BLAST searches between the sequence of PB1-F2 encoded by PR8 and non-influenza virus sequences deposited in GenBank failed to reveal any significant sequence similarities. Lacking such clues regarding PB1-F2 structure, we turned to the software programs PHD (13
) and APSSP (http://imtech.ernet.in/raghava/apssp
) to predict regions of secondary structure (Fig. ). Both programs strongly predict α-helical regions between amino acids 54 to 62 and 73 to 82. The predicted helix at amino acids 73 to 82 was found to contain an 11-amino-acid sequence with obvious similarity to the amphipathic α-helix of the human T-cell leukemia virus type 1 (HTLV-I) p13II
protein, which is responsible for mitochondrial targeting of this protein (5
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 (more ...)
Specifically, LKTRVLKRWRL of PB1-F2 and LRVWRLCARRL of HTLV-I p13II
both have three Leu residues and are rich in basic amino acids, possessing five and four basic residues, respectively. A helical wheel representation (created using the program GCG Lite [Accelrys, San Diego, Calif.]) demonstrates the potential of PB1-F2 to form amphipathic helices (Fig. ). Two other retroviruses encode small proteins with an MTS that are likely to form amphipathic helices: one is the bovine leukemia virus (like HTLV-I, a deltaretrovirus), which encodes G4 protein (10
), and the other is human immunodeficiency virus 1, which encodes viral protein R (VPR) (9
Using the AlignX component of Vector NTI Suite (Informax, Inc., Bethesda, Md.), we aligned the amino acid sequences of predicted PB1-F2 gene products from 74 IAV isolates as a further method of gauging potential functional activities of PB1-F2 domains. Representative sequences encoded by PB1-F2 genes from different IAV subtypes are shown in Fig. . Amino acids that are completely conserved among all 74 isolates are colored red, and those conserved in more than 90% of isolates are colored yellow. Notably, residues within the predicted amphipathic helix are highly conserved, with two Leu residues being among only 10 residues that are completely conserved among the 74 sequences. The basic residues are also highly conserved and, with only a few exceptions, are replaced by other basic residues.
This sequence conservation is consistent with the proposed helical structure for this region, but it is not conclusive, particularly since PB1-F2 overlaps with the PB1 open reading frame, so conserved residues in PB1 may restrict the latitude of acceptable substitutions in PB1-F2. On the other hand, more than 85% of residues in PB1-F2 differ between influenza virus isolates and the protein is much more variable than the overlapping region of PB1.
Plasmid-mediated expression of PB1-F2-EGFP fusion proteins.
To map the MTS of PB1-F2, we generated a panel of cDNAs encoding amino- and carboxy-terminal deletion mutations appended to EGFP as a reporter protein (Fig. ). This approach has been used successfully to identify the MTSs present in retroviral proteins (5
). These constructs were designed to encompass or omit potential secondary structural elements at amino acids 42 to 50, 52 to 64, 67 to 72, and 73 to 83. Using the cytomegalovirus early promoter to control expression of the fusion proteins, proteins were expressed by plasmid-mediated transfection.
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 (more ...)
Transfection of the control plasmid pEGFP, expressing EGFP alone, results in a diffuse green fluorescence in the cytoplasm and nucleus of HeLa cells. This reflects the lack of trafficking signals in EGFP in conjunction with its compact structure, which enables it to freely traverse nuclear pores. In cells expressing full-length PB1-F2 fused with EGFP (1-87-EGFP), by contrast, fluorescence was primarily localized to filamentous cytoplasmic structures that in number and appearance are likely to represent mitochondria (Fig. ). This was confirmed by staining cells with TMRE, which is an inner mitochondrial membrane potential (ΔΨm)-sensitive dye (Fig. and C). Constructs expressing PB1-F2 amino acids 65 to 87 (Fig. to I), 42 to 87, and 52 to 87 also exhibited mitochondrial localization (data not shown), whereas constructs expressing PB1-F2 amino acids 1 to 72 (Fig. to F), 1 to 51, 1 to 41, 73 to 87, and 52 to 72 exhibited EGFP-like diffuse cytoplasmic and/or nuclear fluorescence, demonstrating the loss of the MTS (data not shown).
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 (more ...)
