The authors would like to thank Dr. Gongpu Zhao and Danxia Ke for technical support. We thank Drs. Edward Egelman and Niko Grigorieff for sharing their image processing software. We also acknowledge the staff who support the Structural Biology cryo-EM facility and Beowulf cluster and grid at the University of Pittsburgh School of Medicine. This work was supported by GM082251 and GM085043.

1.	Frozen-hydrated EM specimen preparation
Because HIV-1 capsid protein (CA) assemblies9 are stable only in high salt (1M NaCl) buffer, which contributes strong background noise in cryo-EM images, we use a rapid dilution and back-side blotting method to transiently reduce the salt concentration when preparing the frozen-hydrated EM grid.
<ol>
<li>Glow discharge the carbon side of 200-mesh R2/1 Quantifoil copper grids under 25mA for 25 seconds.</li>
<li>Use a nebulizer to bring the humidity in the environmental chamber, a home-made manual gravity plunger, to 80%.</li>
<li>In the FEI Vitrobot Plunger dewar, cool liquid ethane using liquid nitrogen. Mount the plunge-freezing dewar onto the manual gravity plunger.</li>
<li>Apply 2.5 &mu;l of preassembled CA solution onto the carbon side of the grid, which is mounted on forceps, and load the forceps onto the plunger with the carbon side of the grid facing away from you. </li>
<li>Add 3 &mu;l of low salt dilution buffer (100 mM NaCl) to the back side of the grid and immediately blot the back side of the grid with a piece of filter paper. The whole back surface of the grid should be in close contact with the filter paper for approximately 6 seconds, before removing the filter paper. Immediately plunge the grid into liquid ethane after removing the filter paper.</li>
<li>Remove the forceps from the plunger and quickly transfer the grid into a grid storage box.</li>
</ol>
2.	Cryo-electron microscopy of CA tubular assemblies
<ol>
<li>Load the frozen-hydrated grid into an FEI Polara G2 electron microscope operating at 200kv and equipped with a Gatan 4Kx4K CCD camera.</li>
<li>Under low-dose-search mode, at a magnification of ~200x and with a dose &lt;0.001e-/&Aring;2, screen the whole grid for areas with suitable ice and save the positions of these areas in a stage file.</li>
<li>Recall the saved positions and further screen these areas at a magnification of 3,900x in low-dose-search mode. Select the areas with a uniform, thin layer of ice containing well-separated, long tubes over holes, for data collection. Save the locations of these areas in a second stage file.</li>
<li>Switch to exposure mode at a magnification of 59,000x, inset a 100 &mu;m objective aperture, and adjust the objective stigmatism and beam intensity for a dose of &tilde;15 e-/&Aring;2 per exposure.</li>
<li>Return to the low-dose-search mode, move to a saved position and identify and center a good tube using the CCD camera. Switch to the focus mode, adjust the focus, and set a defocus value, normally between 0.5 to 2.5 &mu;m. Switch to the exposure mode, set an exposure time of 0.3-0.5 seconds, for a dose of 15 e-/&Aring;2, and collect an image. The images are collected on a plate camera, and the films should be allowed to settle for 10 seconds before an exposure is taken. </li>
<li>Move to the next saved position and repeat step 5 to collect more images.</li>
<li>The films are developed in full strength D19 for 12 minutes and digitized using a Nikon super coolscan 9000 ED scanner at a pixel size of 6.35 &mu;m. The format of image files is TIFF.</li>
</ol>
3.	Helical indexing
A helical object can be indexed by two parameters: the Bessel order, n, and layer line number, l. Each layer line in the Fourier transform, as characterized by (n, l), corresponds to a set of lines on the surface lattice of the helical object denoted by (h, k) indices, using the notation from a 2D lattice. For any (h, k), an (nhk, lhk) layer line is a linear combination of two basic vectors (n10, l10) and ( n01, l01), which are the n and l values of the two principal layer lines (1, 0) and (0, 1). l can be obtained from the layer line height measured along Z axis in the Fourier transform. The value of n can be estimated using the following equation10
