INTRODUCTION

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Open reading frame 6 of the E4 region (E4orf6) of the group C adenoviruses (Ad) encodes a multifunctional protein that promotes virus replication and acts as an oncoprotein (reviewed in reference 34). As part of a complex with the E1B 55-kDa protein, the E4orf6 protein promotes the export of late viral mRNA from the nucleus to the cytoplasm and concomitantly inhibits the transport of most host cell mRNA (4, 35, 46). In addition, the E4orf6 protein enhances viral DNA replication, late protein synthesis, and the stability of unprocessed late viral RNA in the nucleus (3, 4, 24). Furthermore, the E4orf6 protein, as well as the product of the E4 open reading frame 3 (E4orf3), stimulates the accumulation of viral mRNA containing the tripartite leader (38) and affects splicing patterns of late viral RNA (41, 42). Finally, the E4orf6 protein has recently been shown to act as an oncoprotein by binding the tumor suppressor p53 and inhibiting its interaction with the transcriptional initiation complex (10, 47).

Genetic analyses have demonstrated that the E4orf6 and E4orf3 proteins share partially redundant and overlapping functions during lytic infection. Ad mutants that fail to express the E4orf6 protein show a modest reduction in virus yield and plaquing efficiency compared to the wild-type virus (4, 24). By contrast, mutant Ads that lack the entire E4 region or fail to express the E4orf6 and E4orf3 proteins are severely restricted for growth (4, 24, 26). However, E4 deletion mutants that express the E4orf6 protein in the absence of all other E4-encoded proteins replicate to near-wild-type levels (1, 24). Replication of a complete E4 deletion virus could be restored by transfecting the mutant-virus-infected cells with a cDNA expressing the E4orf6 protein (32).

A portion of the E4orf6 protein associates with the E1B 55-kDa protein in the cell. Physical evidence for this complex derives from the ability of E1B 55-kDa-specific antibodies to indirectly immunoprecipitate the E4orf6 protein from Ad-infected cells (46, 48, 49), after coexpression of the proteins by transfection (21), and from cellular lysates reconstituted in vitro (49). Immunofluorescence and electron microscopy have demonstrated that the E4orf6-E1B 55-kDa protein complex localizes to the peripheries of the sites of viral transcription and RNA processing within the nucleus of Ad type 5 (Ad5)-infected cells (43). When transiently expressed, the E4orf6 protein localizes to the nucleus and directs the E1B 55-kDa protein to the nucleus. In the absence of the E4orf6 protein, the E1B 55-kDa protein is retained in the cytoplasm (21). In addition, expression of the E4orf6 protein relieved the apparent cytoplasmic retention of the E1B 55-kDa protein in primate cells but not in mouse or rat cells (21). Thus, a primate-specific cellular factor may be required for the E4orf6 protein-directed nuclear localization of the E1B 55-kDa protein. It has been proposed that the E4orf6-E1B 55-kDa protein complex modulates mRNA transport by sequestering a cellular factor required for mRNA export, thus inhibiting host mRNA export and directing this factor to the sites of viral transcription, where it is available for viral mRNA export (43). It is also possible that such a factor enables shuttling of the E4orf6-E1B 55-kDa protein complex as part of the mechanism of mRNA transport control (9). The identity of this hypothetical cellular factor is unknown, but recent studies have demonstrated that overexpression of a cellular protein related to hnRNP-U/SAF-A in Ad-infected cells overcomes the inhibition of host cytoplasmic mRNA accumulation, implicating this cellular protein as a potential factor used by the E4orf6-E1B 55-kDa protein complex (18).

