This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol>
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The authors have nothing to disclose.

<html> O conhecimento da estrutura de glicoproteína pico viral no virião membrana é essencial para a replicação do vírus a compreensão e desenvolver agentes terapêuticos para tratar e prevenir a infecção. Electron crio-microscopia combinado com média única partícula surgiu como o método mais utilizado para resolver as estruturas de partículas de vírus envelopados, incluindo os picos de glicoproteínas. No entanto, este método está limitado a icosahedrally vírus simétricas. Aqui, através da aplicação da eletrônica de crio-tomografia e subtomogram médias na Jsubtomo, nós descrevemos um protocolo geral para determinar pontos de glicoproteínas em pleomorphic envolto vírus que não são passíveis de outros métodos de biologia estrutural atuais. Nossos resultados representativos demonstrar que a resolução deste método é suficiente para revelar insights sobre arquitetura de domínio, oligomerização e maior organização a fim de spikes glicoproteína em virions intactas. jove_content &quot;&gt; O passo mais importante dentro deste protocolo está construindo dois modelos de partida de confiança que são estatisticamente independentes uma da outra execução. bem-sucedida desta etapa pressupõe que os picos de glicoproteínas são suficientemente grandes e não embalados uns contra os outros com muita força, de modo que os picos individuais podem ser visualmente reconhecido e manualmente pegou no tomografias, e dois modelos independentes média. Se isso não for possível, duas modificações no protocolo pode ser tentada. Primeiro, dois modelos aleatórias independentes pode ser construído definindo primeiro dois subconjuntos aleatórios de subtomograms e depois média das subtomograms dentro destes subconjuntos 30. Em segundo lugar, se uma estrutura do pico isolado foi obtido por outros meios, por exemplo por cristalografia de raios-X, que pode ser utilizado como um modelo de partida. No entanto, deve ser tomado cuidado para baixo teor Filtro passa este modelo usando uma baixa resolução de corte (50-70 Å), como os dois modelos resultantes na próxima rodada de refinamento iráser estatisticamente independentes para além desse resolução. Devido a esta ressalva, é recomendável a abordagem anterior. A resolução obtida a partir deste protocolo depende de quatro fatores principais: i. estratégia de recolha de dados e a qualidade dos dados de entrada, ii. número das subtomograms, iii. a precisão do alinhamento das subtomograms, e iv. heterogeneidade das estruturas. Embora a primeira e segunda limitação pode ser largamente ultrapassado usando detectores de sinal de alta-ruído directa de electrões, combinados com CTF corrigido tomografia e recolha automática de dados, a precisão do alinhamento é mais afectada pelo tamanho e forma da estrutura do próprio interesse. Ao aplicar este protocolo em pequenas pontas que faltam características proeminentes, pode ser vantajoso para ligar fragmentos Fab para o pico para melhorar a precisão do alinhamento e, portanto, resolução 31. Finalmente, se as estruturas a serem médios exibem várias conformações, sub-Tomogrmétodos de classificação am pode ser utilizado para calcular a média conformações diferentes separadamente. Para o efeito, Jsubtomo integra com o pacote Dynamo, oferecendo classificação poderoso subtomogram 9. O protocolo anterior é complementar a cristalografia de raios-X de glicoproteínas virais isoladas. Estruturas cristalográficas pode ser montada em médias de sub-tomogram para obter a orientação precisa da glicoproteína com respeito à membrana viral. A aplicação desta metodologia, sem dúvida, continuar a lançar luz sobre a estrutura do vírus envelopado e pathobiology.

