Freeze-Fracture of Electron Microscopy of HIV-1 p24 capsid protein interaction with the Viral Envelope.Eric R. Newman

Project Summary

The goal of this research is to further understand how HIV incorporates/attaches the host cell's lipid membrane as a viral envelope to its RNA enveloping capsid. HIV and other similar enveloped virus’s biggest competitive advantage is their ability to evade the host's immune system by hiding behind host cell's membrane complete with host cell recognition proteins. Research into how this viral envelope recognizes and attaches to non-infected cells and the mechanism of budding through hijacking of the ESCRT complex has been extensive. However, research into the actual attachment of the viral envelope to the capsid is relatively new as it is not a conventional target for treatment. The goal of this research is to visualize the underlying structure between the viral envelope and capsid by freeze-fracture electron microscopy to further elucidate the attachment of the viral envelope to the capsid. Of specific interest is the C-terminal region of the CA capsid protein that has been shown in previous studies to bind to lipids. Understanding of this binding event could lead to methods to disrupt this process, thereby inhibiting infection by HIV and other enveloped viruses.

Specific Aims

The long term goal of this research is to further understand the mechanism of HIV infection in human cells. Knowledge of how the viral envelope attaches/remains secure to the capsid enables the possibility of utilizing this complex as a potential drug or treatment target to prevent and inhibit HIV infection through stripping the virus of its ability to hide from the host immune system. Without an attached viral envelope the HIV capsids would be vulnerable to the traditional immune response pathways.

The specific aim of the proposed research is to directly visualize the attached face of the HIV capsid and the corresponding bottom layer of the viral envelope for qualitative and visual analysis of the connective elements between the two.

This aim will be carried out through arresting viral bud release by introducing a premature stop codon into the Gag protein coding for the p6 domain of Pr55Gag, Freeze-fracture coupled electron microscopy will then be employed to visualize the arrested and forming viral buds in- and cross-plane with the fractured membrane for a qualitative look at the capsid/envelope interaction.

Background and Significance

Human immunodeficiency virus (HIV) (Figure 1.) is an enveloped lentivirus responsible for acquired immunodeficiency syndrome (AIDS). AIDS is a syndrome designated by a low CD4+ T cell count and/or specific diseases and infections correlated to HIV infection. The HIV virion is spherical with a ~120nm diameter, it is composed of the viral envelope (phospholipid bi-layer) with its associated membrane proteins (host and viral) and a protein capsid within which two copies of HIV ssRNA are located. The capsid is conical and made up of an estimated 2000 copies of capsid protein monomer. HIV is particularly difficult to treat due to its ability to infect non dividing cells, genetic variability, and acquisition of host cell membrane during its budding process. The characteristic of HIV as an enveloped virus is likely the largest challenge faced by the host immune system in recognizing and eliminating it. As HIV completes its replication cycle it forms a viral capsid containing two copies of the HIV ssRNA genome (Moss, 2013). Through the production of proteins coded for by the pro-viral genetic material integrated into the host cell genome and hijacking of native proteins, this capsid begins to bud through the host cell membrane. The viral bud releases from the host cell enveloped in the host’s cell membrane with its associated proteins (Figure 1.). The integration of the hosts membrane bound proteins help the virion avoid detection by the immune response. Before budding, bud sites are proliferated with glycoprotein 41 (gp41) and glycoprotein 120 (gp120) proteins responsible for the mature virions ability to bind and fuse to a host cell. HIV’s genetic variability is a product of its rate of replication ~1010 virions daily, mutation rate of 3 x10-5 per nucleotide base pair per replication cycle and the genetic recombination ability imparted to it through its reverse transcriptase-mediated into the host’s genetic material (Moss, 2013). This rate of

mutation helps keep the few elements that might be recognized by a host’s immune system (e.g., gp120 and any degraded protein fragments picked up by MHC class 1 molecules) too variable to elicit any effective immune response. The same challenges the immune system faces in targeting HIV are faced by any attempted medical intervention. Though there are conserved regions

Figure 1. Mature HIV-1 VirionUsed without permission (http://www.scistyle.com/).

within the virus’s proteins and genome, much of it is variable and changes rapidly making a vaccine unattainable so far, and anti-viral modification only moderately effective. Issues such as these put constraints on what can be used as viable treatment targets and force modern science to look at alternate non-conventional approaches to treat HIV.

