Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.National Institutes of Health (U.S.

Site-specifically labeled HA-Srt protein is incorporated into virions.

<p>(<b>A</b>) <b>Experimental setup.</b> Confluent monolayers of MDCK cells were infected with an MOI = 0.5 during 4.5 hours after which cells were starved and pulse-labeled with [^S35]Cysteine/Methionine for 20 minutes. After a 2 hour chase, a second pulse-labeling was performed using 100 µM sortase and 250 µM biotin probe to label surface accessible HA-Srt. At indicated timepoints, both cell supernatant as well as cell lysate was analyzed for presence of viral proteins. (<b>B</b>) <b>Surface behavior HA on infected MDCK cells analyzed via affinity adsorption to neutravidin-agarose.</b> At indicated timepoint, cells were lysed in 0.5% NP40 buffer and biotin labeled HA-Srt remaining at the cell surface recovered via affinity adsorption on neutravidin-agarose. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. (<b>C</b>) <b>Accumulation of HA-biotin in supernatant analyzed by affinity adsorption to neutravidin-agarose.</b> Accumulation of biotin-HA-Srt in the supernatant of the cells analyzed in 4B was measured via immunoprecipitation on neutravidin-agarose. Supernatant was lysed via addition of NP40 buffer prior to biding to beads. Proteins were eluted with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography. (<b>D</b>) <b>Quantification of HA loss from the cell surface.</b> Densitometric quantification of radioactivity was performed on autoradiographs from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002604#ppat-1002604-g004" target="_blank">figure 4B and 4C</a>. Total levels of HA-Srt were quantified relative to the levels at the cell surface at t = 0 hrs (top graph). To quantify the kinetics of budding, loss of HA-Srt from the cell surface was quantified as percent reduction relative to the t = 0 timepoint at the cell surface. The rate of accumulation in the cell supernatant was quantified relative to the maximal amount recovered at the t = 10 hrs timepoint. (<b>E</b>) <b>Accumulation of whole virus particles analyzed by affinity adsorption to chicken erythrocytes.</b> Accumulation of complete virus particles in the cell supernatant was measured via affinity adsorption on chicken erythrocytes. Supernatant from cells analyzed in 4B was removed at indicated timepoints and mixed with chicken erythrocytes for 30 minutes at 4°C. Cells and bound viral particles were lysed in 2× SDS sample buffer, proteins resolved on 12.5% SDS-PAGE and visualized via autoradiography. (<b>F</b>) <b>Kinetcs of virus accumulation as analyzed by adsorption to neutravidin-agarose versus erythrocytes.</b> Densitometric quantification of radioactivity was performed to compare the rate of HA-Srt accumulation in the supernatant compared to whole viral particles (4C versus 4E). Numbers were normalized at t = 1 hrs at which both methods were used.</p

Visualization of HA-Srt behavior by site-specific pulse labeling using sortase A.

<p>(<b>A</b>) <b>Time course of surface appearance of HA-Srt protein.</b> Experimental setup is depicted on top. MDCK cells were infected with HA-Srt and labeled with 250 µM biotin probe using 100 µM sortase followed by labeling with Qdot655. At indicated timepoints after the initial labeling, a second round of labeling was performed using sortase and Alexafluor 488 probe for 60 minutes at 4°C. Images were acquired immediately after labeling. Scale bars = 5 µm. (<b>B</b>) <b>Overlap coefficients.</b> The overlap coefficients for Qdot655 signal with the AF488 signal and the AF488 signal with the Qdot655 signal are displayed for the images shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002604#ppat-1002604-g006" target="_blank">figure 6A</a> as well as the images from Supporting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002604#ppat.1002604.s001" target="_blank">Fig. S1</a> (n = 10). (<b>C</b>) <b>Intracellular appearance of Qdot655 conjugated HA-Srt.</b> Spinning disc confocal microscopy images were taken of the samples from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002604#ppat-1002604-g006" target="_blank">figure 6A</a> (stained with AF488 and Qdot655) at a focal plane midway through the cells in order to visualize both plasma-membrane as well as the intracellular space, where Qdots accumulate at later timepoints (t = 3 h). Scale bars = 5 µm.</p

Surface distribution of HA-Srt measured via pulse-chase analysis.

