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.).

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The authors have nothing to disclose.

Knowledge of viral glycoprotein spike structure on the virion membrane is essential for understanding virus replication and developing therapeutics to treat and prevent infection. Electron cryo-microscopy combined with single particle averaging has emerged as the most utilized method to solve the structures of enveloped virus particles, including the glycoprotein spikes. However, this method is limited to icosahedrally symmetric viruses. Here, through the application of electron cryo-tomography and subtomogram averaging in Jsubtomo, we have outlined a general protocol for determining glycoprotein spikes on pleomorphic enveloped viruses that are not amenable to other current structural biology methods. Our representative results demonstrate that the resolution of this method is sufficient to reveal insights into domain architecture, oligomerization, and higher order organization of glycoprotein spikes on intact virions.

The most critical step within this protocol is constructing two reliable starting models that are statistically independent from one another. Successful execution of this step assumes that glycoprotein spikes are sufficiently large and not packed against each other too tightly, so that individual spikes can be visually recognized and manually picked in the tomograms, and two independent models averaged. If this is not feasible, two modifications to the protocol can be attempted. First, two independent random models can be constructed by first defining two random subsets of subtomograms and then averaging the subtomograms within these subsets30. Second, if a structure of the isolated spike has been derived by other means, for example by X-ray crystallography, it can be used as a starting model. However, care must be taken to low-pass filter this model using a low-resolution cut-off (50-70 &#197;), as the two resulting models in the next round of refinement will be statistically independent only beyond this resolution. Due to this caveat, the former approach is recommended.

The obtainable resolution from this protocol depends on four major factors: i. data collection strategy and the quality of the input data, ii. number of the subtomograms, iii. alignment accuracy of the subtomograms, and iv. heterogeneity of the structures. While the first and second limitation can be largely overcome by using high signal-to-noise direct electron detectors combined with CTF corrected tomography and automated data collection, the alignment accuracy is further affected by the size and shape of the structure of interest itself. When applying this protocol on small spikes lacking prominent features, it may be advantageous to bind Fab fragments to the spike to improve the alignment accuracy and thus resolution31. Finally, if the structures to be averaged exhibit multiple conformations, sub-tomogram classification methods may be used to average different conformations separately. To that end, Jsubtomo integrates with the Dynamo package, offering powerful subtomogram classification9.

The above protocol is complementary to X-ray crystallography of isolated viral glycoproteins. Crystallographic structures can be fitted into sub-tomogram averages to obtain the precise orientation of the glycoprotein with respect to the virion membrane. Application of this methodology will undoubtedly continue to shed light onto enveloped virus structure and pathobiology.

Electron cryo-tomography is an electron cryo-microscopy imaging technique allowing the calculation of a three-dimensional (3D) reconstruction of complex biological specimens. Suitable specimens range from purified macromolecular complexes1, filaments2, coated vesicles3, and pleomorphic membrane viruses4 to whole prokaryotic cells5 and even thin areas of whole eukaryotic cells6. Following the data collection of a tilt-series, 3D tomographic volumes, or tomograms, may be calculated using several established software packages, including Bsoft7 and IMOD8.
Two aspects inherent to the study of biological specimens by electron cryo-tomography limit the biological interpretation of the corresponding tomographic volumes. First, due to the limited electron dose that can be applied to biological materials before introducing significant radiation damage, signal-to-noise ratios in tomographic data are typically very low. Second, as a result of limited sample tilt geometry during data collection, some views of the object remain absent, leading to a so-called &#8216;missing wedge&#8217; artifact in the tomographic volume. However, both of these limitations can be overcome if the tomographic volume contains repeating identical structures, such as macromolecular complexes, that can be successfully averaged9-12.
Prior to averaging structures from tomogram reconstructions, objects of interest must be found and aligned to the same orientation. Locating such structures may be achieved by cross-correlation of a template structure in the tomographic volume using an approach often referred to as template matching13. The template used in this matching process can be derived from electron cryo-microscopy or electron cryo-tomography combined with 3D reconstruction, or it can be a density map simulated from an atomic structure. Several computational packages have been developed to carry out these tasks11.
Averaging of glycoprotein spikes of membrane viruses, such as HIV-1, has been a particularly successful approach for studying their structure14-16. An understanding of the structure is integral for revealing both the molecular basis of virus&#8211;host interactions and guiding antiviral and vaccine design development. While macromolecular crystallography is the technique of choice for high-resolution (usually better than 4 &#197;) structural analysis of individual viral glycoproteins and their complexes, the X-ray structures resulting from this method are of proteins isolated from the natural membranous environment on the virion. Thus, important details such as the higher order architecture of viral glycoproteins, in the context of the virion, remain lacking. On the other hand, electron cryo-microscopy and single particle reconstruction of entire enveloped viruses is restricted to virions with icosahedral symmetry17,18. Electron cryotomography combined with sub-volume alignment has thus emerged as a complementary technique allowing the study of glycoprotein spikes of irregularly shaped, pleomorphic viruses in situ.
We have developed software named Jsubtomo (www.opic.ox.ac.uk/jsubtomo) for the detection, alignment, and averaging of tomographic sub-volumes. Jsubtomo has been utilized in the structure determination of a number of cellular and viral structures19-26. Here, we outline a detailed protocol, which enables the determination viral-surface spike structures. To circumvent over-refinement of averaged structures by correlating noise, the &#8216;gold-standard&#8217; refinement scheme is adopted10,27. Finally, strategies for visualization and interpretation of typical results are discussed.

