Name: determination of molecular structures of hiv envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging
Uploaded: 2011-12-01T12:00:00
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Description: Watch this Scientific Journal Video about Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging at JoVE.com

We thank the Biological Products Section of the AIDS and Cancer Virus Program, SAIC Frederick, Inc, for providing purified, AT-2 treated HIV-1 viruses, Steven Fellini and colleagues for assistance with use of the high-performance computational capabilities of the Biowulf Linux cluster at NIH, Bethesda, MD <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>, FEI Company for assistance with electron microscopy, and Ethan Tyler for expert assistance with figures. This work was supported by funds from the Center for Cancer Research at the National Cancer Institute, National Institutes of Health, Bethesda, MD.

The approach described here was developed using a specific set of instruments, tools and software. Because all labs will not be using this same experimental setup, effort was made to generalize the approach where possible, and provide alternatives where this was not possible.
1. Preparing vitrified virus samples
In the following section vitrified grids are prepared using the FEI Vitrobot Mark III, a blotting and plunge freezing robot. See Iancu, et al. for a detailed protocol on using this system 7. As an alternative to a robotic plunger, a guillotine-style or gravity plunger is perfectly sufficient 6.
In Sections 1.2. and 1.3. a Gatan Solarus 950 glow discharge unit is used to clean sample grids and increase their hydrophilicity, though any glow discharge device designed for use with electron microscopy grids can be substituted.
Though the amount of sample volume applied to the grid before blotting and plunge freezing should be in the range of 2 &mu;L, the virus and protein-A gold colloid volume in the sample mixture will vary depending on the source. As such, it is likely that a range of virus and gold dilutions or ratios will have to be tested and imaged in the microscope, and an ideal mixture determined empirically. Note that when image data is acquired in Section 3, between 10 and 20 gold fiducial markers should be present for proper tilt series alignment.
Caution: This section describes the use of gaseous ethane, hydrogen, and oxygen which are highly flammable. Proper caution should be taken when using these gases. Additionally, care should be taken to handle virus samples in accordance with biosafety recommendations. Finally, always wear protective eyewear and clothing when handling liquid nitrogen (N2) and ethane.
<ol>
<li>Prepare FEI Vitrobot Mark III by turning on its N2 gas supply, installing the humidifier cartridge and then filling the cartridge with filtered deionized water. Power on Vitrobot and set its climate chamber temperature to 22 &deg;C, target humidity to 100%, blotting time to 6 sec, and blot offset to -2 mm.</li>
<li>Power on the Gatan Solarus 950 glow discharge unit and open H2 and O2 canisters that supply the unit. Pre-clean specimen chamber by running glow discharge for 1 min at 50 Watts.</li>
<li>Arrange the desired number of holey carbon grids on a glass coverslip, carbon side up, and place inside glow discharge specimen chamber through the top loading door. Clean grids by running glow discharge for 6-10 sec at 25 Watts. (Note that the carbon side of the Quantifoil brand grids used in this protocol are matte in appearance. This will not be true for all commercial grids so it is best to obtain this information from the manufacturer.)</li>
<li>Place grid box(es) inside coolant container bowl, and thermal diffuser spindle on the central cup that sits within the coolant bowl. Slowly pour liquid N2 into the bowl until vigorous bubbling subsides, indicating equilibrium between bowl and liquid. Maintain liquid N2 level throughout the remainder of the procedure, taking care to keep it out of the central cup.</li>
<li>Attach one end of a plastic hose to the ethane gas canister, and the other end to a glass pipette. Hold glass pipette tip to bottom of central cup, establish a slow, steady ethane flow, and ethane will begin condensing. Continue filling until the central cup is full. Remove spindle from central cup.</li>
<li>Prepare sample mixture by adding 2 &mu;L protein-A gold colloid fiducial to 10 &mu;L AT-2 inactivated HIV-1 virus suspension. Gently pipette mixture to bring fiducials into solution. Keep on ice.</li>
<li>Grasp the outer rim of a grid with the specialized Vitrobot tweezers. Apply 2 &mu;L of sample mixture to carbon side of grid. Immediately instruct robot to load sample into humidified climate chamber, blot and plunge freeze in liquid ethane.</li>
<li>Transfer vitrified grid to grid box. Repeat step 1.7. to produce the desired number of grids. When finished, tighten screws on grid box lid(s) then quickly transfer box(es) to small liquid nitrogen transport dewar.</li>
</ol>
2. Loading samples into transmission electron microscope
Throughout this section, the liquid N2 level in the loading chamber should be monitored vigilantly, and liquid replenished as needed, to ensure that grid boxes remain immersed. Before bringing a tool into contact with a grid, cool it by submerging it in liquid N2 until it equilibrates with the liquid. After each use, tools should be defrosted using a hot air gun.
