INTRODUCTION

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The adenovirus type 5 (Ad5) early region 1B 55-kDa oncoprotein (E1B 55-kDa) functions to overcome restrictions imposed on Ad growth by the cell cycle. In a randomly cycling population of cells, the E1B 55-kDa-deletion virus dl338 is restricted for growth such that it produces progeny virus in only a fraction of infected cells. By contrast, the wild-type virus produces progeny in nearly every infected cell. By analyzing virus growth in synchronized populations of cells, we demonstrated that the growth of the mutant virus was severely restricted in cells infected during G₁ whereas this restriction was partially relieved in cells infected at the onset of S phase (26). Furthermore, we have recently demonstrated that the cell cycle restriction of the E1B 55-kDa-mutant virus can be overcome by infecting cells at 39°C (27). Because the E1B 55-kDa-mutant virus is not defective for viral DNA synthesis, we concluded that the role of the E1B 55-kDa protein in promoting cell cycle-independent virus growth resides in the late phase of viral replication (26).

At late times in the lytic infection, the E1B 55-kDa protein facilitates the transport of late viral transcripts and a subset of cellular transcripts while inhibiting the transport of most cellular transcripts (4, 6, 42, 54, 76). The E1B 55-kDa protein mediates viral and cellular mRNA transport in a complex with the early region 4 (E4) orf6 protein and possibly primate cell-specific cellular factors (10, 28, 30, 52). Consequently, viral mutants that fail to express either the E1B 55-kDa or the E4 orf6 gene products are defective for late viral gene expression and viral replication (10, 14, 30, 54). The transport of several cellular messages also requires the E1B 55-kDa protein late in Ad infection. These include the heat shock protein 70, -tubulin, and the interferon-inducible Mx-A and 6-16 mRNAs. Transport of this subset of cellular mRNAs correlates with activation of these genes late in infection (47, 76). The ability of the E1B 55-kDa-mutant virus to grow selectively in S-phase cells suggests the possibility that a host cell factor compensates for the defect in virus-mediated mRNA transport in the absence of the E1B 55-kDa protein (26). Furthermore, other viral proteins that function in the control of mRNA transport may also contribute to cell cycle-independent Ad growth.

The E4 orf6 and orf3 proteins have redundant and overlapping functions, some of which are related to functions of the E1B 55-kDa protein (10, 11, 14). Each of these E4 gene products independently augments viral DNA synthesis, late viral gene expression, shutoff of host protein synthesis, and production of progeny virus (10, 11, 30, 34). Both the orf3 and orf6 proteins stabilize late mRNAs in the nucleus and thus enhance cytoplasmic accumulation of late Ad mRNAs (10, 11, 65, 66). Furthermore, the orf3 and orf6 proteins have been shown to affect mRNA splice site selection (50, 51).

The E1B 55-kDa and E4 orf6 proteins contribute to cellular transformation in cooperation with E1A. Both the E1B 55-kDa and E4 orf6 proteins independently bind and inhibit transcriptional activation mediated by the cellular growth suppressor, p53 (16, 37, 48, 77, 79). Inhibition of p53-mediated transactivation by the E1B 55-kDa protein is required for transformation by both the weakly oncogenic group C and the highly oncogenic group A Ads (37, 77, 80, 81). In cooperation with the E1A and E1B proteins, the E4 orf6 protein transforms baby rat kidney cells (48), converts the nontumorigenic 293 cell line (29) into a tumorigenic cell line in nude mice, and blocks p53-dependent apoptosis (46). Furthermore, coexpression of the E1B 55-kDa and E4 orf6 proteins decreases the stability of p53 in both transformed and productively infected cells (46, 48, 60, 70). Despite the relationship between these viral proteins and p53, the failure of the E1B 55-kDa-mutant virus to replicate independently of the cell cycle is not due to a failure to abrogate p53 function (27, 64). Recently, the E4 orf3 gene has been shown to cooperate with E1A, E1B, and E4 orf6 in transforming nonpermissive primary rat cells (49).