The structural features of the Ad E4orf6-E1B 55-kDa protein interaction have been partially elucidated (48). The association of the E4orf6 protein with E1B 55-kDa proteins bearing small, in-frame insertions was measured by an immunoprecipitation assay. This analysis suggested that the integrity of two distinct segments of the E1B 55-kDa protein are required for association with the E4orf6 protein in vivo. The portions of the E1B 55-kDa protein important for the interaction with the E4orf6 protein map to amino acid 143 and the region between amino acids 262 and 326 (48). The investigators also showed that the amino terminus of the E4orf6 protein, expressed in bacteria as a translational fusion to glutathione S-transferase, could bind the E1B 55-kDa protein in a protein blot assay. This study provided additional evidence for an interaction between the amino terminus of the E4orf6 protein and the E1B 55-kDa protein by using 293 cells transfected with an E4orf6/7 cDNA. The E4orf6/7 protein is composed of the amino-terminal 58 residues of E4orf6 and 92 residues encoded by E4orf7. A portion of the E4orf6/7 protein expressed by transfection was indirectly recovered upon immunoprecipitation of the endogenous E1B 55-kDa protein, suggesting that the amino-terminal 58 residues of E4orf6 mediated binding between the two proteins. However, this result stands in contrast to prior results that failed to reveal an interaction between the E4orf6/7 protein and the E1B 55-kDa protein in Ad-infected cells (8, 24). The significance of an interaction between the E1B 55-kDa protein and the amino terminus of the E4orf6 protein is unclear at this time because the Ad E4orf6/7 mutant virus is essentially wild type in growth and the E4orf6/7 protein cannot replace the function of the E4orf6 protein during Ad infection.

Here we examine the structural elements of the E4orf6 protein required for the functional interaction with the E1B 55-kDa protein by analyzing intracellular localization of the viral proteins and virus replication. We show that a domain in the E4orf6 protein near the carboxy terminus is required for the nuclear colocalization of the E1B 55-kDa protein. This domain is an arginine-faced, amphipathic  helix, and the integrity of this domain in the E4orf6 protein is required for E1B 55-kDa protein nuclear colocalization. Disruption of the E4orf6 arginine-faced, amphipathic  helix not only abolishes E1B 55-kDa protein nuclear colocalization but also disrupts the function of the E4orf6 protein in an Ad infection. These findings suggest that the arginine-faced  helix near the carboxy terminus of the E4orf6 protein is essential for the function of the E4orf6 protein during a productive viral infection.

	MATERIALS AND METHODS

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Cell culture and viruses. Cell culture media, cell culture supplements, and serum were obtained from Life Technologies (Gaithersburg, Md.) through the Tissue Culture Core Laboratory of the Comprehensive Cancer Center of Wake Forest University. HeLa (CCL2.2) and W162 cells (53) were maintained in Dulbecco modified Eagle's minimal medium supplemented with 10% newborn calf serum as previously described (20).

Plasmids and site-directed mutagenesis. The plasmids carrying the E4orf6 and E1B 55-kDa genes were previously described (21). The cDNA encoding the E4orf6/7 protein was originally described by Freyer et al. (16). The coding region was subcloned from this construct by standard methods with PCR and placed under control of the T7 promoter in a pGEM plasmid (Promega, Madison, Wis.). The E4orf6 carboxy-terminal-truncation mutations were constructed in a pGEM plasmid (Promega) by removal of an appropriate restriction fragment encompassing the carboxy terminus of the E4orf6 protein. The mutations were confirmed by DNA sequencing.

Indirect immunofluorescence. Indirect immunofluorescence and photomicroscopy of whole cells was conducted as previously described (43). Double-label immunofluorescence was performed with the mouse monoclonal antibody (MAb) 3 (38) specific for the amino terminus of the E4orf6 protein and the rat MAb 9C10 (Oncogene Science, Uniondale, N.Y. [54]) specific for the E1B 55-kDa protein. The secondary antibodies (Jackson ImmunoResearch, West Grove, Pa.) were multiple-label-qualified goat antibodies conjugated to dichlorotriazinylamino fluorescein and lissamine rhodamine sulfonyl chloride. Samples were examined with a Leitz Dialux 20 EB microscope fitted for epifluorescent illumination and photographed with TMax film (Eastman Kodak, Rochester, N.Y.) developed to an exposure index of 1600 ASA. Prints were prepared on high-contrast (no. 5) paper (Eastman Kodak), scanned at 300 dpi, and then cropped and assembled with Canvas 5.0 software (Deneba, Miami, Fla.) operating on a Macintosh microcomputer.

