するために制限酵素ベースのクローニング方法<em&gt;インビトロ</em&gt; HIV-1サブタイプC Gag特異MJ4キメラウイルスの複製能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

治療標的としてのタウロコール酸ナトリウム共輸送ポリペプチドを介したB型肝炎ウイルス侵入を調べるコンピテント肝細胞モデル

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

細胞種特異的局在の解析のためのヘテロカリオンのテクニック

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

生物学

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

低コピーの検出数は B 型肝炎感染培養によって形成された統合ウイルス DNA

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

A Cell Culture Model for Producing High Titer Hepatitis E Virus Stocks

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is predominantly transmitted by blood transfusions, inadequate sanitary conditions and contaminated food products. To date the off-label drug ribavirin (RBV) is the treatment of choice for many patients. Nonetheless, a specific HEV treatment remains to be identified. So far, the knowledge about the HEV life cycle and pathogenesis has been severely hampered because of the lack of an efficient HEV cell culture system. A robust cell culture system is essential for the study of the viral life cycle which also includes the viral pathogenesis. With the method described here one can produce viral titers of up to 3 x 106 focus forming unit/mL (FFU/mL) of non-enveloped HEV and up to 5 x 104 FFU/mL of enveloped HEV. Using these particles, it is possible to infect a variety of cells of diverse origins including primary cells and human, as well as animal cell lines. The production of infectious HEV particles from plasmids poses an infinite source, which makes this protocol exceedingly efficient.

Described here is an effective method on how to produce high viral titer stocks of hepatitis E virus (HEV) to efficiently infect hepatoma cells. With the presented method, both non-enveloped, as well as enveloped viral particles can be harvested and used for inoculating various cell lines.

高力子型E型肝炎ウイルス株を生産するための細胞培養モデル

Hepatitis E virus is the leading cause of liver cirrhosis and liver failure with increasing prevalence worldwide. The single-stranded RNA virus is ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

ヒト骨髄性U937細胞におけるフローサイトメトリーによるSAMHD1制限の解析

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...