In this work, a series of mutant Ads were analyzed to determine if other Ad gene products related to the E1B 55-kDa protein contributed to cell cycle-independent virus growth. Mutant viruses that fail to express the E4 orf6 protein were restricted by the cell cycle for growth in a manner similar to that of the E1B 55-kDa-mutant virus. Therefore, the cell cycle restriction to virus growth may be linked to the defect in virus-mediated mRNA transport shared by the E1B 55-kDa- and E4 orf6-mutant viruses. Consistent with this suggestion, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G₁ cells infected with the E1B 55-kDa- or E4 orf6-mutant viruses compared to that in G₁ cells infected with the wild-type virus. By contrast, this ratio, which serves as an indirect measure of mRNA transport, was equivalent among cells infected during S phase with the wild-type or mutant viruses. Additionally, cells infected during S phase with the mutant viruses synthesized more late viral protein than did cells infected during G₁. Nevertheless, comparison of total cytoplasmic levels of late transcripts suggested that the cell cycle restriction may not result entirely from the inability to accumulate late viral transcripts in the cytoplasm. Finally, the growth restriction of mutant viruses that failed to express both the E4 orf3 and orf6 proteins could not be overcome by infecting cells during S phase. These results demonstrate a requirement for E4 orf3 and orf6, as well as the E1B 55-kDa protein, in promoting cell cycle-independent virus growth.

	MATERIALS AND METHODS

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Cell culture. 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 (ATCC CCL 2; American Type Culture Collection, Manassas, Va.), 293 (29), and W162 (72) cells were maintained as monolayers in Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented with 10% newborn calf serum, 100 U of penicillin, and 100 µg of streptomycin per ml. Cells were maintained in subconfluent adherent cultures in a 5% CO₂ atmosphere at 37°C by passaging them twice weekly at a 1:10 dilution.

Viruses. The viruses used in these studies are described in Table 1. The phenotypically wild-type Ad5 parent virus dl309 used in these studies lacks a portion of the region encoding the E3 region that has been shown to be dispensable for growth in tissue culture (35). The E1B-mutant virus dl338 contains a 524-bp deletion in the 55-kDa protein coding region (54). Mutant virus dl1520D contains an 827-bp deletion in the region encoding the 55-kDa protein in combination with a stop codon to ensure that a truncated 55-kDa product cannot be expressed (5). The E1B 55-kDa-mutant virus dl110 contains a 472-bp deletion between nucleotide sequence positions 2333 and 2804 of the E1B coding region (4). E1B 55-kDa mutants S380, R443, and A143 were constructed by making in-frame 12-bp insertions in the DNA sequence of the E1B gene (78). The E1B 19-kDa-mutant virus dl337 contains a 146-bp deletion in the E1B 19-kDa coding region between nucleotide sequence positions 1770 and 1960 (53).

Electron microscopy. Infected HeLa cells were processed for transmission electron microscopy at a time when virus production had reached a maximum and the integrity of the cell was well preserved. For most virus infections, cells were processed 24 h postinfection; for viruses that suffer delays in viral DNA synthesis, cells were processed 36 h postinfection. The infected HeLa cells were mechanically removed from the culture dish and pooled with the supernatant medium before harvesting to ensure quantitative recovery of the infected cells. The cells were washed with 0.2 M sodium cacodylate, pH 7.2, and fixed with 2.5% gluteraldehyde in the same buffer. The fixed cells were prepared for transmission electron microscopy as previously described (26).

Plaque assays for viral yields. Detailed methods for Ad plaque assays have been described elsewhere (36). In brief, virus was harvested from cells in culture medium 48 to 72 h postinfection by multiple cycles of freezing and thawing. The cell lysates were clarified by centrifugation and serially diluted in infection medium for infection of 293 and W162 cells for plaque assay of E1B 55-kDa- and E4-mutant viruses, respectively. After incubation with the diluted virus for 1 h, the infected cells were overlaid with 0.7% SeaKem ME agarose (FMC BioProducts, Rockland, Maine) in DMEM supplemented with 0.75% sodium bicarbonate and 4% newborn calf serum. The cells were overlaid with additional agarose in growth medium on the third day after infection. Plaques were visualized by staining with neutral red in an agarose overlay on the seventh day after infection. Data were typically collected from three dilutions in each series of dilutions. The virus yield (PFU per milliliter) was determined by linear regression and expressed as the number of PFU per infected cell. Replicate samples were compared with the two-tailed Student t test.