Protein structure. Peptides (E4orf6 peptide [NH₂-ARRTRRLMLRAVRIIAE-COOH] and L₂₄₅P peptide [NH₂-ARRTRRPMLRAVRIIAE-COOH]) were synthesized on an Applied Biosystems model 430A automated peptide synthesizer, and the amino and carboxy ends were left unblocked. Peptide concentrations were determined by quantitative amino acid analysis. Each peptide (200 µl) at a concentration (c) of 200 µM in 50 mM Na₂PO₄-150 mM NaCl (pH 7.0) in 0 to 80% trifluoroethanol (TFE) was dispensed in a stoppered optical cell with a path length (l) of 0.05 cm. The temperature of the sample was maintained at 25°C by circulation of water through a jacket surrounding the cell. Spectra were obtained on a Jasco 600 spectrophotometer by taking readings every 1 nm with a bandwidth of 1 nm. An average of four independent scans were baseline corrected and smoothed by using a third-order least-squared polynomial. Mean residue molar ellipticity (^[]mean) in units of degrees times square centimeters per mole was calculated from the equation ^[]mean = ^[]observed × 1,000 × MRW/(10 × l × c). ^[]observed is the observed ellipticity in millidegrees, and MRW is the mean residue weight of the peptide. Structural and sequence analysis was performed with the assistance of the Wisconsin Package version 9.1 software (Genetics Computer Group, Madison, Wis.).

Complementation analysis and protein expression. HeLa cells were infected with dl309 (30) or dl1014 (3) and simultaneously transfected with the E4orf6 variant cDNA constructs (see Fig. 7 for analysis). For these experiments, 4 × 10⁵ cells in a 65-mm-diameter dish were exposed to 1 µg of plasmid DNA, 3 µg of Lipofectin, and 4 × 10⁶ PFU of virus in a 2-ml volume of OptiMEM (Life Technologies). After 6 h at 37°C, the virus and plasmid mixture was replaced with normal growth medium and the cells were returned to normal growth conditions. After 48 h, the cells were collected, resuspended in urea sample buffer (7.5 M urea, 50 mM Tris [pH 6.8], 1% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, 5% 2-mercaptoethanol, 0.05% bromophenyl blue) and heated for 10 min at 65°C. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to nitrocellulose, and the E4orf6-related proteins were visualized on an E4orf6 Western blot with MAb 3 and chemiluminescence (Pierce, Rockford, Ill.). Replicate cultures of infected and transfected cells were harvested at 48 h postinfection to quantify virus yields. Detailed methods for Ad plaque assays have been described elsewhere (29). In brief, virus was harvested from HeLa cells by multiple cycles of freezing and thawing. The cell lysates were clarified by centrifugation and serially diluted for infection of W162 cells grown in six-well tissue culture dishes for plaque assays. After incubation with diluted lysates for 1 h, the W162 cells were overlaid with 0.7% SeaKem ME agarose (FMC BioProducts, Rockland, Maine) in Dulbecco modified Eagle's minimal medium supplemented with 0.75% sodium bicarbonate and 4% newborn calf serum. The cells were overlaid with additional agarose in growth medium on the third and sixth day after infection. Plaques were visualized by staining with neutral red in an agarose overlay on the ninth day. Typically, data were collected from three dilutions in each series of dilutions. The virus yield was determined by linear regression and expressed as the number of plaques per milliliter of initial lysate. Replicate samples were compared with the two-tailed Student's t test, where a probability of <0.05 was considered to represent a significant difference.