Crio-tomografia de electrões é uma técnica de imagiologia de electrões crio-microscopia permitindo o cálculo de uma (3D) a reconstrução tridimensional de amostras biológicas complexas. Amostras adequadas variam de complexos purificados macromoleculares 1, filamentos 2, vesículas revestidas 3 e vírus membrana pleomórficos 4 para células procariotas todo 5 e até áreas finas de células eucarióticas inteiros 6. Após a coleta de dados de uma série de inclinação, os volumes de tomografia 3D, ou tomografias, pode ser calculado utilizando vários pacotes de software estabelecidos, incluindo bsoft 7 e 8 Imod. Dois aspectos inerentes ao estudo de amostras biológicas por limite de electrões crio-tomografia a interpretação biológica dos volumes tomográficos correspondentes. Em primeiro lugar, devido à dose de electrões limitado que pode ser aplicado a materiais biológicos antes de introduzir os danos da radiação significativa, signal-ruído proporções em dados tomográficos são tipicamente muito baixos. Em segundo lugar, como resultado da geometria inclinação amostra limitada durante a coleta de dados, alguns pontos de vista do objeto permanecer ausente, levando a uma chamada artefato &#39;cunha perdido &quot;no volume tomográfica. No entanto, ambas as limitações podem ser superadas se o volume tomográfica contém repetição de estruturas idênticas, tais como complexos macromoleculares, que pode ser com sucesso em média 9-12. Antes da média de estruturas de reconstruções tomogram, objetos de interesse devem ser encontrados e alinhado com a mesma orientação. A localização das referidas estruturas pode ser obtida por correlação cruzada de uma estrutura de modelo no volume de tomografia por uma abordagem muitas vezes referida como modelo correspondente 13. O molde utilizado neste processo de harmonização pode ser derivada a partir de electrões ou microscopia crio-electrão crio-tomografia combinada com a reconstrução em 3D, ou pode ser um mapa de densidade de simuladouma estrutura atômica. Vários pacotes computacionais foram desenvolvidos para realizar estas tarefas 11. Calculando a média dos picos de glicoproteína do vírus de membrana, tal como o HIV-1, tem sido uma abordagem particularmente bem sucedido para o estudo da sua estrutura 14-16. Uma compreensão da estrutura é parte integrante para revelar tanto a base molecular das interações vírus-hospedeiro e orientar o desenvolvimento do projeto antiviral e vacina. Enquanto cristalografia de macromoléculas é a técnica preferida para a alta resolução (geralmente melhor do que 4 Â) a análise estrutural de glicoproteínas virais individuais e os seus complexos, as estruturas de raios-X resultantes deste método são de proteínas isoladas a partir do ambiente natural do membranoso virião . Deste modo, detalhes importantes, tais como a arquitectura de ordem superior de glicoproteínas virais, no contexto do virião, permanecem escassos. Por outro lado, de electrões e microscopia crio-reconstrução de partícula únicade vírus envelopados inteiras se restringe a partículas virais com simetria icosaédrica 17,18. Electron cryotomography combinado com alinhamento sub-volumes foi assim que surgiu como uma técnica complementar que permite o estudo de spikes glicoproteína de forma irregular, vírus pleomórficos in situ. Nós desenvolvemos software chamado Jsubtomo (www.opic.ox.ac.uk/jsubtomo) para a detecção, alinhamento e nivelamento de sub-volumes tomográficas. Jsubtomo tem sido utilizado na determinação da estrutura de um número de estruturas virais e celulares 19-26. Aqui, nós descrevemos um protocolo detalhado, que permite a determinação de estruturas de pico viral de superfície. Para contornar o excesso de refinamento de estruturas em média, correlacionando ruído, o esquema de refinamento &#39;padrão-ouro&#39; é adotado 10,27. Por fim, são discutidas estratégias para a visualização e interpretação dos resultados típicos.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