Late viral protein synthesis. Late viral protein synthesis was analyzed by pulse-labeling infected HeLa cells for 1 h with 0.1 mCi of ³⁵S-labeled amino acids (Tran³⁵S-label; ICN Biochemicals) per ml in cysteine- and methionine-free DMEM supplemented with 2% fetal bovine serum at 32 h postinfection. Cells were then scraped into PBS, pelleted, and lysed in 2% sodium dodecyl sulfate (SDS)-125 mM Tris (pH 6.8)-20% glycerol-5% 2-mercaptoethanol-100 mM dithiothreitol-0.01% bromophenol blue. Protein from 1 × 10⁵ to 2 × 10⁵ cell equivalents was separated by SDS-polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel with a 36:1 ratio of acrylamide to N,N'-methylenebisacrylamide (Polysciences, Warrington, Pa.). PAGE gels were fixed in 15% glacial acetic acid-7.5% methanol. Proteins were quantified with the use of a Molecular Dynamics PhosphorImager and ImageQuant analysis software (Molecular Dynamics, Sunnyvale, Calif.). Ad late proteins were identified by using virion standards that were synthesized in the presence of ¹⁴C-labeled mixed amino acids (Amersham, Arlington Heights, Ill.) and gradient purified from 293 cells infected with the wild-type Ad, dl309, as described previously (36).

Cell fractionation and RNA isolation. All solutions used for RNA purification and in vitro transcription were prepared from diethyl pyrocarbonate-treated water as described in Ausubel et al. (3). RNA was isolated from the nucleus and cytoplasm of Ad-infected HeLa cells at 16 to 18 h postinfection. Approximately 4 × 10⁶ infected cells were scraped in ice-cold PBS, pelleted at 1,000 × g at 4°C, and then suspended in 0.15 ml of isotonic buffer (140 mM NaCl, 10 mM Tris-Cl [pH 7.4], 1.5 mM MgCl₂) containing 10 mM vanadyl-adenosine complex. An equal volume of isotonic buffer with 1% (vol/vol) Nonidet P-40 (Calbiochem) was added to the cells on ice with gentle mixing. After 5 min, the nuclei were pelleted at 1,000 × g and briefly washed with isotonic buffer containing 0.5% Nonidet P-40. After again the nuclei were again pelleted, the supernatant liquid was pooled as the cytoplasmic fraction.

RNase protection assays. DNA fragments obtained by thermal cycle amplification corresponding to Ad5 DNA spanning the polyadenylation site of the L3 and L5 gene were subcloned into pGEM7z(+) (Promega) for transcription in vitro. The primers used for the L3 gene (5'-TTCCTAACTTTGACGCGGTA-3' and 5'-TTCGACAGGAAACCGTGTG-3') generated a 442-bp thermal cycle product corresponding to bases 27811 through 28252 of the Ad5 genome. The primers used for the L5 gene (5'-TTCAGCTTATCCAAAATCTCACG-3' and 5'-TTCGCCTTGGTTTGCTT-3') generated a 513-bp product corresponding to bases 32545 through 33058 of the Ad5 genome. Radiolabeled RNA complementary to the sequences surrounding the L3 and L5 polyadenylation site was synthesized in vitro with SP6 RNA polymerase as recommended by the manufacturer (Promega) with [-³²P]CTP (ICN; 10 mCi/ml, >400 Ci/mmol). The DNA template was removed by digestion with RNase-free DNase (RQ1 DNase; Promega), and the radioactive RNA was purified by sequential ethanol precipitation from 0.125% SDS, 0.5 M ammonium acetate, 25 µg of tRNA carrier per ml, and then 2.5 M ammonium acetate. The RNA was resuspended in 0.04 ml of 0.01 mM sodium acetate, and the amount of radioactive transcript was quantified by liquid scintillation counting.

Computer-assisted graphics. Radioactive samples were visualized with the use of a Molecular Dynamics PhosphorImager and ImageQuant analysis software. Radioactive images were obtained as 16-bit gray-scale images and then converted to 8-bit gray-scale images. The density ranges were adjusted for printing without further contrast or image enhancement. The digitized images were imported at 300 dpi into the graphic software, Canvas (Deneba Software, Miami, Fla.), operating on a Macintosh microcomputer to create the final figures.