<p>(<b>A</b>) <b>Experimental setup.</b> Confluent layers of MDCK cells were infected at an MOI of 0.05 for 13 hrs, starved for 60 minutes and labeled with [^S35]Cysteine/Methionine for 20 minutes. Cell surface accessible HA-Srt was labeled for 30 minutes with 100 µM sortase and 250 µM biotin probe at the indicated timepoints. (<b>B</b>) <b>Pulse-chase analysis of virus budding shortly after labeling.</b> Cells were lysed in 0.5% NP-40 lysis buffer and biotin labeled HA-Srt was recovered via immunoprecipitation on streptavidin-agarose for 3 hrs. A 5% aliquot of the total cell lysate was removed prior to the immunoprecipitation (left panel). Bound complexes were eluted from beads with 2× SDS sample buffer, resolved by 12.5% SDS-PAGE and visualized via autoradiography (right panel). (<b>C</b>) <b>Glycosidase treatment of the cell lysate.</b> A fraction of the total cell lysate at the 90 minute timepoint was denatured in glycoprotein denaturing buffer and digested with either PNGase F or EndoH. Proteins were resolved by 12.5% SDS-PAGE and visualized via autoradiography. (<b>D</b>) <b>Experimental setup.</b> Confluent layers of MDCK cells were infected with an MOI = 0.5 for 4.5 hrs, starved for 60 minutes and labeled with [^S35] Cysteine/Methionine for 20 minutes. Cell surface accessible HA-Srt was labeled for 30 minutes with 100 µM sortase and 250 µM biotin probe at the indicated timepoints. (<b>E</b>) <b>Surface behavior of HA-Srt analyzed via pulse chase analysis.</b> MDCK cells were lysed at the indicated timepoints and analyzed as in 3B. Cells infected with HA-Srt for 3 h but not treated with sortase and uninfected cells were similarly analyzed (right two lanes). Densitometric quantification of radioactivity was performed to calculate the ratio of HA0 over HA1 and HA2. (<b>F</b>) <b>Quantification of surface HA-Srt</b> Densitometric quantification of radioactivity was performed to quantify the total levels of HA-Srt at the cell surface. The amount at t = 4 hrs was set at 100% to and other levels compared in…</p

3-D reconstruction of HA-Srt dynamics on the cell surface.

<p>(<b>A</b>) <b>Real time imaging of the disappearance of HA-strep-Qdot655 from the cell surface.</b> MDCK cells were infected with HA-Srt, labeled with 100 µM sortase and 250 µM biotin probe followed by 20 nmQdot655 as described. Images were acquired every three minutes in Z-stack series of 8 µm in 21 images. Imaris software was used to create 3D images. Top figure displays the entire frame while the bottom image is focused on a single cell. Scale bars = 5 µm. (<b>B</b>) <b>Real time imaging of CD154/CD40L on the cell surface.</b> CD154/CD40L expressing MDCK cells were labeled with 100 µM sortase and 250 µM biotin probe and subsequently with 10 nm Qdot 655. Images were acquired every three minutes in Z-stack series of 8 µm in 21 images. Imaris software was used to create 3D images. Top figure displays the entire frame while the bottom image is focused on a single cell. Scale bars = 5 µm. (<b>C</b>) <b>Sum of pixel intensities in 7A and 7B.</b> The sum of pixel intensities at every timepoint was analyzed with imaris software and percent reductions quantified relative to the first timepoint.</p

Engineered viruses are sortase substrates.

