A SNAP-tagged derivative of HIV-1--a versatile tool to study virus-cell interactions

Abstract

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches.Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.

Figure 1. Construction and characterization of an HIV derivative carrying a SNAP-tag within Gag.

(A) Scheme of the HIV-1 gag open reading frame indicating the snap-tag gene inserted between the MA and CA coding regions. Expanded regions display the junction between Gag (bold) and SNAP-tag (grey boxes) amino acid sequences, separated by short linker sequences (italics) for HIV^SNAP and HIV^iSNAP constructs, respectively. Arrowheads indicate cleavage sites for HIV-1 PR. (B) Immunoblot analysis of wt and modified virions. Particles released into the supernatant of 293T cells transfected with the indicated proviral plasmids were purified by ultracentrifugation and subjected to immunoblot analysis using the indicated antisera as described in methods. Positions of molecular mass standards are indicated at the left (in kDa). (C, D) Morphology of HIV and HIV^SNAP virions. 293T cells transfected with pNLC4-3 (C) or pNLC^SNAP (D), respectively, were fixed at 24 h post transfection and embedded for EM analysis as described in methods. Samples were analyzed by thin section electron microscopy.

Figure 2. Replication capacity of SNAP-tagged HIV derivatives.

(A) Influence of the SNAP-tag insertion on distinct steps in HIV replication. 293T cells were transfected with pCHIV or pNLC4-3 (HIV), pCHIV^SNAP or pNLC^SNAP (HIV^SNAP), an eqimolar mixture of both plasmids (HIV^SNAP+HIV), or with pNLC^iSNAP (HIV^iSNAP), respectively. Virions were harvested from the supernatant at 40 h post transfection. Values were normalized against the mean of the respective control. White bars: Entry efficiency of pCHIV-derived particles, purified by ultracentrifugation through a sucrose cushion, was measured on JC53 cells using the β-lactamase virion fusion assay as described in methods. Mean values and standard deviation from triplicate samples are shown. n.d. = not determined. Light grey bars: Infectivity (in r.l.u. per ng CA) of pNLC4-3-derived particles was determined by titration of viurs supernatants on TZM-bl reporter cells followed by analysis of luciferase activity as described in methods. Bars represent mean values and standard deviation from 6 independent experiments. Black bars: For determination of release efficiency, the amount of p24 CA released into the supernatant at 40 h post transfection was measured by quantitative immunoblot and divided by the sum of p24 in the supernatant and cell lysates. Mean values and standard deviations from three independent experiments are shown. (B) Replication capacity of HIV derivatives. Equal amounts of virus (0.5 ng p24 equivalent) produced from 293T cells transfected with pNLC4-3 (HIV; open circles), pNLC^SNAP (HIV^SNAP; filled circles), pNLC^iSNAP (HIV^iSNAP; filled triangles), or pNLC^eGFP (HIV^eGFP; filled squares), respectively, were used to infect MT-4 cells. At the indicated time points post infection the amount of CA released into the supernatant was determined by a quantitative dot blot analysis. Mean values and standard deviations from six replicate infections are shown. (C) Retention of the SNAP-tag after several rounds of replication. Particles from tissue culture supernatants of infected MT-4 cells at day 18 post transfection were analyzed by immunoblot using the indicated antisera. The position of molecular mass standards is indicated at the left (in kDa).

Figure 3. Specific intracellular staining of SNAP-tagged Gag protein.

(A) Intracellular detection of SNAP-tagged Gag protein in transfected HeLa cells. HeLa cells transfected with an equimolar mixture of pCHIV^eGFP and pCHIV^SNAP were labeled at 24 h post transfection using BG-TMR-Star as described in methods; nuclei were stained with Hoechst 33258. The figure shows confocal images of a mid-section through a representative cell in the green channel (i), red channel (ii), and as an overlay of green, red and blue channel (iii). Scale bars: 18 µm. Pearson's coefficient for green and red channels equals 0.754. The image in (iv) represents an enlarged section from (iii). (v) Fluorescence intensity profiles from the green and red channel along the white line shown in (iv). (B, C) Intracellular detection of SNAP-tagged Gag protein in infected C8166 T-cells. C8166 cells were infected with HIV (B) or HIV^SNAP (C), respectively, produced from transfected 293T cells. At 15 days post infection, cells were stained with BG-TMR-Star as well as by immunofluorescence using an antiserum raised against HIV-1 CA; nuclei were stained with Hoechst 33258. The figure shows confocal images of mid-sections for immunofluorescence (left panels), TMR-Star (middle panels) and an overlay of αp24, TMR-Star and Hoechst signals (right panels). Scale bars: 8 µm. White asterisks: non-infected cell.

Figure 4. Visualization of HIV^SNAP virions and budding sites by super-resolution TIRF microscopy.

(A–C) High-resolution microscopy of stained HIV^SNAP particles. HIV^SNAP particles were purified by ultracentrifugation from the supernatant of transfected 293T cells, bound to glass coverslips and stained using SNAP-surface 647. Summed-molecule TIRF (A) and dSTORM (B) fluorescence images of the same region of interest are shown. The white box indicates the particle magnified in (C). Scale bar: 1 µm. (C) Cross sectional profile derived from the dSTORM image of an individual HIV particle. Scale bar: 200 nm. (D–F) HIV budding sites at the plasma membrane of an HIV^SNAP expressing T-cell. A3.01 cells were nucleofected with pCHIV^SNAP, stained with SNAP-surface 647 and imaged as described in materials and methods. Summed-molecule TIRF (D) and dSTORM (E) fluorescence images of the same cell are shown. (F) Magnified view of the boxed regions from the dSTORM image shown in (D). Scale bar: 200 nm.