Imaging HIV Particles at the Virological Synapse
Using Optical Nanoscopy to Resolve the Transfer of HIV
- Photo: Spectral-Design/Shutterstock
- Fig. 1: Scheme of the experimental outline. (a) An illustration of a HIV particle where two viral proteins, Env glycoprotein complex (composed of gp41 and gp120 subunits) and Gag, are displayed. (b) Drawing of the three used sample sets. sfGFP is fused to gp120 (labeling Env) and mCherry to Gag, and these are the first two sample sets. Additionally, both proteins are labeled at the same time. (c) Scheme of the preparation process and assumed model of the transmission through a T cell to T cell VS.
- Fig. 2: 3D-SIM images of the different stages of the HIV VS transfer process (green – Env, magenta – Gag, blue – nucleus, white – plasma membrane). (a) Fluorescently labeled HIV expressing Jurkat-T cell where Gag can be predominantly localized at the plasma membrane and Env is mostly accumulating in the cytoplasm. (b) Virological Synapse. Three primary CD4+ T cells are attached to the HIV+ Jurkat-T cell, but no HIV particles can be seen transferred in the target cells. Again, the distribution of Env and Gag is as expected (mostly cytoplasm and plasma membrane at the contact area respectively). (c) Transferred virus particles are observed in primary T cells. On the right side, apparently single internalized virus particles can be observed as magenta spots. The accumulation and therefore assembly at the VS adhesive structures is clearly visible. Z-stacks, 2.5 µm (a, c), and 2.75 µm (b). Scale bars, 3 µm.
- Fig. 3: 3D-SIM image of the transmission of the virus. Three primary T cells are attached to the Jurkat-T cell (one is in the back). Z-stack, 15 µm. Scale bar, 5 µm.
The Acquired Immune Deficiency Syndrome (AIDS) is caused by the Human Immunodeficiency Virus (HIV) and currently affects over 36 million humans worldwide with over a million deaths in 2015 alone. HIV has been first isolated in 1983 by Barré-Sinoussi et al. and efforts towards the development of a cure began shortly thereafter . The heterogeneity of HIV and the mechanism behind the capacity of the virus to evade the human immune system, and therefore not induce an effective immune response, are poorly understood. Thus, a thorough basic understanding of the infection process is still needed for the understanding of HIV infection and dissemination pathways .
In the last years, research has focused on the infectious pathways of which HIV makes effective use. First, the virus can enter the host cell when a cell-free virus particle binds to the CD4 molecules at the plasma membrane of a T cell. In recent years, it has, however, become increasingly clear that the virus can also be directly transferred from infected cells to uninfected cells via an adhesive structure dependent on viral proteins, called the virological synapse (VS) . In the case of T cell to T cell transfer, the VS is initiated through the interaction of the viral envelope glycoproteins (Env) at the surface of the infected cell and the CD4 receptor expressed by the target cell. The virus then assembles specifically at the VS, buds and is endocytosed by the previously uninfected cell . The infection is thought to occur after maturation of the virus particles within the endocytic compartments [5, 6].
The direct transfer by cell to cell contact allows for multiple infection events of a single host cell and thus is much more efficient at transfering viral antigen to target cells than the free particle plasma membrane fusion mechanism [4, 7]. The VS mediated transfer process is rather difficult to observe by conventional live cell microscopy, because it occurs on spatial length scales of less than 2μm and the cells are highly polarized and motile [4, 5]. Fluorescence microscopy is subject to the famous Abbe resolution limit restricting its spatial resolution to typically 250 nm at best.
Single HIV particles, which are 120 nm in diameter, cannot be resolved with conventional optical methods (fig. 1a).
We use Structured Illumination Microscopy (SIM) which provides twice the resolution compared to conventional optical microscopy. SIM can simultaneously acquire fast, super-resolved 3D images, with multiple color channels . This technique outperforms other super-resolution methods in terms of speed and much lower photo-damage, and therefore it is ideal and often used for live-cell imaging [9,10].
Materials and Methods
The virus can be labeled through fusions of proteins of interest with fluorescent proteins. The structural protein of HIV, Gag, can be labeled through internal insertion of a sequence coding for a fluorescent protein between the sequences corresponding to the Matrix and Capsid domains. This results in an efficient way of fluorescently labeling assembly sites and immature as well as mature viral particles without impairing much of the infectivity rates of such constructs. Double labeling is possible with the use of other fluorescent proteins fused to other viral components. Here, we used an insertion of sequences coding for sfGFP (super folding Green Fluorescent Protein) in the gp120 subdomain combined with an internal mCherry fusion to Gag (Gag-mCherry) [11,12]. For our experiments, we prepare different sample sets, where either the individual proteins are labeled or otherwise both proteins are labeled at the same time (fig. 1b).