Modern treatment of HIV consists of five antiviral drug classes. There are currently 20 approved antiretroviral drugs with the majority being reverse-transcriptase inhibitors and retroviral protease inhibitors (avert.org). The the most recent classes consist of fusion of entry inhibitors and integrase inhibitors. The most recent two classes have had the most success in maintaining virulency against HIV due to the conserved nature of their targets, HIV's membrane bound glycoproteins and integrase. A issue with current treatment is HIV's development of resistance to retroviral treatment leading to a "first line" and "second line" strategy of therapy approach consisting of multiple waves of a combination of drugs (avert.org).

Global funding in response to HIV/AIDS ranging from care & treatment through prevention to research is estimated to be $31.7 Billion dollars for 2016 by The Henry J. Kaiser Family Foundation (Figure 2.). The NIH reported funding for HIV/AIDS research in the U.S. alone at $3 billion in 2014. According to the World Health Organization (WHO) there are an estimate 35 million people currently infected by the HIV virus, with 2.1 million new infections during 2013, and HIV is the leading cause of infectious death

Figure 2. Proposed U.S. Federal funding request for HIV/Aids by category.

worldwide. Numbers such as these emphasize the demand and importance in furthering HIV research.

Synthesis of the capsid protein is carried out through a polyprotein precursor the group-specific antigen protein (Gag) (Figure 3.) that is cleaved by HIV protease into capsid protein, nucleocapsid, mature matrix and Gag proteins.

Within the Gag protein there is a proline rich 6-kDa domain designated p6 located at the proteins C-terminal (Figure 4.). This p6 domain was shown to be integral to viral bud release from the cell membrane (Huang, Et al. 1995). The p6 region consist of several highly conserved domains implying function. A nonsense mutation consisting of a premature stop codon at the very beginning of the p6 domain reduced viral bud release 20 fold (Huang, Et al. 1995).

The long term goal of the proposed research is to exploit the viral coat/capsid complex as a treatment target. Recent experiments have shown the C-Terminal domain of the capsid protein to have lipid binding properties through fluorescence and FTIR measurements and it is believed that this is effect plays a part in the conjoining of the capsid and viral envelope (Figure 5.)(Barrera, Et al. 2006).

Understanding of capsid and viral envelope functional morphology through imaging of the capsid protein's interaction with the fore mentioned envelope is intended to be a building block to further more quantitative research into the strength and perturbations

Figure 3. Organization of the HIV-1 domains Capsid (CA), Matrix (MA), Nucleocapsid (NC), protease p1 & p6 (PR),

integrase (IN) and RT domains of the gag and pol reading frames. Used without permission (Huang, Et al. 1995).

Figure 4. Assembled HIV-1 Capsid composed of ~2000 monomeric copies (Left), Capsid protein with N-terminal domain & C-Terminal domain of interest (Top Right), Capsid Pentamer with NTD

displayed in blue (Middle Right), Capsid Hexamer with NTD displayed in green (Bottom Right). Used without permission (Deshmukh, Et

al. 2013).

of this attachment. The viral envelope is one of HIV’s greatest strengths allowing it to hide from host immune response and an essential component in continuing the HIV life cycle. If the viral coat could be disrupted or stripped from the HIV capsid it would not

cure a prevailing infection but could be utilized much as anti-virals are today, as an inhibitor to current infections and prevention against possible infection by at risk populations. Disruption or removal of the viral envelope would shut down one of HIV’s two pathways to continue its infection of new cells and the only pathway to infection in a new host. Without an intact viral envelope the HIV virion faces multiple issues. First, without the glycoproteins located on and within the viral envelope HIV cannot recognize and bind to the CD4 receptors and CCR5

& CXCR4 co-receptors on the host cell preventing fusion and infection with the inserted viral genetic material (Figure 3.). Second, without the host proteins associated the stolen lipid membrane the virion cannot effectively hide from the host’s immune response leaving it vulnerable.