	RESULTS

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Ad mutants that exhibit a potential cell cycle restriction for growth produce virus in only a fraction of infected cells. The cell cycle restriction for growth of the E1B 55-kDa mutant virus dl338 was initially identified by analyzing Ad-infected HeLa cells by electron microscopy. This approach permits analysis of the Ad infection at the level of the individual cell. Only 20% of the HeLa cells infected with dl338 produced progeny virions, although all cells were infected and synthesized viral DNA. By contrast, the wild-type virus dl309 produced virus in nearly every infected cell (26). In the work presented here, we evaluated other Ad mutants with defects in viral genes related to the E1B 55-kDa gene for a potential cell cycle restriction. These include mutant viruses containing lesions in the E4 orf3 and orf6 genes. Asynchronous HeLa cells were infected with the wild-type virus dl309, an E1B 55-kDa-mutant virus, dl1520, an E4 orf3-mutant virus, inorf3, or an E4 orf6-mutant virus, dl355, at multiplicities of 20 PFU per cell. The cells were then processed for transmission electron microscopy to determine the fraction of infected cells producing virus (Fig. 1 and Table 2).

View larger version (345K): [in this window] [in a new window]   FIG. 1.   E1B 55-kDa- and E4 orf6-mutant viruses, but not an E4 orf3-mutant virus, produce virus in only a fraction of infected cells. Monolayers of HeLa cells were infected with the wild-type virus dl309 (A to D), the E1B 55-kDa mutant virus dl1520 (E to H), the E4 Orf3-mutant inorf3 (I to L), or the E4 orf6-mutant virus dl355 (M to P), at a multiplicity of 20 PFU/cell. At 24 h postinfection, cells were fixed in 2.5% glutaraldehyde, embedded, and sectioned for transmission electron microscopy. Nearly all (>95%) of the cells infected with the wild-type virus contained electron-dense viral particles in the nucleus. Four representative wild-type virus-infected cells are shown in panels A through D. Virions can be easily seen in the high-magnification images, representing a fivefold magnification of the boxed region of the nucleus from the low-magnification image. Of the four representative cells infected with the E1B-mutant virus (E to H), only the cell shown in panel H contains virus particles in the nucleus. Nearly all (>95%) cells infected with the E4 orf3-mutant virus produced virus, as can be seen in the four representative cells shown in panels I through L. Of the five E4 orf6-mutant virus-infected cells (M to P), only the cell in panel P and one of the cells in panel O (marked by an asterisk) contained virus particles in their nuclei, although all cells were infected. Representative viral inclusions are marked VI. A crystalline array of virus particles in panel J is marked by an arrowhead, and crystalline aggregates of viral proteins in panels C and L are marked by dagger (). Bars, 2 µm.

E4-mutant viruses that are restricted by the cell cycle for growth produce greater yields of virus in cells infected during S phase. Virus yields were measured from cells infected during G₁ or S phase by plaque assay to confirm the growth restriction of the E4-mutant viruses deduced by electron microscopy. If growth of these mutant viruses is restricted by the cell cycle, then cells infected during S phase should produce greater yields of virus. Note that it is the stage of the cell cycle at the time of infection that determines the outcome of the E1B 55-kDa-mutant virus infection, since cells no longer continue to progress through the cell cycle shortly after infection (25). For these experiments, synchronized HeLa cells were infected during S phase or G₁ with each E4-mutant virus at a multiplicity of infection of 3 to 10. At 48 to 72 h postinfection, viral yields were determined by titer on W162 cells. The two-tailed Student t test was used to determine if the average yield of virus obtained from S-phase-infected cells and from G₁-infected cells differed. The results shown in Fig. 2 demonstrate a cell cycle restriction for growth of mutant viruses lacking the E4 orf6 gene.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   HeLa cells infected during S phase produce greater yields of E4 orf6-mutant virus, but not E4 orf3-mutant virus, than do cells infected during G1. HeLa cells were synchronized to S phase or G1. Cells were infected with the wild-type virus (dl309), the E4-mutant viruses lacking orf6 (dl355*, inorf6/inorf6/7, and dl366*+orf3), the E4 orf3-mutant virus (inorf3), or the E4-mutant viruses lacking orf3 and orf6 (inorf3/inorf6, dl355*/inorf3, dl366*+or4, and dl366*±orf1-2) at a multiplicity of 3 to 10 PFU per cell. Cells were lysed 48 to 72 h postinfection, and virus yields were measured by plaque assays with W162 cells. For the wild-type virus, 1 PFU measured with W162 cells corresponds to approximately 100 infectious units in HeLa cells. The results shown are averages of three to nine independent infections performed in four independent experiments. Yields are expressed as PFU per cell. Viruses that produced significantly different yields between cells infected during S phase and cells infected during G1 are identified by the associated P values derived from the two-tailed Student t test. All other comparisons of S with G1 were not statistically different, with P values of >0.5. The status of the E4 orf3 and orf6 genes in each mutant virus is indicated, where a plus sign indicates a wild-type gene and a minus sign indicates a functionally null gene.