Throughout this section the FEI Tecnai G2 Polara transmission electron microscope (TEM) is used in conjunction with a Gatan Cryo Workstation. While the principle of loading samples under liquid N2 conditions is applicable to all cryo-electron microscopy work, the steps of this section are specific to the equipment used in the experiment. The steps used for different equipment will vary by manufacturer.
CAUTION: Throughout this section, always wear protective eyewear and clothing when handling liquid N2.
<ol>
<li>Ensure that TEM Compustage does not contain a specimen cartridge, and that the specimen selection rod and insertion rod are below 110 K.</li>
<li>Cool and prepare Cryo Workstation for loading grids into specimen storage block, which is part of the sample selection rod.</li>
<li>Transfer the cooled (&lt; 110 K) selection rod from microscope to Cryo Workstation.</li>
<li>Slide selection rod forward to insert selection block into nitrogen bath on Cryo Workstation. Remove all specimen cartridges from storage block using specialized cartridge-handling forceps. Place grid box(es) in loading workstation and loosen lid(s) with a screwdriver.</li>
<li>Use tweezers to remove grid from grid box and place in cartridge. Use the specialized C-clip placement tool to deposit a C-clip on top of grid, securing grid in cartridge.</li>
<li>Repeat step 2.5. until desired number of grids are in cartridges. Transfer cartridges to specimen selection block.</li>
<li>Prepare Cryo Workstation for removal of specimen selection rod. Slide selection rod back to remove selection block from nitrogen bath. Remove selection rod from Cryo Workstation and attach to TEM.</li>
</ol>
3. Acquiring cryo-electron microscopy data
During this section, the FEI Batch Tomography software interface is used to set up a batch file that stores the coordinates of all grid positions of interest. When instructed by the user, the Batch Tomography software will revisit each of the positions and will coordinate tilt series acquisition via Digital Micrograph. If this software is not available, the Leginon software package is viable free, open source alternative 13. Imaging was done at 200 keV, using a Gatan Imaging Filter (GIF) with a post-GIF 2K x 2K CCD camera (Gatan).
<ol>
<li>Position Compustage such that electron beam passes through vacuum. Switch to high magnification (34kX) Exposure Mode and perform direct alignments.</li>
<li>Align zero loss peak on energy filter. (This provides the energy filter with a reference for electrons with zero energy loss.)</li>
<li> Move the Compustage to a grid area featuring carbon. In low magnification (4.5kX) Search Mode, use the Wobbler function to tilt the stage back and forth between +/-15&deg;. Meanwhile adjust the Z-axis stage position until minimal stage shift is observed. (This positions the stage approximately at eucentric height). Disable Wobbler when no stage shift is observed.</li>
<li>Use the automated Eucentric Height function to refine the eucentric height. (This routine uses cross correlation to obtain a more accurate eucentric height than in step 3.3). Eucentric height is stored automatically for later reference by Batch Tomography software.</li>
<li>Find an area of interest featuring approximately three to six virions, and no less than ten fiducial markers. Add this grid position to the batch tomography file.</li>
<li>Repeat steps 3.4. and 3.5. until desired number of positions are designated. Define batch parameters (&plusmn;60&deg; with 2&deg; tilt increments, 1-2 e-/&Aring;2/tilt view) and run the batch.</li>
</ol>
4. Reconstructing cryo-electron tomograms
<ol>
<li> Align tilt series using automatic fiducial-based alignment in RAPTOR 1.</li>
<li>Reconstruct tomograms using R-weighted back projection in IMOD 8. The standard file format for the resulting data volumes is the MRC file, with file extension .mrc.</li>
</ol>
5. Segmenting virion subvolumes and identifying spikes
This section uses a customized extension of IMOD that allows the user to designate virion subvolumes within the tomogram. In Section 6, the user defined virion boundaries are used as a surface along which spikes are automatically selected.
<ol>
 <li>Open IMOD and load a tomogram populated with virions. </li>
 <li>Navigate along the Z-axis of the tomogram until the central slice through one of the virions is located. Verify that the virion centroid has been identified. After doing so, deposit a virion marker. This marker will be used by the automated virion segmentation utility in Section 6. </li>
 <li>Continue centroid designation until all virion volumes are identified. </li>
 <li>Close tomogram and save marker set defining virion centroids. Repeat until virions have been designated in all tomograms. </li>
 <li>Using IMOD, down-sample virion subtomograms by a factor of four. </li>
 <li>Denoise virions using edge-enhancing anisotropic diffusion as implemented in IMOD. </li>
 <li>Subject virions to unsupervised membrane segmentation using an energy-based three-dimensional approach (detailed in Bartesaghi et al. 2005) 3. </li>
 <li>Spikes are identified at locations on the segmented virion surfaces corresponding to the local maxima of cross correlation between the particular location and a synthetic symmetric spike-like volume. Those locations above a specified threshold are added to a list of coordinates for putative spike subvolumes. </li>
</ol>
6. Classifying and averaging the particles
<ol>