To study T cell to T cell transfer via virological synapses, we used Jurkat-T cells as an HIV expressing cell line. These are obtained after transfection of the different full replication competent viral genomes containing the fusions to fluorescent proteins present on a single plasmid (fig. 1c).
The HIV expressing Jurkat-T cells are mixed with an equal amount of primary CD4+ T cells isolated from HIV negative donors. The cells are chemically fixed three hours after co-incubation. The cell mixture will represent most of the stages of viral transfer at the VS. While a majority will not have engaged in forming a VS, 10 to 20% will show a strong adhesion between HIV+ Jurkat-T cell and one or more primary CD4+ T cells.
We used a Delta Vision OMX v4 from GE Healthcare to acquire 3D-SIM super-resolution images of the VS. This commercial setup allows for SIM with up to four different fluorophores. We recorded 3D images with 3 - 15 µm thickness. The images were reconstructed to super-resolution images with the manufacturer-supplied software. Maximum intensity z-projections are shown in figures 2 and 3.
As counterstains we used DAPI for nuclear DNA staining and membrane dyes such as FastDiI (fig. 2c and 3) or Wheat Germ Agglutinin bound to the chemical fluorophore AlexaFluor-647 (WGA647) (fig. 2a). Both dyes have specific affinities for membranes on fixed samples, most importantly including the plasma membrane of the cells used here. The samples are mounted after staining in a higher than water refractive index solution such as Vectashield.
Results and Discussion
The transfected Jurkat-T cells will express different fluorescently labeled HIV constructs. While the sfGFP protein fused to gp120 will appear mainly internally as well as at the extra-cellular surface in the Env complex, the mCherry labeling Gag will be present predominantly bound intracellularly at the plasma membrane were viral particles assemble (fig. 2a). As expected, the occurrence of labeled Env complex at the plasma membrane appears very rare.
In the case of a virus particle, gp120-sfGFP will remain at surface of the viral membrane while mCherry is trapped within the particle either bound or unbound to Matrix and Capsid domains as the virus matures.
After mixing the Jurkat-T cell culture with primary CD4+ T cells for three hours, a fraction of cell conjugates can be observed to have formed VSs. The virus dependent cell to cell adhesion increases the localization and assembly of Gag at the contact area. Transfer of the virus could possibly be recorded. As seen in figure 2b there are three primary T cells which form this adhesive structure with one Jurkat-T cell. Gag seems to strongly accumulate and assemble at the VS, but there is no visible viral fluorescence at the target cells.
Nevertheless, we observed transferred, possibly endocytosed, virus particles in primary target cells (fig. 2c). The VS is here clearly defined by the bright fluorescence of mCherry at the adhesive structure. Acquired 3D images visualize that mCherry, sfGFP and WGA647 fluorescence can be found at that spot together (data not shown).
An attachment of target cells to an infected Jurkat-T cell does not lead necessarily to a transmission of the virus. As seen in figure 3 there are three primary T cells attached to one host cell but in only one primary T cell single virus particles at the plasma membrane are visible.
Conclusion and Further Steps
The different stages of T cell to T cell HIV transfer at the VS can be imaged by 3D-SIM with significant resolution gain. This makes it possible to resolve single virus particles which have a diameter of about 120 nm.
Uncertainties remain nevertheless even with such a resolution increase. While single particles which assemble and bud from a cell would carry several hundreds of fluorescent proteins and therefore are recorded by 3D-SIM, single molecules are probably not visible. In the case of no fluorescence transfer observed in target cells, the resolution gain by 3D-SIM is either too low for a low fluorescence signal or the transmission did not happen.
A 3D-SIM approach has a definite advantage for future studies: It is live cell imaging compatible .
We thank the Boehringer Ingelheim Fonds for their financial support.
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Alice Wilking1, Lili Wang2, Benjamin K. Chen2, Thomas Huser1, Wolfgang Hübner1
1 Biomelcular Photonics, Faculty of Physics, University of Bielefeld, Bielefeld, Germany
2 Icahn School of Medicine at Mount Sinani, New York, USA