Cell cycle restriction for growth of the E1B 55-kDa-mutant viruses correlates with the defect in virus-mediated mRNA transport. The E1B 55-kDa and the E4 orf6 proteins function as a complex to mediate viral and cellular mRNA transport in the infected cell. Since both the E1B 55-kDa- and E4 orf6-mutant viruses are defective for mRNA transport and cell cycle-independent growth, we sought to determine if the cell cycle restriction may be linked to the defect in virus-mediated mRNA transport. This notion was further supported by the analysis of three viruses bearing in-frame insertions in the E1B 55-kDa gene. These mutant viruses direct the synthesis of a stable E1B 55-kDa protein that is defective predominantly in one of three properties attributed to the E1B 55-kDa protein (37, 78). A143 is primarily defective for late viral gene expression, which may stem from the defect in virus-mediated mRNA transport. S380 is primarily defective for binding p53 and transformation, and R443 is defective for transcriptional silencing. The insertion mutants were evaluated for their ability to replicate independently of the cell cycle to determine if the loss of a specific property of the E1B 55-kDa protein correlates with cell cycle-restricted virus growth. The fraction of infected HeLa cells producing these mutant viruses was determined by electron microscopy (Table 3), and yield of mutant virus from HeLa cells infected during S phase or asynchronous HeLa cells were infected at a multiplicity of 20 PFU per cell. Cells were processed for transmission electron microscopy at 24 h postinfection. For the measurements of virus yields, S-phase or asynchronous HeLa cells were infected at a multiplicity of 3 PFU per cell. Cells were lysed at 48 h postinfection, and viral yields were determined by titer on 293 cells.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   E1B 55-kDa-mutant viruses defective for late gene expression produce greater yields of virus in S-phase cells than in asynchronous cells. Asynchronous or S-phase HeLa cells were infected with either the wild-type virus (dl309), and E1B 55-kDa-mutant virus (dl338, A143, R443, or S380), or an E1B 19-kDa-mutant virus (dl337) at a multiplicity of 3 PFU per cell. Cells were lysed 48 h postinfection, and virus yields were measured by plaque assays with 293 cells. Yields are averages from multiple experiments and are expressed as PFU per cell. Asynchronous values are derived from five experiments, and S-phase values were derived from three experiments, except for dl337. Both asynchronous and S-phase values for dl337 were derived from three experiments. The standard errors of the means are shown. The probabilities indicated above each set of bars derived from a two-tailed Student t test and represent the probabilities that the two averages are from the same population.

Late viral gene expression is partially restored in HeLa cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses. The E1B 55-kDa- and the E4 orf6-mutant viruses are defective for late gene expression due, in part, to reduced transport of late viral messages (14, 30, 43, 54). If the defects in mRNA transport and late gene expression contribute to the cell cycle restriction for growth of the E1B 55-kDa- and E4 orf6-mutant viruses, then late viral gene expression should be at least partially restored in cells infected during S phase with either of these mutant viruses. The results shown in Fig. 4 and Table 4 are consistent with this idea. The gel shown in Fig. 4 is representative of four independent experiments that are summarized in Table 4. For these experiments, cells were infected as an asynchronous population or synchronized and infected at the onset of S phase with dl309, dl1520, dl355, or inorf3 or mock infected. At 32 h postinfection, protein synthesis was evaluated by metabolically labeling the cells with ³⁵S-amino acids for 1 h. Protein from equivalent numbers of cells was separated by SDS-PAGE and analyzed by phosphorescence imaging.