 <li>Computationally extract the tomogram subvolumes (100 x 100 x 100 voxels) at each location that was identified in step 5.8. (this is done without denoising or binning). The resulting collection of subvolumes contain the spike proteins that will be classified and averaged in the following steps. </li>
 <li>Determine the orientations of the long axis of the spike using the normal to the automatically segmented membrane at the location of each spike. This approach provides initial estimates for two of the three Euler angles. </li>
 <li>Randomize the remaining in-place rotation to prevent any possible bias in subsequent alignments. </li>
 <li>Apply Euler angles to subvolumes, then translationally align the subvolumes to their cylindrically averaged global average to ensure they all share the same center of mass. </li>
 <li>Remove the 10% of subvolumes that correlate most poorly with the updated global average. This is done to remove densities at greatest risk of being erroneously identified spikes. </li>
 <li>Align and classify spike volumes without using external references and with proper accounting of the missing wedge (detailed in Bartesaghi et al. 2008) 4. Progressively refine subvolume alignments and classify spike volumes at each iteration. </li>
 <li>Three-fold symmetry should be clearly observed in the early stages of classification, and at the fourth iteration, 3-fold symmetry is imposed. At each round, the most well-defined classes are averaged and used as the reference for the next round. </li>
 <li>Typically &tilde;4000 spikes should be selected for each dataset. Final density maps are obtained after &tilde;5-12 refinement rounds and will include contributions from &tilde;50% of sub-volumes in each dataset. </li>
</ol>
7. Coordinate fitting
<ol>
 <li>The density maps resulting from step 6.8 are opened in UCSF Chimera 12. </li>
 <li>Produce a simulated 20 &Aring; map of the X-ray coordinates using Chimera or EMAN 10. </li>
 <li>Dock coordinates into random orientations. Using Chimera's built in steepest-ascent local optimization, fit coordinates by performing multiples of 100 steepest ascent steps until convergence is obtained.</li>
</ol>
8. Representative Results 
Using 3D averaging and coordinate fitting, a variety of HIV and SIV Env spike structures have been resolved. Presented in Figure 3A is the structure of the Envelope spike from the HIV-1 strain, BaL (purple). The spike has three-fold symmetry with dimensions of &tilde; 120 &Aring; as measured from the membrane to the spike apex, and a maximal width of &tilde; 150 &Aring; which tapers down to &tilde; 35 &Aring; at the base of the spike. As seen in Figure 3B, by combining the density map for trimeric Env determined using cryo-electron tomography with the crystallographically determined structure for monomeric gp120 (red, derived from PDB ID, 2NY7), we were able to obtain a working molecular model for the trimeric envelope glycoprotein complex (PDB ID, 3DNN) 9. 
Our approach also allows for the structural analysis of complexes formed between trimeric Env and a variety of gp120-specific protein fragments and antibodies. An example of this, in which Env is complexed with the broadly neutralizing b12 Fab is shown in Figure 4A (the entire complex is shown in purple). In this figure, the extra density corresponding to b12 is seen projecting outward from the spike, and parallel to the membrane. Structural interpretations of such complexes can be extended to the molecular level by fitting the density maps with atomic coordinates for portions of the spike, bound to the corresponding ligand. As seen in Figure 4B, when this density map is fitted with coordinates of the gp120 spike protein (red, derived from PDB ID, 2NY7) bound by b12 Fab (white), the relative orientations of the three gp120 cores can be discerned (PDB ID, 3DNL). These conformational relationships are instructive for understanding how such ligands interact with the spike and interfere with virus activity. Some other structures of unliganded and liganded Env that have been resolved by the method presented here include those of HIV-1 R3A, SIV CP-MAC, SIVmneE11S and SIVmac239, the ternary complex between HIV-1 Bal Env, sCD4 and the 17b Fab, and SIV CP-Mac Env complexed to the 7D3 antibody 9,14.
<img src="/files/ftp_upload/2770/2770fig1.jpg" align="Figure 1" /> 
Figure 1. From viral suspension to 3D structure: conceptual steps in cryo-electron microscopy and 3D reconstruction.
<img src="/files/ftp_upload/2770/2770fig2.jpg" align="Figure 2" /> 
Figure 2. (A) A representative slice from a tomogram of HIV-1 virions. (B) A single HIV-1 virion, with the core and surface spikes shown schematically.
<img src="/files/ftp_upload/2770/2770fig3.jpg" align="Figure 3" /> 
Figure 3. (A) Three-dimensional structure of the HIV-1 BaL Envelope glycoprotein (surface spikes) as displayed on the surface of the viral membrane. (B) Molecular model for the trimeric spikes determined by placing three copies of the structures for monomeric gp120 (red) derived by X-ray crystallography into the density map 9.
<img src="/files/ftp_upload/2770/2770fig4.jpg" align="Figure 4" /> 
Figure 4. (A) Three-dimensional structure of HIV-1 BaL Envelope glycoprotein in complex with the Fab fragment of a broadly neutralizing antibody, b12. (B) Molecular model for the complex determined by placing three copies of the structures for monomeric gp120-B12 Fab complex derived by X-ray crystallography into the density map 9.