	RESULTS

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The carboxy terminus of the E4orf6 protein is required for E1B 55-kDa protein nuclear colocalization. In Ad-infected cells, the E4orf6 protein directs the E1B 55-kDa protein to the peripheries of the viral transcription centers within the nucleus (43). Although the E1B 55-kDa protein is restricted to the cytoplasm when transiently expressed, it is directed to the nucleus when coexpressed with the E4orf6 protein (21). To determine if a discrete region of the E4orf6 protein is required for the nuclear localization of the E1B 55-kDa protein, a series of carboxy-terminal-truncation variants was created in E4orf6 cDNA. The E4orf6 sequences of these truncation variants are represented in Fig. 1, as well as the amino acids derived from vector sequences that are expressed in these variants, the E4orf7 sequence for the E4orf6/7 splice variant, and the MAb 3 (38) antibody recognition site, which is preserved in each of these constructs. These truncation variants were coexpressed with the E1B 55-kDa protein in HeLa cells with the vaccinia virus/T7 RNA polymerase infection-transfection expression system (17). The E4orf6 truncation variants (Fig. 2, left column) and the E1B 55-kDa protein (Fig. 2, center column) were visualized by indirect immunofluorescence. The nucleus was visualized by staining DNA with DAPI (4',6-diamidino-2-phenylindole) (Fig. 2, right column). Unless otherwise noted, typical staining patterns are represented in Fig. 2 and 6.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the carboxy-terminal-truncation variants of the E4orf6 protein and E4orf6/7 protein. The 294-residue E4orf6 protein is represented by the bar at the top of the figure. The solid portion of the bar represents the postulated extent of the arginine-faced, amphipathic  helix described in this report. Truncation variants are named according to the number of E4orf6-derived amino acids included in the variant, where the open bar represents E4orf6 amino acids. Amino acids encoded by vector sequences are represented by shading. Amino acids encoded by E4orf7 are indicated by cross-hatching. The solid black line over residues 16 through 22 indicates the epitope recognized by the E4orf6-specific antibody, MAb 3.

View larger version (68K): [in this window] [in a new window]   FIG. 2.   A region near the carboxy terminus of the E4orf6 protein is required to direct the E1B 55-kDa protein to the nucleus. HeLa cells were infected with the recombinant vaccinia virus vTF7.3 to establish expression of the T7 RNA polymerase and then transfected with cDNA under control of the T7 promoter encoding an E4orf6-related protein (indicated on the left) and the E1B 55-kDa protein. The Ad proteins were visualized by double-label immunofluorescence at 12 to 14 h posttransfection, and representative cells are shown except as noted in the text. E4orf6 proteins were visualized with the mouse MAb 3 (left column; E4), E1B 55-kDa protein was visualized with the rat MAb 9C10 (center column; E1B), and DNA was visualized with DAPI (right column; DNA). The asterisks in panels D, E, and F identify cells that express both the N276 E4orf6 variant and the E1B 55-kDa protein; the other cell in each field expresses only the N276 protein. The arrowheads in panels M, N, and O identify cells displaying the typical localization seen in approximately 90% of cells expressing the N108 E4orf6 protein.

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Carboxy-terminal truncation variants of the E4orf6 protein are expressed to comparable levels in HeLa cells. The E1B 55-kDa protein and E4orf6 variant proteins depicted in Fig. 1 were expressed by the vaccinia virus/T7 RNA polymerase system as described in the legend to Fig. 2. Total cell protein was isolated 14 h post-infection-transfection, separated by SDS-PAGE, and transferred to a solid support. The E4orf6-related protein was visualized by immunoblotting with MAb 3 (38). The approximate positions of molecular mass standards are indicated in kilodaltons on the left.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   The region of the E4orf6 protein required to direct E1B 55-kDa protein to the nucleus is predicted to contain an arginine-faced, amphipathic  helix. (A) The E4orf6 protein between amino acids 194 and 294 of the E4orf6 protein was analyzed for hydrophilicity by the method of Kyte and Doolittle (33), for surface probability by the method of Emini et al. (14), and for predicted  helices by the methods of Chou and Fasman (6) and Garnier et al. (19). Regions of striking hydrophilicity and high surface probability are highlighted by shading. The hatched region indicates predicted -helical segments that are analyzed further below. (B) The hydrophobic moment of the E4orf6 protein between amino acids 225 and 260 is shown for all possible angles of rotation. Contour lines are plotted for values of 0.35 and 0.45 as determined by the method of Finer-Moore and Stroud (15) with a window of 11 amino acids. The dashed lines flank the region of the plot corresponding to the angle of rotation associated with an  helix (100° ± 5°). (C) A helical-wheel presentation of amino acids 239 through 255 of E4orf6 depicts an amphipathic structure dominated by arginine residues on one face and hydrophobic residues on the other face. Charged amino acids are indicated by the charge at neutral pH, and hydrophobic amino acids are boxed.