<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

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

We demonstrate the application of the sub-tomogram averaging workflow outlined above for the envelope glycoprotein complex of Bunyamwera virus (Orthobunyavirus, Bunyaviridae) using a previously published data set24. Data collection and refinement parameters are listed in Table 1. One representative tomogram is shown in Figure 2.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Parameter (unit)</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Data collection</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltage (kV)</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Calibrated magnification (X)</td>
			<td>111,000</td>
		</tr>
		<tr>
			<td>Pixel size (&#197;)</td>
			<td>5.4</td>
		</tr>
		<tr>
			<td>Estimated dose (e&#8211;/ &#197;2)</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Underfocus (&#956;m)</td>
			<td>4.0&#8211;4.5</td>
		</tr>
		<tr>
			<td>First CTF zero (&#197;)a</td>
			<td>26&#8211;30</td>
		</tr>
		<tr>
			<td>Tilt range (&#176;)</td>
			<td>&#8211;60&#8211;60</td>
		</tr>
		<tr>
			<td>Tilt sampling (&#176;)</td>
			<td>3</td>
		</tr>
		<tr>
			<td>Data and refinement</td>
			<td></td>
		</tr>
		<tr>
			<td>Tomograms</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Virions</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Seeds per virion</td>
			<td>106</td>
		</tr>
		<tr>
			<td>Total number of seeds</td>
			<td>3,074</td>
		</tr>
		<tr>
			<td>Spikes detected</td>
			<td>1,346</td>
		</tr>
		<tr>
			<td>Spikes after removing overlaps</td>
			<td>1,401</td>
		</tr>
		<tr>
			<td>Spikes after cross-correlation based selection</td>
			<td>1,022</td>
		</tr>
		<tr>
			<td>Spikes included in the final average</td>
			<td>1,022</td>
		</tr>
		<tr>
			<td>Symmetry</td>
			<td>C3</td>
		</tr>
		<tr>
			<td>Angular sampling (&#176;)</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Resolution range used in the refinement (&#197;)</td>
			<td>42&#8211;334</td>
		</tr>
		<tr>
			<td>Final resolution estimate (&#197;)b</td>
			<td>35</td>
		</tr>
	</tbody>
</table>
Table 1: Bunyamwera data collection and refinement statistics. 
a CTF, contrast transfer function. 
b Calculated using Fourier shell correlation between two independently refined structures at a threshold of 0.143.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 
Figure 2: Slice through a tomogram of Bunyamwera virions. Several spike side views evident in the periphery of each virion are indicated with arrowheads. The tomogram has been low-pass filtered to 60 &#197;. Scale bar 100 nm.
First, we refined an initial model using 205 manually picked spikes (Figure 3). Three-fold symmetry of the center-most spike was evident without applying any symmetry (Figure 3B) and was imposed in the subsequent rounds of refinement (Figure 3C). For detecting all the spikes automatically on the virion surfaces, we generated 106 seeds for each virion at the radius of 43 nm and spacing of 20 degrees (Figure 4A) and iteratively refined their positions relative to the membrane (Figure 4B).
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 
Figure 3: Refinement of the initial template structure. (A)&#160;Cylindrically averaged (C100) template constructed from manually defined positions of spikes. (B)&#160;Averaged density after five rounds of refinement without any symmetry (C1) imposed displays a spike with three-fold symmetric features. The resolution of the model is 48 &#197;. (C) Average of the spike was resolved at 41 &#197; after five rounds of refinement with three-fold symmetry.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 
Figure 4: Refinement of the seeds. A-B) A subset of seeds before (A) and after (B) refinement are shown on one virion density from Figure 2. Seeds in (B) have been color-coded based on the respective cross-correlation coefficients (blue, low correlation; red, high correlation).
The best correlating glycoprotein spike patches (top 75% after removing overlaps; ~1,000 spikes) were used to calculate the final average. The average was resolved to 35-&#197; resolution (Figure 5). It revealed a trimeric spike structure in the middle, in addition to some contribution from six neighboring spikes. Composite models of the virions, calculated by placing the structure in the known positions, revealed placement of the spikes on the virion surface (Figure 6A). Occasionally, locally ordered patches of spikes were evident (Figure 6B).
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 
Figure 5: Refined structure of a patch of the glycoprotein spike layer after template matching. (A-B) Two maps &#8216;even&#8217; and &#8216;odd&#8217;, reconstructed from two halves of the data are shown. The two maps show a remarkable degree of similarity, verifying the validness of the approach. The orientation around the spike long axis is different as the two maps have been reconstructed fully independently from each other. (C) Average of the two maps is shown at the final resolution of 35 &#197;.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 
Figure 6: Placement of the spikes on the Bunyamwera virion. (A) Composite model of a virion. View vectors (sticks) indicate the orientations of the spikes. Colors indicate the cross-correlation between each spike and the template structure (blue, low correlation; red, high correlation). (B) A close-up of an ordered patch of 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.