View larger version (117K): [in this window] [in a new window]   FIG. 4.   The E1B 55-kDa-mutant and E4 orf6-mutant viruses synthesize greater levels of viral late proteins in HeLa cells infected during S phase than in asynchronous (Async) cells. Asynchronous or S-phase HeLa cells were mock infected or infected with the wild-type virus dl309, the E1B 55-kDa-mutant virus dl1520, the E4 orf6-mutant virus dl355, or the E4 orf3-mutant virus inorf3 at a multiplicity of 20 PFU per cell. At 32 h postinfection, cells were labeled with 35S-labeled amino acids for 1 h. Proteins from 105 cells (per lane) were separated by SDS-PAGE. Proteins were visualized and quantified by phosphorescence imaging. The migrations and masses (in kilodaltons) or molecular weight standards are indicated to the right of the gel. The positions of six Ad late proteins were determined with Ad virion standards labeled with 14C-amino acids and are indicated to the left of the gel. The positions of the E2A 72-kDA protein and the cellular actin protein are also indicated. The gel shown is representative of four independent experiments.

Cytoplasmic levels of L3 and L5 late viral mRNAs are greater in cells infected during G₁ than in cells infected during S phase. Late viral protein synthesis was partially restored in cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant virus. To determine if this reflected greater levels of late viral mRNA in the cytoplasm of cells infected during S phase, cells were synchronized and infected and RNA was isolated at 16 h postinfection. Levels of processed late viral transcripts in the cytoplasmic and nuclear fractions were measured by RNase protection assays with probes that spanned the polyadenylation sites of the L3 and L5 families of late viral transcripts.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Relative levels of cytoplasmic (Cyt) and nuclear (Nuc) L3 and L5 late viral transcripts determined by RNase protection assays. S-phase or G1 HeLa cells were mock infected or infected with the wild-type virus dl309 or the E1B 55-kDa-mutant virus dl338 at a multiplicity of 10 PFU per cell. At 16 h postinfection, cells were fractionated, and total RNA was isolated from both cytoplasmic and nuclear fractions. L3 and L5 transcript levels were determined by RNase protection assays with RNA probes that span the polyadenylation site of the L3 and L5 families of transcripts, respectively. Protected hybrids were resolved on polyacrylamide-urea minigels. Results of a representative protection assay for S-phase and G1 cells infected with dl309 or dl338 are shown. The positions of readthrough transcripts that extended beyond the L3 (filled arrowhead) and L5 (open arrowhead) polyadenylation sites and the mature transcripts (L3 or L5) are indicated to the left of the gel. The nucleotide length (bases) of each product is indicated to the right of the gel. Duplicate RNase protection results are in adjacent lanes.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Cytoplasmic fractions from cells infected during G1 contain greater levels of L3 and L5 viral transcripts than do those from cells infected during S phase. S-phase or G1 HeLa cells were infected with the wild-type virus dl309, the E1B 55-kDa-mutant virus dl338 or dl1520, the E4 orf6-mutant virus dl355, or the E4 orf3-mutant virus inorf3. Total RNA was isolated from cytoplasmic fractions at 16 h postinfection. L3 and L5 transcript levels were determined by RNase protection assay as described in Fig. 5. The cytoplasmic levels of L3 and L5 transcripts from S-phase cells infected with dl309 were normalized to 100 to determine the relative percentages of L3 (A) and L5 (B) transcripts in cytoplasmic fractions from cells infected during S phase with the mutant viruses or infected during G1 with the wild-type or mutant viruses. The results summarized in panels A and B are averages obtained from 10 experiments for dl309, 5 experiments for dl338, 8 experiments for dl1520, 6 experiments for dl355, and 2 experiments for inorf3. The standard errors of the means for each experiment are shown.

Late viral mRNA transport is partially restored in cells infected during S phase with the E1B 55-kDa- and E4 orf6-mutant viruses. Although cells infected during G₁ contained more late viral mRNA in the cytoplasm than did cells infected during S phase, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant virus appeared to exhibit enhanced mRNA transport compared to cells infected during G₁ with these mutant viruses. To serve as an indirect measure of mRNA transport, we determined the cytoplasmic-to-nuclear ratio of L3 and L5 mRNA. The amount of viral mRNA in the cytoplasm and nucleus was determined by RNase protection experiments such as that presented in Fig. 5. Because this RNase protection assay identifies mRNA only by the polyadenylation event, it is possible that some of the nuclear mRNA included incompletely processed forms, whereas all of the specific product in the cytoplasm was most likely to be mature mRNA. Consequently, the measure of the cytoplasmic-to-nuclear RNA ratio may underestimate the ratio of mature cytoplasmic mRNA to mature nuclear mRNA.