<p>(<b>A</b>) <b>NA-Srt virus is a sortase substrate.</b> NA-Srt virus bearing a sortase cleavage site appeneded to the C-terminus of neuramindase, followed by an HA epitope, was pelleted from tissue culture supernatant and incubated with 150 µM sortase A and 5 mM of the indicated probe at 37°C for 1 hour, with agitation. Streptavidin-HRP immunoblot (top panel) shows incorporation of a G₃K(Biotin) probe with concomitant loss of the HA epitope (middle blot). A glycine based tetramethylrhodamine probe can also be incorporated (bottom panel, fluorescence gel). (<b>B</b>) <b>Highly purified NA-Srt virus is labeled with a glycine based AlexaFluor 647 probe.</b> NA-Srt virions were purified through a 20% sucrose cushion and further banded on a continuous 15–60% sucrose gradient before labeling with sortase (200 µM) and an AlexaFluor 647 probe (500 µM) for 2 hours at 37°C. The labeling reaction was fractionated by 12.5% SDS-PAGE and scanned by a Typhoon Imager. (<b>C</b>) <b>NA-Srt protein incorporated into virions is quantitatively forms disulfide bonded dimers.</b> NA-Srt virus was purified over a sucrose gradient and loaded on a 12.5% SDS-PAGE gel in the presence or absence of reducing agent (2-mercaptoethanol). Anti-HA epitope immunoblot displays the presence of the NA-Srt protein. (<b>D</b>) <b>Neuraminidase on infected MDCK cell surfaces is selectively labeled by sortase A.</b> MDCK cells were infected at MOI = 0.5 and at the indicated times post infection, were incubated for 30 min with 200 µM SrtA and 500 µM biotinylated nucleophile. Cells were collected, subjected to 12.5% SDS-PAGE, and immunoblots with streptavidin-HRP (top), anti-HA (middle), and anti-p97 (bottom, loading control) were performed. (<b>E</b>) <b>Glycosidase digestions of cell surface labeled HA-Srt protein.</b> MDCK cells were infected at an MOI of 0.4 overnight and labeled for 1 hour at 37°C with 100 µM sortase and 500 µM biotin probe. Cells were then lysed in glycoprotein denaturing buffer and digested with either PNGase F or EndoH, resolved by 12.5% SDS-PAGE, transferred to nitrocellulose, and used for immunoblotting with the indicated antibodies.</p

Visualization of HA-Srt protein by site-specific sortase labeling followed by streptavidin-Qdot staining.

<p>(<b>A</b>) <b>Budding of Qdot labeled virus analyzed via flow cytometry.</b> Confluent MDCK cells or CD154/CD40L expressing MDCK cells were infected with HA-Srt or Wild-type virus at an MOI = 1 for 4 hrs. Cell surface accessible HA-Srt or CD154 was labeled for 30 minutes with biotin using 100 µM sortase and 250 µM biotin probe. Biotin labeled surface molecules were further labeled via a 5 minute incubation with 20 nM Qdot655 or 10 nM Qdot655 in the case of uninfected CD154/CD40L expressing MDCK cells. Total fluorescent intensity of the cells was measure via flow cytometry at the indicated timepoints. (<b>B</b>) <b>HA-Srt colocalizes with lipid rafts at the cell surface.</b> Confluent layers of MDCK cells were infected at an MOI of 0.35–0.5 for 4.5 hours. Cell surface molecules of infected MDCK cells or CD154/CD40L expressing MDCK cells were labeled with 100 µM sortase and 250 µM biotin probe for 30 minutes. Biotin labeled HA-Srt of CD154 were further labeled with 20 nm and 10 nm Qdot655 respectively and lipid rafts were stained with 20 µg/ml CT-B conjugated to Alexa fluor 594. Images were acquired immediately after staining. Scale bars = 5 µm.</p

Generation of sortase compatible influenza viruses.

<p>(<b>A</b>) <b>Structural representation of sortase cleavage site mutations in hemagglutinin and neuraminidase.</b> Sortase cleavage sites including residues that remain with the labeled species (green) and those lost after sortase-medaited transpeptidation (red) were engineered into the indicated positions in hemagglutinin (left) and neuraminidase (right). Cartoons of crystal structures are depicted on top (PDB ID for HA: 1HA0, NA: 2HTY). (<b>B</b>) <b>NA-Srt multistep replication assay.</b> NA-Srt virus and wild-type WSN virus (n = 3) were used to infect MDBK monolayers at an MOI = 0.001 and viral supernatant was plaqued on MDCK cells at the indicated times. (<b>C</b>) <b>HA-Srt multistep replication assay</b> HA-Srt and wild-type WSN virus (n = 3) were used to infected MDCK monolayers at an MOI = 0.001 and viral supernatant was analyzed via standard hemaggutination assays. Shown are the last dilution factors at which hemagglutination of red blood cells still occurs. (<b>D</b>) <b>Infectivity of engineered viruses.</b> Mice (n = 4 in each group) were inoculated with 40000 pfu of the indicated virus and body weight was monitored at the indicated intervals. Shown is the mean and SEM.</p