An arginine-faced, amphipathic  helix exists within the E4orf6 protein. To test if the amino acids of the predicted E4orf6 -helical region can adopt an -helical secondary structure, a synthetic peptide corresponding to amino acids 239 to 255 of the E4orf6 protein was prepared and tested for secondary structure by circular dichroism (CD) spectroscopy. CD and proton magnetic resonance studies of peptides derived from proteins of known secondary structure show that the conformational preference of the peptides correlates well with the secondary structures of the respective regions in the proteins (11). In near-physiological conditions, the CD spectrum of the E4orf6 peptide resembles spectra correlated with the atypical conformation (Fig. 5A), a conformation previously identified as random coil, consisting of a large negative band around 200 nm and a peak or shoulder with a small negative value at 220 nm (23). This is the expected behavior for a peptide composed of over 50% hydrophobic residues (9 of 17) in an aqueous environment. TFE, an organic polar solvent, stabilizes -helical structure by strengthening intermolecular hydrogen bonding and fostering peptide-peptide dimerization of amphipathic peptides (28, 39). Although TFE-induced multimerization of the E4orf6 peptide (data not shown), the spectra of the peptide in 30% or less TFE were not characteristic of any known secondary structures (Fig. 5A). However, the E4orf6 peptide spectra obtained in 30% or less TFE intersect at an isobestic point at 209 nm, which is indicative of a biphasic secondary-structure transition. By contrast, in greater than 30% TFE, the CD spectra of the E4orf6 peptide resembled the spectra of an  helix containing a positive peak around 190 to 195 nm and two negative peaks at 208 and 222 nm (Fig. 5A) (23, 25). In addition, the E4orf6 peptide spectra in 30% or greater TFE cross at an isobestic point at 201 nm that is indicative of a second biphasic transition (Fig. 5A). Although the magnitudes of the minima at 208 and 222 nm exceed the theoretical values for a monomeric -helical peptide (5, 25), recent studies have demonstrated that multimers of amphipathic peptides produce CD spectra with minima at 222 and 208 nm comparable to those observed for the E4orf6 peptide in Fig. 5A (44, 52).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Peptides corresponding to the predicted arginine-faced, amphipathic  helix of the E4orf6 protein exhibit -helical spectra when analyzed by CD spectroscopy in the presence of TFE. CD spectra of the E4orf6 peptide (A) and L245P peptide (B) at a concentration of 200 µM in 50 mM Na2PO4-150 mM NaCl (pH 7.0) at 25°C in various concentrations of TFE. The measured CD values were converted to mean residue molar ellipticities, []mean, and plotted as a function of the incident wavelength. The arrowheads above the x axis identify the minima associated with an -helical spectrum. (C) Changes in []mean measured at 222 nm in increasing concentrations of TFE reveal a triphasic transition in the secondary structure of the E4orf6 peptide. The value for []mean measured at 222 nm is plotted on the y axis as a function of the percent TFE on the x axis.

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The integrity of an arginine-faced, amphipathic  helix is required for E1B 55-kDa protein nuclear localization. To test whether the arginine-faced, amphipathic  helix of the E4orf6 protein is important for the nuclear localization of the E1B 55-kDa protein, a small in-frame deletion and proline replacement or insertion mutations were introduced into this region (Table 1). These E4orf6 variants were then tested for their ability to direct the E1B 55-kDa protein to the nucleus. The E4orf6 variants were coexpressed with the E1B 55-kDa protein by the vaccinia virus/T7 RNA polymerase infection-transfection system (17), and the localization of the E4orf6 variants and the E1B 55-kDa protein was determined by indirect immunofluorescence. The variant bearing the internal deletion, 241-250, localized to the nucleus to the same extent as the wild-type E4orf6 protein (Fig. 6D) but failed to direct the E1B 55-kDa protein to the nucleus (Fig. 6E), thus supporting the importance of this region of the E4orf6 protein for nuclear localization of the E1B 55-kDa protein.