Vírus envelopados utilizar glicoproteínas da membrana na sua superfície para mediar a entrada nas células hospedeiras. Análise estrutural tridimensional dessas glicoproteína &#39;picos&#39; é muitas vezes um desafio técnico, mas importante para a compreensão da patogênese viral e na concepção de medicamentos. Aqui, um protocolo é apresentado para a determinação da estrutura viral pico através da média computacional de dados crio-tomografia eletrônica. Crio-tomografia eletrônica é uma técnica em microscopia eletrônica utilizada para obter reconstruções tridimensionais de tomografia de volume, ou tomografias, de espécimes biológicos pleomórficos, como vírus de membrana em um estado quase original, congelados hidratado. Estas radiografias revelam estruturas de interesse em três dimensões, embora com baixa resolução. Média Computacional de sub-volumes, ou sub-tomografias, é necessário obter maior detalhe resolução de repetir motivos estruturais, tais como picos de glicoproteínas virais. A abordagem computacional detalhado para aligning média e sub-tomografias utilizando o pacote de software Jsubtomo está delineado. Esta abordagem permite a visualização da estrutura de picos de glicoproteínas virais para uma resolução da ordem de 20-40 Å e estudo de estudo de interacções de ordem superior pico-a-pico na membrana viral. Os resultados típicos são apresentados por vírus Bunyamwera, um vírus com invólucro a partir da família Bunyaviridae. Esta família é um grupo estruturalmente diverso de patógenos que representam uma ameaça para a saúde humana e animal.

Nós demonstrar a aplicação do fluxo de trabalho média sub-tomograma descrito acima para a glicoproteína do envelope do vírus complexo Bunyamwera (Orthobunyavirus Bunyaviridae) utilizando dados publicados anteriormente definido 24. Recolha de dados e refinamento parâmetros listados na Tabela 1. Uma tomografia representativo é mostrado na Figura 2. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Parâmetro (unidade) </td><td> Valor </td></tr><tr><td> A coleta de dados </td><td></td></tr><tr><td> Tensão (kV) </td><td> 300 </td></tr><tr><td> Ampliação calibrado (X) </td><td> 111000 </td></tr><tr><td> O tamanho do pixel (Å) </td><td> 5.4 </td></tr><tr><td> Dose estimada (e - / a 2) </td><td> 100 </td></tr><tr><td> Underfocus (im) </td><td> 4,0-4,5 </td&gt; </tr><tr><td> Primeiro de zero CTF (A) um </td><td> 26-30 </td></tr><tr><td> Faixa de inclinação (°) </td><td> -60-60 </td></tr><tr><td> Amostragem Tilt (°) </td><td> 3 </td></tr><tr><td> Dados e requinte </td><td></td></tr><tr><td> Tomogramas </td><td> 11 </td></tr><tr><td> Virions </td><td> 29 </td></tr><tr><td> Sementes por virion </td><td> 106 </td></tr><tr><td> Número total de sementes </td><td> 3074 </td></tr><tr><td> Spikes detectado </td><td> 1346 </td></tr><tr><td> Spikes após eliminar sobreposições </td><td> 1401 </td></tr><tr><td> Spikes após seleção com base correlação cruzada </td><td> 1022 </td></tr><tr><td> Spikes incluídos na média final </td><td> 1022 </td></tr><tr><td> Simetria </td><td> C3 </td></tr><tr><td> A amostragem angular (°) </td><td> 8 </td></tr><tr><td> Resoluçãogama utilizada no refinamento (Å) </td><td> 42-334 </td></tr><tr><td> Estimativa resolução final (a) b </td><td> 35 </td></tr></tbody></table> Tabela 1: coleta de dados e estatísticas Bunyamwera refinamento. FTC, a função de transferência de contraste. b Calculado utilizando a correlação de shell Fourier entre duas estruturas de forma independente refinados em um limite de 0.143. <img alt="Figura 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> Figura 2:. Fatia através de uma tomografia de viriões Bunyamwera Diversas vistas laterais pico evidente na periferia de cada vírus são indicadas por pontas de seta. A tomografia tem sido baixa-pass filtrada a 60 Å. A barra de escala de 100 nm. Primeiro, temos refinado um modelo inicial usando 205 pontos escolhidos manualmente ( <strong&gt; A Figura 3). Simetria Trina do centro-maior pico foi evidente, sem aplicar qualquer simetria (Figura 3B) e foi imposta nas rodadas posteriores de refinamento (Figura 3C). Para a detecção de todos os picos automaticamente sobre as superfícies de virião, geramos 106 sementes para cada virião no raio de 43 nm e um espaçamento de 20 graus (Figura 4A) e iterativamente refinado suas posições em relação à membrana (Figura 4B). <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> Figura 3: O refinamento da estrutura do modelo inicial. (A) cilíndrica média (C100) modelo construído a partir de posições definidas manualmente de spikes. (B) densidade médio depois de cinco rodadas de refinamento sem qualquer simetria (C1) imposta exibe um pico com três dobrascaracterísticas simétricas. A resolução do modelo é de 48 Å. (C) Média do pico foi resolvido em 41 Å após cinco rodadas de refinamento com simetria tripla. <img alt="Figura 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> Figura 4:. Refinamento das sementes AB) Um subconjunto de sementes antes (A) e após (B) requinte são mostrados em uma densidade virion na Figura 2 Sementes em (B) foram codificados por cores com base no respectivo cruzada. coeficientes de correlação (azuis, baixa correlação; vermelho, alta correlação). Os melhores correlacionando glicoproteína manchas Spike (75% superior após eliminar sobreposições; ~ 1.000 pontos) foram utilizados para calcular a média final. A média foi resolvido a 35 uma resolução (Figura 5). Ele revelou uma estrutura pico trimeric no meio, emAlém de alguma contribuição de seis picos vizinhos. Modelos compostos dos viriões, calculadas através da colocação da estrutura nas posições conhecidas, revelado colocação dos espigões na superfície do virião (Figura 6A). Ocasionalmente, patches de ordenados localmente de spikes eram evidentes (Figura 6B). <img alt="Figura 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> Figura 5: estrutura refinada de um patch da camada de glicoproteína pico após casamento de modelos. (AB) Dois mapas &#39;even&#39; e &#39;estranho&#39;, de reconstrução de duas metades de os dados são mostrados. Os dois mapas mostram um notável grau de similaridade, verificando a válido até da abordagem. A orientação em torno do eixo longitudinal é diferente espiga como os dois mapas foram reconstruídos completamente independente um do outro. (C) Média dos dois mapas é mostrado naa resolução final de 35 Å. <img alt="Figura 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> Figura 6: Colocação dos picos sobre o virion Bunyamwera. (A) modelo composta de um viral. Exibir vetores (varas) indicam as orientações das espigas. As cores indicam a correlação cruzada entre cada ponto ea estrutura do modelo (azul, baixa correlação; vermelho, alta correlação). (B) Um close-up de um tapa-ordenada de spikes.