View larger version (22K): [in this window] [in a new window]   FIG. 7.   S-phase cells infected with the E1B 55-kDa-mutant or the E4 orf6-mutant virus yield greater cytoplasmic (Cyt)-to-nuclear (Nuc) ratios for L3 and L5 transcripts than do G1 cells. S-phase or G1 HeLa cells were mock infected or infected with the wild-type virus dl309, the E1B 55-kDa-mutant virus dl338 or dl1520, or the E4 orf6-mutant virus dl355 at a multiplicity of 10 PFU per cell. At 16 h postinfection, cells were fractionated and total RNA was isolated from both cytoplasmic and nuclear fractions. L3 and L5 transcript levels were determined by RNase protection assays with RNA probes that span the polyadenylation sites of the L3 and L5 families of transcripts, respectively. Relative levels of cytoplasmic and nuclear L3 and L5 mRNAs were determined by phosphorescence imaging and were normalized to total RNA to account for inconsistencies in RNA recovery. The cytoplasmic-to-nuclear ratios from representative S-phase (A) and G1 (B) infections are expressed as percentages of the ratios for the wild-type infections. The standard errors of the means are shown.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have demonstrated a role for the E4 orf6 protein similar to that of the E1B 55-kDa protein in overcoming cell cycle restrictions imposed on virus growth. E1B 55-kDa- and E4 orf6-mutant viruses that are defective for virus-mediated mRNA transport were restricted by the cell cycle for growth (Fig. 1 and 2; Tables 2 and 3). Cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses had greater cytoplasmic-to-nuclear ratios of L3 and L5 late transcripts (Fig. 7) and synthesized greater levels of late proteins (Fig. 4 and Table 4) than did cells infected with these mutant viruses during G₁ or asynchronous growth. Furthermore, these mutant viruses produced greater yields of virus from cells that were infected during S phase (Fig. 2 and 3). Nevertheless, factors other than late viral gene expression are involved in permitting cell cycle-independent replication, since accumulation of late viral messages in the cytoplasm of mutant-infected cells did not correlate with enhanced growth in cells infected during S phase (Fig. 6). An E4 orf3-mutant virus was not restricted by the cell cycle for replication (Fig. 1 and 2 and Table 2). However, enhanced growth of the E4 orf6-mutant viruses in cells infected during S phase required expression of the E4 orf3 protein (Fig. 2 and Table 2). These studies have demonstrated novel roles for the E4 orf6 and orf3 proteins in promoting cell cycle-independent Ad growth in cooperation with the E1B 55-kDa protein.

In addition to promoting cell cycle-independent growth, the E1B 55-kDa and E4 orf6 proteins function as a complex to mediate both viral and cellular mRNA transport (4, 6, 30, 42, 54, 76). The function of the E1B 55-kDa and E4 orf6 proteins in mediating viral and cellular mRNA transport, and thus gene expression, late in infection may underlie cell cycle-independent Ad growth. When cells were infected during S phase with the E1B 55-kDa- or E4 orf6-mutant virus, the defect in late viral gene expression was partially restored such that cells infected during S phase with the mutant virus synthesized greater levels of late viral proteins than did cells infected during asynchronous growth (Fig. 4 and Table 4). Interestingly, the defect in late gene expression and growth can also be ovecome by infecting and maintaining cells at elevated temperatures during the infection (27, 32, 42, 74). Furthermore, the cell cycle restriction was alleviated in cells infected at elevated temperatures in a manner resembling that observed in cells infected during S phase such that mutant virus was produced in approximately 70% of the infected cells (27). These findings suggest that a common mechanism may underlie cell cycle restriction and virus-mediated mRNA transport, since both defects exhibit a cold-sensitive phenotype.

We measured cytoplasmic and nuclear levels of the L3 and L5 late viral transcripts in cells infected during S phase or G₁ to more directly analyze the link between virus-mediated mRNA transport and the cell cycle restriction (Fig. 5). The cytoplasmic-to-nuclear ratio of viral transcripts reflects the combined contributions of transport from the nucleus to the cytoplasm and stability of these transcripts in the respective cellular compartment (54). Because the stability of late viral mRNA did not vary with the infecting virus or stage of the cell cycle (data not shown), we conclude that the cytoplasmic-to-nuclear ratio provides a reasonable measure of viral mRNA transport. Cells infected during G₁ with the wild-type virus had greater cytoplasmic-to-nuclear ratios of L3 and L5 mRNA than did cells infected during S phase with either wild-type or mutant virus (Fig. 7). By contrast, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant virus had greater cytoplasmic-to-nuclear ratios of L3 and L5 mRNA than did cells infected during G₁ with these mutant viruses. In addition, this ratio was equivalent among S-phase cells infected with either wild-type or mutant virus. Therefore, the apparent ability of the mutant viruses to transport viral mRNA in cells infected during S phase with wild-type-like efficiency results from an increase in transport of late viral mRNA in cells infected with the mutant viruses and a decrease in transport of late viral mRNA in cells infected with the wild-type virus during S phase. These results indicate that the transport of late viral mRNA is differentially regulated in response to the stage of the cell cycle at the time of infection.

