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. 2003 Jul;77(13):7214-24.
doi: 10.1128/jvi.77.13.7214-7224.2003.

The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function

James S Gibbs  1 , Daniela Malide, Felicita Hornung, Jack R Bennink, Jonathan W Yewdell
Affiliations

The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function

James S Gibbs et al. J Virol. 2003 Jul.

Abstract

The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.

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Figures

FIG. 1.
FIG. 1.
PB1-PB1-F2 sequence comparison and structural predictions. (A) The sequence of PB1-F2 is shown along with structural predictions (H, helix; E, extended) by PHD and APSSP programs with respect to helix formation. (B) Helical wheel representation of a predicted amphipathic helix formed by residues 72 to 82 of PB1-F2. (C) Sequences of PB1-F2 representing all IAV subtypes for which sequences are available. Residues present in all 90 IAV strains examined are highlighted in red; those conserved in >90% of the strains are highlighted in yellow. Strains are indicated as follows: A, Puerto Rico/8/34 (H1N1); B, FM/1/47 (H1N1); C, Equine/London/1416/73 (H7N7); D, Chicken/Korea/38349-p96323/96 (H9N2); E, Goose/Guangdong/1/96 (H5N1); F, Duck/Nanchang/662/98 (H4N6); G, Leningrad/134/47/57 (H2N2); H, Gull/Maryland/704/77 (H13N6); I, Teal/Hong Kong/W312/97 (H6N1); J, NT/60/68 (H3N2).
FIG. 2.
FIG. 2.
Chimeric PB1-F2-EGFP fusion proteins utilized in this study. The residues incorporated in fusion proteins are shown on the left. Green dots signify EGFP. Dark blue segments represent predicted helices. Red segments represent substituted residues. All fusion proteins contained a 7-residue linker (RDPPVAT) between the PB1-F2 sequence and EGFP. The intracellular localization of the fusion proteins is summarized on the right as follows: M, mitochondrial; C, cytoplasmic and/or nuclear; NM, nonmitochondrial.
FIG. 3.
FIG. 3.
Intracellular targeting of PB1-F2-EGFP fusion constructs. HeLa cells were labeled with the ΔΨm-sensitive mitochondrial dye TMRE, and images were acquired live at 16 h posttransfection with plasmids expressing PB1-F2-EGFP fusion proteins as indicated. Fluorescence images are presented as unmerged EGFP (left column), TMRE (middle column), and merged fluorescence (right column; EGFP is shown in green, and TMRE is shown in red). The full-length (1 to 87) chimeric protein (A to C) localizes to mitochondria (arrowheads) but also to the nucleus, nuclear membrane, and cytoplasm (double arrowheads). A deletion mutant (1 to 72) lacking the last 15 amino acids (D to F) displays uniform cytoplasmic and nuclear localization and is clearly absent from mitochondria. Among all the chimeric proteins, that consisting of PB1-F2 residues 65 to 87 exhibits the most complete mitochondrial localization (G to I). Replacing the 5 positively charged residues in the MTS of the full-length construct with Ala (1-87-5A) targeted the fusion protein to the nuclear membrane and another cytoplasmic structure (J to L). Replacing the MTS with the MTS of p13II in the full-length construct (1-71-p13 h-EGFP) targeted the fusion protein to mitochondria (M to O). Bars, 10 μm.
FIG. 4.
FIG. 4.
FRET analysis of 65-87-EGFP-TMRE localization. (A) Using a Leica AOBS-SP2 confocal system, EGFP and TMRE emission spectra were recorded by wavelength scanning (lambda scan) between 500 and 660 nm with a 5-nm detection window. Fluorescence intensity plots show significant overlap between EGFP emission spectra and TMRE excitation spectra (green filled histogram) and sufficient separation between the TMRE excitation and emission spectra. (B) Spot bleaching (area marked in red) of the acceptor was performed as described in Material and Methods. (C) The ratio images of donor-acceptor fluorescence before (upper panel) and after (lower panel) photobleaching are indicated together with the look-up table on the left side of the figure. Notice that the only change is in the region of photobleaching, which demonstrates a large increase in donor signal. (D) Confocal images of the donor acquired using a 488-nm excitation line and emission between 500 and 550 nm obtained pre- and postbleaching reveal dequenching of the donor. Using a 568-nm excitation line and emission between 580 and 620 nm, sequential scan images of the acceptor were also acquired pre- and postbleaching; these reveal effective bleaching of the acceptor. (E) These panels depict the average pixel intensity profiles over time of the bleached region, with the images pre- and postbleaching marked by arrows. Notice the increase in intensity of EGFP (donor; upper graph) signal that occurs concomitantly with the bleaching-induced decrease in the TMRE (acceptor; bottom graph) signal.
FIG. 5.
FIG. 5.
Effect of PB1-F2 MTS-targeted protein on ΔΨm cells as detected by flow cytometry. The results of analysis of transiently transfected are shown. At 16 h posttransfection, HeLa cells were incubated with 50 nM TMRE for 30 min and then analyzed for EGFP expression (x axes) and TMRE accumulation (y axes). Control, mock-transfected cells. CCCP was added to cells to demonstrate that TMRE accumulation was based on maintenance of ΔΨm.
FIG. 6.
FIG. 6.
Effect of PB1-F2 MTS-targeted protein on ΔΨm-microscopy. HeLa cells expressing PB1-F2 65-87-EGFP at 16 h posttransfection were labeled with TMRE, and images were acquired. Images were collected at one frame per 10 s. Selected frames are shown side by side (green, EGFP; red, TMRE) with the cumulative time elapsed (in minutes and seconds) indicated in the upper left corner of each panel. Note the rapid decrease in the mitochondrial TMRE fluorescence (red) of one transfected cell (indicated with an arrow). The entire time-lapse video is provided as supplemental material at http://www.niaid.nih.gov/dir/labs/lvd/bennink.htm.
FIG. 7.
FIG. 7.
Visualization of cell death of PB1-F2 65-87-EGFP-expressing cell. (Top panels) Live HeLa cells expressing PB1-F2 65-87 EGFP at 16 h posttransfection were labeled with TMRE, and images were acquired at 37°C in the presence of annexin V (AnV)-Alexa 647 and PI. (A) A merged image of EGFP (green) and annexin V (cyan) fluorescence shows annexin labeling outlining the periphery of five transfected cells. The vesicular staining probably represents endosomes with internalized annexin V. EGFP fluorescence remained associated with mitochondria in most of the annexin-positive cells. (B) A merged image of TMRE (red) and PI (red) and annexin V (cyan) fluorescence shows a marked decrease of TMRE signal (decreased ΔΨm) in the annexin V-positive cells, while these cells excluded PI from the nucleus, demonstrating the integrity of the plasma membrane (with the same imaging settings, dead cells in other fields exhibited bright nuclear staining). (C) A merged image of differential interference contrast (DIC) in grey and annexin V (cyan) fluorescence shows clear plasma membrane blebbing (arrow) of one of the annexin V-positive cells. Bar, 10 μm. (Bottom panel) HeLa cells at 16 h posttransfection were labeled with TMRE (red) and the cell permeant DNA stain Hoechst 33342 (blue) and imaged in the presence of annexin V-Alexa (cyan) and PI (red) at 15-min intervals up to 2 h. (D) A merged image of a representative field image acquired at 15 min shows the decrease in size of the nuclei due to chromatin condensation in annexin-positive cells. Bar, 10 μm.
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