Among the fusion proteins that localized to mitochondria, there was considerable variation in the degree of mitochondrial localization. All of the fusion proteins that localized to the mitochondria, including 1-87-EGFP, were also present in the nucleus, nuclear membrane, and cytoplasm of a significant fraction of cells. While this is similar to the intracellular localization of PB1-F2 in IAV-infected cells, it differs from PB1-F2 expressed via transfection or a recombinant vaccinia virus, which is much more uniformly distributed in a mitochondrial pattern (4
). It appears, therefore, that fusion with EGFP or interaction with other influenza virus proteins disfavors to a degree the intrinsic MTS in PB1-F2. One thing that is clear, however, is that 65-87-EGFP exhibits an extent of mitochondrial localization superior to 1-87-EGFP or any other construct tested.
Taken together, these results imply that the predicted amphipathic α-helix identified as potentially similar to that of HTLV-I p13II functions as the PB1-F2 MTS. Specifically, amino acids 65 to 87 are sufficient for mitochondrial localization, whereas residues 1 to 72 and 73 to 87 are insufficient for mitochondrial localization. The failure of residues 73 to 87 (which contain the predicted amphipathic helix) to function as an MTS suggested that the short hydrophobic sequence upstream is also important for proper targeting. We decided to explore in detail the contribution of the preceding hydrophobic residues to the targeting.
Fine mapping of the MTS.
Working on the basis of these results, we generated plasmids that express PB1-F2 amino acids 69 to 85, 72 to 82, 69 to 82, or 72 to 85 of PB1-F2 fused at the N terminus of EGFP. Of these partial PB1-F2 gene products, sequences spanning amino acids 69 to 85 (LVFLKTRVLKRWRLFSK) and 69 to 82 (LVFLKTRVLKRWRL) alone targeted EGFP to mitochondria, albeit less efficiently than the sequence spanning amino acids 65 to 87, which demonstrated the most complete mitochondrial targeting of all of the sequences tested (all of the localization data are summarized in Fig. ). Deletion of the three amino terminal residues from 69 to 85 or 69 to 82 completely abolished mitochondrial targeting. Notably, despite the apparently important role of these residues in mitochondrial targeting, various IAV isolates demonstrate nonconservative substitutions at residues 69 to 71, suggesting that a variety of residues can support mitochondrial targeting, though the mitochondrial localization of PB1-F2 from these isolates remains to be established.
We next examined the effect of replacement of basic residues with neutral residues in the predicted amphipathic helix. Replacement of the two Lys residues with Ala reduced MTS function. MTS function was completely abolished by substituting Ala for Lys or Arg at positions 73, 75, 78, 79, and 81 in 1-87-EGFP (construct 1-87 5A-EGFP in Fig. and Fig. to L). Instead, the fusion protein localized to the nuclear membrane and condensed cytoplasmic structures of an uncertain nature. Based on the two predictive programs, these substitutions should not interfere with helix formation, which suggests that mitochondrial targeting requires an amphipathic α-helix and not simply an α-helix. These results imply that the five positively charged residues are required for mitochondrial targeting. Replacement of highly conserved Leu residues at positions 72 and 77 with Ile or Ala did not abolish mitochondrial localization.
Finally, we replaced amino acids 72 through 87 in 1-87-EGFP with the 11-residue MTS from HTLV-I p13II
, LRVWRLCARRL (Fig. M to O). The fusion protein localized to mitochondria, demonstrating the functional interchangeability of the two sequences for mitochondrial targeting. Since there is structural evidence that the p13II
forms an amphipathic helix (6
), this provides additional evidence that the analogous sequence in PB1-F2 behaves similarly.
Localization of PB1-F2 65-87-EGFP within mitochondria.
Having established that residues 65 to 87 serve as an MTS, it was of interest to further characterize the location of 65-87-EGFP within mitochondria. Taking advantage of the autofluorescence of EGFP and the known location of TMRE in the mitochondrial matrix, we used FRET to determine whether EGFP is in close proximity to TMRE. When fluorescent molecules are intimately associated (less than 10 nm apart), the fluorescent emissions of donor fluors can be captured by acceptor fluors with relatively high efficiency. When this occurs, the intensity of emission detected from the donor fluor is decreased, a process known as quenching. This occurrence can be detected by photodestruction (photobleaching) of the acceptor fluor, which results in increased emission detected from the donor fluor. EGFP and TMRE constitute a well-matched FRET pair, since there is considerable overlap between the EGFP emission and TMRE excitation spectra (Fig. ).
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 (more ...)