Freeze-etching is a subset of Freeze-fracturing that employs sublimation after fracturing, both are techniques employed in tandem with electron microscopy. Electron microscopy sample chambers are kept at low pressure to avoid electron scattering by gasses which causes problems with biological samples. Freeze-fracturing freezes the water within and around a biological sample at high pressure to from amorphous ice an isochoric process. Fracturing of the frozen sample is conventionally done with a cold razor blade. The fracture plane commonly splits the inner and outer lipid bilayer but can also create a fracture plane splitting the lipid bilayer from the cell or split a plane directly through a cell (Figure 6). When this is done transmembrane proteins are visible either directly within a separated bi/monolayer or through the void they occupied. Once sample fixation is achieved an additional step designated etching can be done where

Figure 5. Crystal structure of monomeric Capsid protein C- terminal domain with tyrosine & tryptophan residues in stick form. Regions

with predicted lipid membrane affinity are in dark gray including the asterisk indicated alpha-helix. Used without permission from

(Barrera, Et al. 2006).

more details of the fractured sample can be visualized if the surrounding ice is removed through sublimation to maintain sample structure. This etching as done at low temperature and high pressure to minimize ice crystal formation and remnant gas deposition both of which contaminate the sample (Severs, 2007). Once the sample has been prepared it is coated under an angle (~2-8o with rotation for small structure resolution) with a thin film of heavy metal (generally platinum ~7nm) to create shadowing for contrast and subsequently a layer of carbon (~15nm) at a 90o angle to the fracture plane to stabilize the thin metal film. With a simultaneous evaporation of platinum and carbon monoclonal steps of 11Å have been resolved (Bradley, 1959) however more common platinum followed by carbon layering routinely have a resolution of 2.5nm opposed to the 11Å resolution expected out of the methods described later. Following sample shadowing and coating the remaining biological material is removed using SDS or acid digestion after which the sample is ready for analysis (Servers, 2007).

Electron microscopy is a form of microscopy that uses an electron beam to visualize a sample. Due to the extremely short wavelengths of electrons the resolving power is much higher than that of conventional light microscopy. Transmission electron microscopy (TEM) has a reported 50 pm resolution and can reach magnification up to 10,000,000x (Ernie, Et al. 2009). TEM utilizes an accelerated electron beam focused by electromagnetic and electrostatic lenses into a focused beam that gets scattered as it

Figure 6. (Top) Examples of different fracture planes obtained through Freeze-Fracture. Exoplasmic Surface (ES), Plasmatic Fracture Face (PF), Exoplasmatic Fracture Face (EF),

Cytoplasm (Cyt). (Bottom) Freeze-Fracture with a PF and integrated proteins.Used without permission (Source: http://www.leica-microsystems.com/science-lab/brief-

introduction-to-freeze-fracture-and-etching/)

passes through the sample and the beam is subsequently used to expose a photographic plate. (Ernie, Et al. 2009).

Altogether the proposed research is viable due to the following reasons. First, bud arrestment fixing the subject to be studied and increasing the density per area of buds approximately 20-fold will provide a large amount of sample to be studied (Huang, et al. 1995). The mutagenesis to achieve this increased bud density has been performed and verified by previous research (Huang, et al. 1995). Second, the C-terminal region of the capsid protein has displayed lipid binding properties that imply some sort of interaction with the lipid bi-layer viral envelope (Figure 5.)(Barrera, et al. 2006). Third, the long term goal of viral envelope degradation/removal would prevent viral bud infection based on the gp120 protein function and viral envelope location and gp40 location and function within the viral envelope, nonetheless the speculative vulnerability of uncoated HIV virion to a host immune response (Figure 1.)(Maartens, et al. 2014). Fourth, freeze-fracture has been demonstrated to provide fracture planes through and around inter-membrane and membrane bound vesicles ~40 nm smaller than the HIV virion (Zampighi, et al. 1999). Fifth, with freeze-fracture electron microscopy's magnification and a resolving power of 11Å we expect to be able to resolve the most likely culprit of capsid protein viral envelope interaction the C-terminal alpha helix due to the 12Å diameter of alpha helices and the variable length a helix or other subunit could protrude into the viral envelope. We hope to elucidate the membrane bound and intermembrane proteins within, through and into the viral envelope (Figure 5)(Ernie, et al. 2009). Last, there is a distinct need and available funding for further information on HIV especially with pathways leading to conventional and nonconventional treatment targets (Figure 2).