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Calculando a média de Viral glicoproteína do envelope Spikes de Electron Cryotomography reconstruções com Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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Research

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Immunology and Infection

12.0K visualizações.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Vídeo: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Usando um ensaio Microarray Pan-Viral (Virochip) para Tela de amostras clínicas para patógenos virais

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Imunologia e Infecção

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Determinação de estruturas moleculares de glicoproteínas do envelope HIV usando Cryo-Electron Tomografia e Média Sub-tomogram Automated

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Relação custo-benefício Método de Rastreamento Fonte microbiana, utilizando específicas Vírus Humano e Animal

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Separação de Single-stranded DNA, Double-stranded DNA e RNA de uma Comunidade Ambiental Viral Usando Cromatografia hidroxiapatita

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Preparação de DNA viral a partir nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imagem do HIV-1 Synapse Envelope induzida virológica e Sinalização em bicamadas lipídicas sintéticas

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Avaliação conformacional do HIV-1 trimérico glicoproteínas do envelope Usando um ELISA Ensaio Celular

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

O Desenvolvimento da liofilizado Loop-mediadas isotérmicas de amplificação Reagentes para a detecção de<em&gt; Burnetii Coxiella</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Medição de Inibição da Neuraminidase Influenza títulos de anticorpos por Enzyme-linked Lectina Ensaio

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...