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

JoVE Journal

Immunology and Infection

12.0K vistas.  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.

Video: 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...

determination of molecular structures of hiv envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging

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

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...

electron cryotomography of bacterial cells

Electron Cryotomography of Bacterial Cells

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm)...

cryo-electron tomography remote data collection and subtomogram averaging

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197;...

preparing electron microscopy (em) grids for cellular cryotomography

Preparing Electron Microscopy (EM) Grids for Cellular Cryotomography  

sample preparation for in situ cryotomography of mammalian cells

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

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plunge freezing of electron microscopy (em) grids for cryotomography

Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography  

cell seeding and transfection on electron microscopy (em) grids for cryotomography

Cell Seeding and Transfection on Electron Microscopy (EM) Grids for Cryotomography  

conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay

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

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...

production of high-titer infectious influenza pseudotyped particles with envelope glycoproteins from highly pathogenic h5n1 and avian h7n9 viruses

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope...

preparation of viral dna from nucleocapsids

Preparation of Viral DNA from Nucleocapsids

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...

biocytin recovery and 3d reconstructions of filled hippocampal ca2 interneurons

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

Sample Preparation for In Situ Cryotomography of Mammalian Cells

preparing lamellae from vitreous biological samples using a dual-beam scanning electron microscope for cryo-electron tomography

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron...

imaging of hiv-1 envelope-induced virological synapse and signaling on synthetic lipid bilayers

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

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...

high-resolution single particle analysis from electron cryo-microscopy images using sphire

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic...

anatomical reconstructions of the human cardiac venous system using contrast-computed tomography of perfusion-fixed specimens

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography...

using tomoauto: a protocol for high-throughput automated cryo-electron tomography

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological...

a small volume procedure for viral concentration from water

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

using a sandwich elisa to determine the immunogenic glycoprotein content in a vaccine

This video showcases an indirect enzyme-linked immunosorbent assay or ELISA sandwich immunocapture assay designed to assess immunogenic glycoprotein levels in vaccines. The assay plate, containing glycoprotein-specific antibodies, is incubated with serially diluted vaccine samples, including reference and test vaccines, followed by immunoassay to quantify glycoprotein content in the test...

Using a Sandwich ELISA to Determine the Immunogenic Glycoprotein Content in a Vaccine

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.

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 ...

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.

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.

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.

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.

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

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.

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

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.

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

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.

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.

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.

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