Research Design and Methods

First, arresting viral bud release will be achieved through site directed mutagenesis followed by transfection. Viral bud visualization is challenging due to multiple characteristics of the virion. The challenging characteristics of viral bud visulization constituting of the viral bud size and migratory. Through site directed mutagenesis a premature stop codon will be added right after the start codon of the p6 region of the HIV Gag protein (Figure 3.). The p6 region plays a crucial role in the release of the HIV virion from the infected cell membrane once the viral capsid is fully formed and disrupting it has been shown to arrest virion release from the host membrane but still allow virion formation. Using a 1.2-kbp SphI-BalI fragment (nucleotides 1442-2619) from a full length infectious clone of pNL4-3, the most widely used vector for in vitro manipulation of the HIV proviral sequences. The 1.2-kbp SphI-BalI fragment will then be sub-cloned into an M13mp18 single stranded DNA plasmid and directed-oligonucleotide mutagenesis will be performed as proscribed by (Kunkle, et. al., 1987). A 473-bp of ApaI-PpuMI fragment (nucleotides 2006-2483) will carry a premature stop codon mutation in the (PTAP-, residues 7-10) region of the p6 domain of the HIV Pr55Gag protein (Huang, et. al., 1995). This premature stop codon removing the highly conserved PTAP- motif within the p6 region was shown to inhibit viral bud release as designated in a 20 fold decrease in viral reverse transcriptase activity

(Huang. 1995). Arresting bud release will give a higher density of attached viral buds for freeze fracture and subsequent electron microscopy imaging. Due to the small size of retrovirus particles ~100nm and the inconsistent fracture planes of the freeze-fracture technique an increased particle density will correlate to an increase in viral buds fractured along a c-terminal plane while also being visualized in the subsequent electron microscopy. Following the site directed mutagenesis the ApaI-PpuMI fragment will be DNA sequenced as a positive control to verify the directed mutagenesis took place.

Two separate cell lines will be studied during the visualization the HIV capsid stripped of either one or both of the lipid layers constituting its viral envelope in an attempt to obtain different fracture planes during freeze-fracturing. Cell cultures containing HeLa 10-15µm and CEM(12D-7) 5-10µm cells will be prepared and as described in (Freed, et al., 1994). CEM(12d-7) cells are of the T-cell line native targets for HIV. However, the HeLa cell line has been transfected to express Cluster differentiation complex 4 (CD4) to allow infection by HIV. Calcium phosphate precipitation and DEAE-dextran transfection will be used to transfect the HeLa and CEM(12D-7) cells respectively, both methods described in (Freed, et al., 1994). The CEM(12D-7) cell line will then be infected with equal amounts of mutated HIV after normalization for reverse transcriptase activity as described by (Freed, Orenstein, et al., 1994). Reverse Transcriptase assays for the CEM(12D-7) cell line will performed as described by (Willey, et al., 1998) and (Freed, Orenstein, et al., 1994) for the HeLa cell line as controls to determine the successful retardation of viral bud release by the mutated HIV strain and HeLa cells. The afore mentioned normalization for RT activity is executed to maintain a standard activity level for the subsequent RT assay to verify virion release inhibition.