2&pi;Rr &asymp; Jn &asymp; 1.1|n|+0.9 &nbsp;&nbsp;.....................(1)
where Jn is the Bessel function, which determines the intensity of the nth layer line, r is the radius of the helical object, and R is the radius of the maximum amplitude of the layer line. The layer line number, l, is related to n by the selection rule11
 l = tn + um &nbsp;&nbsp;....................(2)
where t and u are constants of the helix. For any given helix, there may be exactly u units in exactly (or very closely) t complete turns.
<ol>
<li>Box out a relatively straight and long tube with uniform diameter using the EMAN's program12 helixboxer and save the image in MRC format.</li>
<li>Determine the helical repeat distance, c, using a cross-correlation based program, such as 'imgccf', in the MRC package13.</li>
<li>Calculate the Fourier transform with a new box length that is an integral of the repeat distance, c.</li>
<li>Choose two principal layer lines that define two basic surface lattice vectors (1,0) and (0,1).</li>
<li>Measure the radius of the tube, r, and the height and radius of the two principal layer lines in Fourier transform, l10, R10, l01 R01, respectively (Fig. 1).</li>
<li>Calculate n10 and n01 according to equation (1).</li>
<li>Since n values are only estimation, a few combinations of n10 and n01 are tested in later steps (See step 4.2) to find the correct helical symmetry using the program package IHRSR.</li>
<li>Calculate the screw symmetry described by two real numbers: the rotation between subunits (&Delta;&#966;) and the axial rise (&Delta;z) of the one-star helix (n=1). Given l10, l01, n10, and n01 values, u and t can be obtained by testing a range of m values (for instance, -50&lt;m&lt;50) according to the selection rule. Finally, &Delta;&#966; and &Delta;z are calculated using &Delta;&#966; =360t/u and &Delta;z =c/u.</li>
</ol>
4.	Three-dimensional reconstruction
<ol>
<li>Particle segmentation<ol>
<li>Open a micrograph containing helical particles, using the graphical program Boxer, which is a program in the EMAN package. </li>
<li>Cut the helical particle into overlapping segments. In the control panel of Boxer, choose Helix mode and set the parameters for boxing: the size of the box should be larger than the diameter of the particle and the value for 'Olap' should be &tilde;90% of the box size.</li>
<li>After left-clicking on either end of the helical particle, Boxer will automatically generate a series of particle boxes along the helix length.</li>
<li>Save boxed segments as well as their coordinates.</li></ol></li>
<li>Initial 3D reconstruction using IHRSR programs<ol>
<li>Invert the contrast of the cryo-EM images and apply low pass filtering14 (optional) prior to processing with the iterative helical real space reconstruction method (IHRSR). </li>
<li>Open the graphical interface of IHRSR program by typing "Generator". Provide the graphical interface with all the information for the boxed particles stack, including the name and path of the stack, number of images in the stack, the values for symmetry parameters, etc. Click the button "Finish" to create the reconstruction script, b25.spi.</li>
<li>Use a solid or hollow cylinder as an initial reference and allow the procedure to cycle until there are no changes in the defined screw symmetry, which usually occurs after a few cycles. A right helical symmetry should give a stably converged reconstruction. The reconstruction generated in the last cycle will be used as an initial reference for further refinement. IHRSR performs 3D reconstruction using SPIDER programs.</li></ol></li>
<li>Reconstruction with iterative refinement15