View this table: [in this window] [in a new window]   TABLE 1.   E4orf6 variants bearing mutations in the vicinity of the arginine faced, amphipathic  helixa

View larger version (78K): [in this window] [in a new window]   FIG. 6.   Disruption of the predicted arginine-faced, amphipathic  helix in the E4orf6 protein abolishes its ability to direct nuclear localization of the E1B 55-kDa protein. Coexpression of the E1B 55-kDa protein and the indicated E4orf6 protein variants (described in Table 1) was established, and the localization of the Ad proteins was determined as described in the legend to Fig. 2. E4orf6 proteins were visualized with the mouse MAb 3 (left column; E4), E1B 55-kDa protein was visualized with the rat MAb 9C10 (center column; E1B), and DNA was visualized with DAPI (right column; DNA). Representative cells are shown for all constructs analyzed.

Disrupting the ability of the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein abolishes E4orf6 function during a lytic Ad infection. Introduction of a proline residue into the predicted arginine-faced, amphipathic  helix of the E4orf6 protein not only reduced the -helical content of this region (Fig. 5) but also disrupted nuclear colocalization of the E1B 55-kDa protein (Fig. 6). To determine if the E4orf6 variants containing proline mutations could complement growth of the E4 mutant virus, dl1014, which expresses only the E4orf4 protein (32), HeLa cells were infected with dl1014 and simultaneously transfected with cDNAs expressing the E4orf6 proline variants. Forty-eight hours after infection, the amount of virus in these cells was quantified by a plaque assay with an E4-complementing cell line, W162 (53).

View larger version (8K): [in this window] [in a new window]   FIG. 7.   E4orf6 protein variants that retain the ability to direct the E1B 55-kDa protein to the nucleus rescue growth of an E4 deletion virus. HeLa cells were infected with the wild-type virus, dl309, or the E4 mutant virus lacking all but orf4, dl1014, at 10 PFU per cell and simultaneously transfected with cDNAs expressing the E4orf6-related constructs indicated at the left (Plasmid). The E4orf6-related proteins were expressed under the control of the major immediate-early promoter of CMV. (A) Virus was harvested after 48 h and quantified by plaque assay on the E4-complementing W162 cell line. The average amount of virus (expressed as PFU per milliliter) from three independent infections is shown with the standard deviations indicated. (B) In a parallel experiment, extracts of the infected and transfected cells were prepared at 48 h post-infection-transfection, and the proteins were separated by SDS-PAGE and transferred to a solid support. The E4orf6-related protein was visualized by immunoblotting with MAb 3 (38); the portion of the membrane containing the E4orf6 protein is shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we identified a structural element of the E4orf6 protein required for its functional interaction with the E1B 55-kDa protein in the cell and demonstrated that this element is required for productive Ad infection. This element is an arginine-faced, amphipathic  helix that is essential for E1B 55-kDa protein nuclear colocalization. E4orf6 protein variants lacking or containing disruptions within this arginine-faced, amphipathic  helix failed to direct the E1B 55-kDa protein to the nucleus when the proteins were transiently expressed. Furthermore, E4orf6 variants that could not direct the nuclear colocalization of the E1B 55-kDa protein also failed to supply E4orf6 function in cells infected with an E4 deletion virus. The severity of the defect of these E4orf6 variants during an Ad infection suggests that the arginine-faced, amphipathic  helix is crucial for the multiple functions of the E4orf6 protein.

A region within the carboxy terminus of the E4orf6 protein is required for the nuclear colocalization of the E1B 55-kDa protein. By contrast, a recent study of Rubenwolf et al. suggests that a region within the amino-terminal 58 amino acids of the E4orf6 protein mediates E4orf6-E1B 55-kDa protein binding in vitro (48). In this study, carboxy-terminal E4orf6 protein fragments failed to bind the E1B 55-kDa protein whereas amino-terminal E4orf6 protein fragments, including the E4orf6/7 protein, bound the E1B 55-kDa protein in vitro. These investigators also were able to indirectly immunoprecipitate the E4orf6/7 protein with E1B 55-kDa protein-specific antibodies after expressing the E4orf6/7 protein by transfection in 293 cells. However, these same investigators, as well as others, failed to detect an interaction between the E4orf6/7 protein and the E1B 55-kDa protein during Ad infection (8, 24, 48). The reason for these discrepancies is not known but may be due to the different experimental approaches used to measure the E4orf6-E1B 55-kDa protein interaction. Further support for the significance of the carboxy terminus of E4orf6 in E1B 55-kDa protein nuclear localization can be derived from an unpublished observation from our laboratory. An E4orf6 protein variant lacking the first 58 amino acids failed to localize to the nucleus and failed to promote E1B 55-kDa protein nuclear colocalization; however, the addition of a nuclear localization signal from the large T antigen of simian virus 40 to this variant promoted the nuclear localization of the protein and concurrently restored its ability to direct the E1B 55-kDa protein to the nucleus. Although we cannot exclude the possibility that the amino terminus of the E4orf6 protein is involved in binding the E1B 55-kDa protein, the results presented in this study suggest that the carboxy terminus of the E4orf6 protein is essential for E1B 55-kDa protein nuclear localization.