To establish whether TMRE quenches 65-87-EGFP, we examined the effect of photobleaching TMRE on the intensity of EGFP fluorescence. Photobleaching of TMRE results in an immediate increase in the EGFP fluorescence of 65-87-EGFP (Fig. to D), which is indicative of close spatial proximity of the two fluors. The measured FRET efficiency of 40% is consistent with a distance of less than 50 Å between EGFP and TMRE. Importantly, we observed no FRET between the TMRE and 1-72-EGFP which was present in the nucleus and/or cytoplasm or enhanced yellow fluorescent protein (EYFP) targeted to the mitochondrial matrix by addition of the MTS of subunit VIII of human cytochrome c oxidase (note that EYFP is an equally suitable FRET partner for TMRE) (data not shown).
In conjunction with previous evidence from immunoelectron microscopy that PB1-F2 localizes to the inner mitochondrial membrane (4
), these data are consistent with the idea that the PB1-F2 MTS targets a substantial fraction of the EGFP fusion protein to the inner mitochondrial membrane in a manner such that the GFP domain is present in the matrix. The enhanced interaction of 65-87-EGFP with TMRE relative to matrix-targeted EYFP might be explained by a higher effective TMRE concentration at the surface of the inner mitochondrial membrane than in the matrix (15
) (L. M. Loew, personal communication).
Effect of the PB1-F2 MTS on mitochondrial function.
We previously reported that the presence of PB1-F2 in mitochondria of IAV-infected or PB1-F2-transfected or -microinjected cells is associated with mitochondrial rounding, swelling, and fragmentation (4
). This was usually accompanied by a loss of mitochondrial potential, as indicated by a decreased accumulation of ΔΨm
-sensitive dyes such as MitoTracker Red. We examined the effect of expression of the chimeric fusion proteins on mitochondrial potential in transfected HeLa cells, as determined by TMRE fluorescence. In the course of examining the patterns of localization of various EGFP-tagged fusion proteins we observed a decrease of the mitochondrial potential of some cells, as indicated by low levels of TMRE staining (Fig. ). We explored this finding in a quantitative manner by both flow cytometry and confocal microscopy.
Via flow cytometry, we found that TMRE accumulation in HeLa cells depends on functional mitochondria, as shown by loss of staining following the collapsing of ΔΨm with the protonophore CCCP (Fig. , bottom right panel). Transient expression of 65-87 EGFP was associated with a decrease in TMRE staining that was related to the level of GFP expression; i.e., decreased TMRE staining correlated with high levels of GFP fluorescence (Fig. ). By contrast, cells expressing 1-87-EGFP (top left panel), 1-72-EGFP (center left panel), or EGFP (top right panel) failed to demonstrate such a relationship.
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 (more ...)
The effects of fusion protein expression on mitochondrial function were confirmed by acquisition of laser-scanning confocal microscopy images of randomly chosen fields of transfected cells grown on coverslips and by determination of the fraction of cells exhibiting low levels of TMRE staining (at least 100 cells were counted for each construct examined). Of the untransfected HeLa cells, 23% spontaneously exhibited low levels of TMRE under these conditions. Expression of 1-87-EGFP resulted in a marginal increase in the number of TMRE cells staining at low levels (33%) over the values obtained with EGFP or 1-72-EGFP (27% of cells were low staining). By contrast, fully 60% of 65-87-EGFP-expressing cells demonstrated low levels of TMRE staining.
These data indicate that in the context of a fusion protein, the PB1-F2 MTS compromises mitochondrial function. We further investigated the effect of 65-87-EGFP on mitochondrial function by time-lapse microscopy. As seen in Fig. , total loss of ΔΨm in a 65-87-EGFP-transfected cell occurred within a span of 2 to 3 min. This seems to be typical, since we observed similar kinetics of loss of TMRE staining in five additional time-lapse movies. Loss of mitochondrial potential eventually leads to cell death which exhibits many of the hallmarks of apoptosis, such as phosphatidylserine exposure on the outer leaflet of the plasma membrane (as determined by annexin V staining), while maintaining membrane integrity (as determined by excluding PI) (Fig. ).
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 (more ...)
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 (more ...)
The behavior of stably transfected cells expressing PB1-F2-EGFP fusion proteins is consistent with this conclusion. Although it is possible to create cell lines expressing 65-87-EGFP, cells exhibit progressive loss of gene expression. In addition, cells expressing 65-87-EGFP at high levels demonstrate a marked tendency toward loss of TMRE staining and cell death compared to surrounding cells expressing lower levels of protein (data not shown).
Taken together, these findings demonstrate that the PB1-F2 MTS can induce mitochondrial dysfunction and cell death in the context of a chimeric protein. This is not strictly related to the amount of the chimeric protein present in mitochondria, as cells expressing smaller amounts of protein are often observed to die surrounded by cells which remain viable and express larger amounts of protein.