The 3D reconstruction generated by IHRSR is now used as an initial reference for additional refinement. During the refinement, the helical symmetry is fixed at &Delta;&#966; and &Delta;z, which are determined from the IHRSR procedure.<ol>
<li>Determine the defocus and astigmatism present in the micrograph using programs CTFFIND3 and CTFTILT16.</li>
<li>Multiply particle segments by the CTF using SPIDER programs17. The operation termed FT in the SPIDER suite is used to compute the Fourier transform (FFT) of the 2D image. Thereafter, the FFT is multiplied by CTF values, determined in the previous steps, using the operation termed MU within the SPIDER suite. The value obtained is then inverse transformed back to form a new image (CTF-corrected) in real space using the SPIDER operation, FT again.</li>
<li>Perform projection matching by comparing projections of the reference volumes with the CTF-corrected images, using multi-reference alignment. The variation in the out-of-plane tilt angle is limited to +/-10&deg; and sampled in 1&deg; steps. Introduce constraints, such as high correlation coefficients, in-plane angles near 0&deg; or 180&deg;, and limited x-shifts, for the alignment parameters of each segment. Include in the reconstruction only those segments that satisfy the constraints.</li>
<li>After each iterative refinement cycle, a 3D reconstruction is generated using back projection and divided by sum over the CTF&circ;2. </li>
<li>Impose the helical symmetry to generate a symmetrized volume. The iterative refinement is terminated when no further improvement in the resolution of the new 3D reconstruction occurs.</li>
<li>The image processing package SPIDER is used for most of the refinement steps. A series of operations are controlled by SPIDER scripts, which are user-created batch control files containing sequences of operations and parameter values. The final reconstruction is calculated using programs in SPIDER suite.</li></ol></li>
</ol>
5. Representative Results:
A single HIV-1 CA A92E tube (Fig. 1a) was boxed out and its Fourier transform (Fig. 1b) was calculated for helical indexing. For layer lines (1, 0) and (0, 1), l10=28, l01=37, R10=55, R01=44. Given a tube radius of 211.57&Aring;, we approximated n10=-12, n01=11 (here, the handedness was predetermined). With a repeat distance of 5195.48&Aring;, the screw symmetry of the tube was determined as &Delta;z=6.8093&Aring;, &Delta;&#966;=328.88&deg;. &Delta;z and &Delta;&#966; were refined to 7.1321&Aring; and 328.86&deg; using IHRSR (Fig. 2a) and the initial reconstruction is shown in Fig. 2b. The final reconstruction (Fig. 3), after iterative refinement, improved the density map significantly from the initial model calculated with IHRSR (Fig. 2b).
<img src="/files/ftp_upload/3041/3041fig1.jpg" alt="Figure 1"/> Figure 1. Indexing of HIV-1 CA helical tube. (A) A single HIV-1 CA A92E tube image. Scale bar, 30 nm. (B) The Fourier transform of the tube shown in (A). The helical indices (n10=-12, n01=11) are denoted. The arrowhead points to the layer line at 23&Aring; resolution.
<img src="/files/ftp_upload/3041/3041fig2.jpg" alt="Figure 2"/> Figure 2. An initial reconstruction using IHRSR. (A) Screw symmetry determination for each iterative cycle. &Delta;&#966; and &Delta;z, starting from the initial values, converge to the stable values after 10 iterative refinement cycles. (B) The initial 3D density map after 10 iterative cycles.
<img src="/files/ftp_upload/3041/3041fig3.jpg" alt="Figure 3"/> Figure 3. The 3D density map after the iterative refinement. (A-C) The density map of CA tubes is displayed as three orthogonal slices: parallel to the tube axis and close to the surface (A), perpendicular to the tube axis (B), and parallel to and through the tube axis (C). Scale bars, 10 nm. (D) Surface rendering of the 3D density map contoured at 1.8s enclosing 100% volume.