The region of the E4orf6 protein required for the nuclear localization of the E1B 55-kDa protein is an arginine-faced, amphipathic  helix. A synthetic peptide corresponding to this region adopted an -helical structure as measured by CD spectroscopy. Although the shapes of the L₂₄₅P peptide CD spectra resembled the shapes of the spectra for the wild-type E4orf6 peptide in the presence of TFE, the magnitudes of the minima of the L₂₄₅P peptide at 208 and 222 nm are less than that of the wild-type E4orf6 peptide. The behavior of the L₂₄₅P peptide suggests that the L₂₄₅P protein may retain some -helical structure in this region. Nonetheless, this mutant protein was unable to promote the nuclear localization of the E1B 55-kDa protein. Similarly, E4orf6 protein variants containing proline residues at amino acids 239 and 241 also failed to direct the E1B 55-kDa protein to the nucleus. However, an E4orf6 protein variant encoding proline after amino acid 255 promoted the nuclear localization of the E1B 55-kDa protein as well as the wild-type E4orf6 protein. These results are consistent with the possibility that an  helix extends from amino acids 239 through 245, but not beyond amino acid 255. A hydrophobic-moment plot of this region reveals that an  helix between amino acids 239 and 253 would exhibit a strong hydrophobic moment (Fig. 4B). This observation leads us to propose the existence of an  helix through residue 253. In addition, amino acid 251 of the E4orf6 protein is an arginine. Therefore, if the arginine face of this  helix is critical for E4orf6 function, it seems likely that the  helix would include arginine 251. Since amino acids 252 and 253 of this region are -helix-favoring isoleucine residues, it also seems likely that these residues would be part of the arginine-faced amphipathic  helix. Because the proline-containing variants, RC₂₃₆RPC and A₂₃₉P, were defective at directing nuclear localization of the E1B 55-kDa protein, we cannot definitively identify the amino terminus of the arginine-faced, amphipathic  helix. However, it seems reasonable that amino acid 239 is the amino-terminal residue of this  helix, since amino acid 239 is an -helix-favoring alanine residue and amino acids 237 and 238 are non--helix-promoting cysteine residues. From these considerations, we suggest that an arginine-faced amphipathic  helix exists between amino acids 239 and 253 and that this  helix lies at an interface between the hydrophobic core of the protein and a solvent-exposed face.

Most amphipathic  helices that have been described are membrane associated and frequently contribute to the aqueous pore of a protein channel through a membrane (reference 51 and references therein). However, an arginine-rich, amphipathic  helix that mediates protein-protein interaction has been identified in the human immunodeficiency virus (HIV) Nef protein (2). The Nef protein is dispensable for virus growth in vitro but is a key factor in the pathogenesis of HIV (31, 36). In HIV-infected cells, the Nef protein modulates the activity of kinases involved in T-cell activation pathways; the arginine-rich, amphipathic  helix in the amino-terminal region of Nef is required for Nef binding to an uncharacterized serine kinase and the Lck tyrosine kinase (2, 7, 50). To our knowledge, the Nef and E4orf6 proteins are the only two examples of proteins that use an arginine-rich amphipathic  helix to mediate protein-protein interaction.