Forty-eight hours post infection, infected cells will be harvested and suspended in distilled water to a resting culture at 4o-6o C with the sample being utilized before 12 hours has passed to minimize sample degradation. Obtaining and freezing of the HIV sample by liquid nitrogen at -160oC will be executed as described by (Gross, Kuebler., 1978). Following sample preparation, freeze-etching, platinum-carbon evaporation, platinum-carbon shadow coating, and carbon backing will all be executed as described in (Gross, Kuebler. 1978). Electron microscopy and image processing will also be done using the procedure as designated by (Gross, Kuebler, 1978). Freeze fracturing will be done on a modified high vacuum Balzers BA 350 U UHV-Freeze-Fracture apparatus operating at ~10-9 torr and -196oC as opposed to the conventional ~10-6 torr and -100oC for improved topographic resolution. The ultrahigh vacuum (UHV) technique was shown to lessen structural distortion and contamination of the fracture plane with condensed gasses hiding and create structures. Lowering the temperature during freeze-fracture reduces distortion of fractured structures, damage caused during radiation heating and surface diffusion by condensing metal atoms. The low vacuum helps alleviate the one non-temperature dependent aspect of freeze fracturing, contamination of the sample by gas molecules 1000-fold (Gross, Bas. 1978). After coating samples will be imaged with a Siemens 102 electron microscope coupled with an anticontamination device with pictures taken on electron image plates at all followed by detailed image analysis encompassed in methods proscribed by (Gross, Kuebler., 1978).

References

Barrera, F. Hurtado-Gomez, E. Lidon-Moya, M. Neira, Jose. (2006). Binding of the C-terminal domain of the HIV-1 capsid protein to lipid membranes: a biophysical characterization. Biochem J. 394:345-353

Bradley, D.E. (1959). High-resolution shadow-casting technique for the electron microscope using the simultaneous evaporation of platinum and carbon. Br. J. Appl. Phys. 10(198)

Deshmukh, L. Schwieters, C. Grishaev, A. Ghirlando, R. Baber, J. Clore, M. (2013). Structure and Dynamics of Full-Length HIV-1 Capsid Protein in Solution. Journal of the American Chemical Society. 135:16133-16147

Erni, R. Rossel, M.D. Kisielowski, C. Dahmen, U. (2009). Atomic-resolution imaging with a sub-50-pm electron probe. Phys Rev Lett. 102(9):096101

Freed, E. O., Martin, M.A. (1994). Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type1 envelope glycoprotein gp120. J. Virol. 68:2503–2512.

Freed, E. O., Orenstein, J.M. Buckler-White, A.J. Martin, M.A. (1994). Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J. Virol. 68:5311–5320.

Gross, H. Bas, E. Moor, H. (1978). Freeze-fracturing in ultrahigh vacuum at -196 degrees C. J Cell Biol. 76(3):712-28.

Gross, H. Kuebler, O. Bas, E. Moor, H. (1978). Decoration of specific sites on freeze-fractured membranes. J. Cell Biol.79:646–656

Huang, M., Orenstein, J., Et. al. (1995). p6Gag is Required for Particle Production from Full-Length Human Immunodeficiency Virus Type 1 Molecular Clones Expressing Protease. Journal of Virology. 69(11): 6810-6818

Kunkle, T. A., J. D. Roberts, and R. A. Zakour. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol.154:367–382.

Maartens, G. Celum, C. Lewin, S.R. (2014). HIV infection: epidemiology, pathogenesis, treatment, and prevention. Lancet. 384(9939):258-271

Moss, J.A. (2013). HIV/AIDS Review. Radiol Technol. 84(3):247-267

Severs, N.J. (2007). Freeze-Fracture electron microscopy. Nat Protoc. 2(3):547-576

Sleytr, U. B., and W. Umrath. (1974). A simple fracturing device for obtaining complimentary replicas of freeze-fractured and freeze-etched suspensions and tissue fragments. J. Microse. (Oxf.). 101:177-186

Willey, R. L., D.H. Smith, L.A. Lasky, T.S. Theodore, P. L. Earl, B. Moss, D.J. Capon, and M.A. Martin. (1988). In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J. Virol. 62:139–147.

Zampighi, G. Loo, D. Kreman, M. Eskandari, S. Wright, E. (1999). Functional and Morphological Correlates of Connexin50 Expressed in Xenopus laevis Oocytes. J. Gen Physiol. 113(4):507-524