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No conflicts of interest declared.

The cryo-electron microscopy and three-dimensional classification and averaging methods presented here allow for the determination of three-dimensional structures of envelope glycoprotein complexes to &tilde; 20 &Aring; resolution. Fitting of X-ray coordinates of the monomeric components to the density maps allows for structural interpretation at the molecular level. Continuing improvements in low-cost computing equipment combined with developments in open source scientific tools and the proliferation of network infrastructure make possible previously unimaginable scientific collaborative endeavors. We take advantage of these developments and show that advanced scientific techniques can be brought within the reach of students of many ages.

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Multi-A Holey Carbon Grids</td>
			<td>Quantifoil</td>
			<td>-</td>
			<td>200 mesh</td>
		</tr>
		<tr>
			<td>Protein A gold colloid</td>
			<td>University of Utrech - The Netherlands</td>
			<td>-</td>
			<td>10 nm diameter</td>
		</tr>
		<tr>
			<td>HIV-1 BaL</td>
			<td>NIH, NCI, Frederick, MD</td>
			<td>-</td>
			<td>~ 1011 virions/mL</td>
		</tr>
		<tr>
			<td>Cryo-EM storage box</td>
			<td>Pacific Grid Tech</td>
			<td>GB-4R</td>
			<td>4-hole, round</td>
		</tr>
		<tr>
			<td>Vitrobot Mark III</td>
			<td>FEI</td>
			<td>-</td>
			<td>6 second blot time, -2 mm offset, 100% humidity</td>
		</tr>
		<tr>
			<td>Tecnai G2 Polara transmission electron microscope</td>
			<td>FEI</td>
			<td>-</td>
			<td>200 keV</td>
		</tr>
		<tr>
			<td>Gatan Imaging Filter</td>
			<td>Gatan</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gatan Post-GIF CCD</td>
			<td>Gatan</td>
			<td>-</td>
			<td>2K x 2K pixels</td>
		</tr>
		<tr>
			<td>Gatan Cryo Workstation</td>
			<td>Gatan</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gatan Solarus 950 Plasma Cleaning System</td>
			<td>Gatan</td>
			<td>-</td>
			<td>Oxygen and Hydrogen plasmas</td>
		</tr>
		<tr>
			<td>C - clips</td>
			<td>FEI</td>
			<td>ZIT0634</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biowulf computing cluster</td>
			<td>National Institutes of Health</td>
			<td>-</td>
			<td><a href="http://biowulf.nih.gov/">http://biowulf.nih.gov/</a></td>
		</tr>
		<tr>
			<td>UCSF Chimera</td>
			<td>University of California - San Francisco</td>
			<td>-</td>
			<td><a href="http://www.cgl.ucsf.edu/chimera/">http://www.cgl.ucsf.edu/chimera/</a></td>
		</tr>
		<tr>
			<td>IMOD</td>
			<td>University of Colorado - Boulder</td>
			<td>-</td>
			<td><a href="http://bio3d.colorado.edu/imod/">http://bio3d.colorado.edu/imod/</a></td>
		</tr>
		<tr>
			<td>EMAN</td>
			<td>Baylor College of Medicine</td>
			<td>-</td>
			<td><a href="http://blake.bcm.tmc.edu/eman/">http://blake.bcm.tmc.edu/eman/</a></td>
		</tr>
		<tr>
			<td>RAPTOR</td>
			<td>Stanford University</td>
			<td>-</td>
			<td><a href="http://www-vlsi.stanford.edu/TEM/index.htm">http://www-vlsi.stanford.edu/TEM/index.htm</a></td>
		</tr>
	</tbody>
</table>

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

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

Watch this Scientific Journal Video about Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging at JoVE.com

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 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 ...

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Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

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 ...

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.

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.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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 ...

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

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

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

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

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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.

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 ...

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 ...