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No conflicts of interest declared.

We present a set of protocols to provide a straightforward approach to obtain 3D structures of helical objects. Using the described procedures, we acquired a 3D structure of HIV-1 capsid assembly from a single tube image (176 segments). Higher resolution structures can be achieved by including more image data. 
There are several critical points for optimal data collection and analysis: First, during preparation of a cryo-EM specimen, the sample solution should be blotted away, leaving a uniform, thin layer of solution that is slightly thicker than the sample size. There are several different ways to blot the sample. For bacterial cells and tubular specimens, such as HIV-1 CA assembly, blotting from one-side, particularly from the back-side, is most appropriate. 
Second, the handedness of the helix needs to be determined, as this cannot be done by helical indexing or reconstruction. A common practice is to use freeze-etching, followed by rotary shadowing18 to determine handedness. Handedness can also be determined post-reconstruction when the resolution of the density map is sufficiently high; the 3D atomic models of individual components should fit well into the density map when a correct handedness is assumed. Otherwise, the opposite handedness should be assumed.
Third, a Wiener filter should be used during image processing, for both phase and amplitude correction, to reduce noise amplification. Since the CTF from a single image always has zero crossings, part of the information in reciprocal space is lost. Therefore, it is necessary to have multiple projection data sets included for 3D reconstruction, each imaged at different defocus values.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Source</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Glow-discharge device 100X</td>
			<td>Glow-discharge device 100X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tecnai Polara F30 microscope with a Field Emission Gun</td>
			<td>FEI, Hillsboro, OR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gatan 4K x 4K CCD camera</td>
			<td>Gatan, Pleasanton, CA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plunge-freezing device</td>
			<td>&nbsp;</td>
			<td>Home-made manual gravity plunger</td>
		</tr>
		<tr>
			<td>Quantifoil R2/1 200 mesh holely-carbon copper grids</td>
			<td>Quantifoil Micro Tools, Jena, Germany</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EM software EMAN</td>
			<td><a href="http://blake.bcm.edu/EMAN/">http://blake.bcm.edu/EMAN/</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EM software IHRSR</td>
			<td><a href="http://people.virginia.edu/~ehe2n/">http://people.virginia.edu/~ehe2n/</a></td>
			<td>Programs available from Edward H. Egelman</td>
		</tr>
		<tr>
			<td>EM software Spider</td>
			<td><a href="http://www.wadsworth.org/spider_doc/spider/docs/spider.html">http://www.wadsworth.org/spider_doc/spider/docs/spider.html</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MRC based helical processing software</td>
			<td><a href="http://www.riken.jp/biostrmech/index.html">http://www.riken.jp/biostrmech/index.html</a></td>
			<td>Programs available from Koji Yonekura</td>
		</tr>
		<tr>
			<td>CTFFIND3/CTFTILT and Real-space helical refinement software</td>
			<td><a href="http://emlab.rose2.brandeis.edu/software">http://emlab.rose2.brandeis.edu/software</a></td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

structure of hiv-1 capsid assemblies by cryo-electron microscopy and iterative helical real-space reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

Watch this Scientific Journal Video about Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction at JoVE.com

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

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do's and don'ts of cryo-electron microscopy: a primer on sample preparation and high quality data collection for macromolecular 3d reconstruction

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

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...

a robust single-particle cryo-electron microscopy (cryo-em) processing workflow with cryosparc, relion, and scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

cryo-electron microscopy specimen preparation by means of a focused ion beam

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a...

streptavidin-affinity grid fabrication for cryo-electron microscopy sample preparation

Author Spotlight: Enhancing Cryo-EM Sample Preparation with Streptavidin-Biotin Approach

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron...

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

fast grid preparation for time-resolved cryo-electron microscopy

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved...

visualization of atp synthase dimers in mitochondria by electron cryo-tomography

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological...

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...

nanoscale characterization of liquid-solid interfaces by coupling cryo-focused ion beam milling with scanning electron microscopy and spectroscopy

Nanoscale Characterization of Liquid-Solid Interfaces by Coupling Cryo-Focused Ion Beam Milling with Scanning Electron Microscopy and Spectroscopy

Cryogenic Focused Ion Beam (FIB) and Scanning Electron Microscopy (SEM) techniques can provide key insights into the chemistry and morphology of intact solid-liquid interfaces. Methods for preparing high quality Energy Dispersive X-ray (EDX) spectroscopic maps of such interfaces are detailed, with a focus on energy storage...

extracting modified microtubules from mammalian cells to study microtubule-protein complexes by cryo-electron microscopy

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron...

low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron...

optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological...

preparation and cryo-fib micromachining of saccharomyces cerevisiae for cryo-electron tomography

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

studying the supramolecular organization of photosynthetic membranes within freeze-fractured leaf tissues by cryo-scanning electron microscopy

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam...

enhancing density maps by removing the majority of particles in single particle cryogenic electron microscopy final stacks

Author Spotlight: Optimizing Cryo-EM Analysis with CryoSieve for Enhanced Particle Selection Efficiency

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world...

Enhancing Cryo-EM Map Quality with CryoSieve Particle Selection

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micropatterning transmission electron microscopy grids to direct cell positioning within whole-cell cryo-electron tomography workflows

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell...

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

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

In vitro Uncoating of HIV-1 Cores

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

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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.

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

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...