Previous studies in our laboratory suggest that the interaction between the E4orf6 protein and the E1B 55-kDa protein in vivo is indirect and that a primate-specific cellular factor is required for the E4orf6-mediated nuclear localization of the E1B 55-kDa protein. Perhaps the arginine-faced, amphipathic  helix of the E4orf6 protein interacts with such a cellular factor. Additional support for the existence of an interaction between this  helix and unknown cellular factors derives from unpublished observations on the long-term expression of the E4orf6 protein and the L₂₄₅P protein. We found that expression of the wild-type E4orf6 protein in HeLa or 293 cells could not be sustained for greater than 4 weeks, whereas expression of the L₂₄₅P protein could be maintained indefinitely (42a). Perhaps both the Nef and E4orf6 viral proteins recruit cellular factors to modulate host activities through an arginine-rich, amphipathic  helix.

A recent report from Dobbelstein et al. suggests that the arginine-faced, amphipathic  helix of the E4orf6 protein acts as a nuclear retention signal (9). These investigators failed to observe shuttling of the wild-type E4orf6 protein between nuclei within a heterokaryon formed of HeLa and mouse cells, whereas an E4orf6 variant containing a glutamic acid in place of arginine 248 was observed to shuttle. However, in previous studies, we found that the wild-type E4orf6 protein does shuttle between nuclei within heterokaryons formed of HeLa or rat cells alone (21) or within heterokaryons of HeLa and rat cells (unpublished data). The reason for these differences is unclear but may reflect the use of different cell lines or different experimental conditions. Although we did not directly measure shuttling of the E4orf6 variants in the experiments reported here, we failed to detect a difference between the intracellular distribution of any E4orf6 proteins bearing mutations in the arginine-faced  helix and the wild-type E4orf6 protein. However, carboxy-terminal truncations that removed the  helix as well as 80 or more amino acids on the amino-terminal side of the  helix created proteins that partitioned more strongly in the cytoplasm than the wild-type E4orf6 protein. These results suggest that sequences within the E4orf6 protein indeed affect the extent to which the protein partitions between the nucleus and cytoplasm; however, these sequences appear to be amino terminal to the  helix identified in this report.

Expression of the E4orf6 protein restored growth of the E4 deletion virus, dl1014, to near-wild-type levels (Fig. 7). Two previously published observations led us to anticipate this result. Ketner et al. (32) demonstrated that ectopically expressed E4orf6 protein restored growth of a larger E4 deletion mutant. Similarly, E4 mutant viruses that express only the E4orf6 protein grow to near-wild-type levels (1, 3, 26). Strikingly, the L₂₄₅P E4orf6 variant, which failed to direct nuclear localization of the E1B 55-kDa protein (Fig. 6), completely failed to enhance replication of dl1014 (Fig. 7). By contrast, expression of the R₂₄₁P variant provided a slight but significant (P < 0.05) enhancement to dl1014 growth over untransfected or L₂₄₅P-transfected cells. Unlike the L₂₄₅P variant, the R₂₄₁P variant displayed a limited ability to direct nuclear localization of the E1B 55-kDa protein when expressed from the CMV immediate-early promoter (data not shown). The reason for this limited ability is not clear, but it may, for example, be indicative of a truncated, arginine-faced, amphipathic  helix. Nonetheless, this result reinforces the correlation between the ability of the E4orf6 protein to direct the E1B 55-kDa protein to the nucleus and the ability of the E4orf6 protein to function during Ad lytic growth.

In conclusion, we have demonstrated that an arginine-faced  helix near the carboxy terminus of the E4orf6 protein is essential for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. Furthermore, we suggest that the arginine-faced  helix is critical for the function of the E4orf6 protein during a productive viral infection. These findings are consistent with the possibility that in the absence of the E4orf3 protein, the E4orf6 protein can enhance virus growth only through its interaction with the E1B 55-kDa protein. Alternatively, the arginine-faced, amphipathic  helix of the E4orf6 protein may be important for the multiple functions of the E4orf6 protein during an Ad infection. Perhaps the basic face of the amphipathic  helix mediates interactions with multiple cellular factors in a manner similar to that described for the Nef protein of HIV. The identity of such factors should provide valuable insight into the means by which the E4orf6 protein can mediate mRNA export, promote viral mRNA accumulation, modulate viral mRNA splicing, enhance viral DNA synthesis and act as an oncoprotein.