Although the transport of late viral mRNA was restored to wild-type efficiency in S-phase cells infected with the E1B 55-kDa- or E4 orf6-mutant viruses (Fig. 7), greater cytoplasmic levels of late viral transcripts were measured in cells infected during G₁ with either the wild-type or mutant viruses than in cells infected during S phase (Fig. 6). The basis for enhanced accumulation of late viral mRNA in the G₁-infected cell is not known, but the accumulation may, for example, reflect differential rates of viral transcription as a function of the cell cycle. Nonetheless, the enhanced synthesis of late viral proteins and enhanced virus growth seen for the S-phase-infected cell was unique to the cell cycle-restricted mutant viruses analyzed here. Perhaps other factors contribute to an increased translational efficiency of the late viral messages in S-phase cells infected with the RNA transport-defective viruses (Fig. 4 and Table 4). Also, since the differential transport of mRNA that occurs as a function of the cell cycle did not impact steady-state levels of cytoplasmic late viral mRNA, we suggest that this control may have a greater significance in regulating expression of cellular genes that impact Ad growth.

Perhaps cells infected during S phase are more promiscuous for the transport of all mRNA. As a result, late viral transcript levels are reduced by the increase in cellular message transport and the resulting competition for limited transport machinery. This suggestion is consistent with the finding that cells infected during S phase with the wild-type or mutant Ads synthesize more cellular proteins than do cells infected during G₁ (Fig. 4). If growth of the wild-type virus is facilitated by host cell shutoff, then the reduced shutoff seen in S-phase-infected cells may hinder replication of the wild-type virus (Fig. 3 and 4 and Table 4). The RNA transport-defective viruses, on the other hand, may derive greater benefit from the promiscuous RNA transport apparent in the S-phase-infected cell. This benefit may lie not so much in the transport of late viral genes as in the enhanced transport and expression of a subset of cellular genes. Conceivably, expression of these cellular genes requires the E1B 55-kDa-E4 orf6 protein complex in cells infected during stages of the cell cycle other than S phase. Transcripts of several cellular genes, such as the hsp-70 and -tubulin genes and the interferon-inducible genes Mx-A and 6-16, are selectively transported late in Ad infection. The selective transport of host transcripts depends on the E1B 55-kDa protein and stimulation of their transcription in the late phase of infection (47, 76). Perhaps the transport of host transcripts such as these is facilitated for the purpose of viral replication.

It has been previously suggested that a positively acting factor may be recruited to the viral inclusions that are nuclear compartments of viral RNA processing by the E1B 55-kDa-E4 orf6 complex to facilitate mRNA transport and cell cycle-independent virus growth (52). Perhaps this factor is abundant in S-phase cells and can promote the transport of RNA in the absence of the E1B 55-kDa-E4 orf6 complex. As suggested by our previous work, such a factor may be unique to primate cells (28). Several cellular proteins function in both mRNA transport and cell cycle regulation such that these processes may be linked to some extent (21, 23, 41, 62, 63, 67, 69). These proteins may be targets of the E1B 55-kDa or E4 orf6 proteins. For example, a novel cellular heterogeneous nuclear ribonucleoprotein, E1B AP5, associates with the E1B 55-kDa protein and overcomes the E1B 55-kDa-dependent inhibition of cellular mRNA export when overexpressed (24). It would be of interest to know if the expression or function of E1B AP5 is differentially regulated through the cell cycle.

In contrast to the E1B 55-kDa- and E4 orf6-mutant viruses, the E4 orf3-mutant virus inorf3 produced virus in nearly every infected cell and thus was not restricted by the cell cycle for growth (Fig. 1). Nevertheless, in the absence of the E4 orf6 gene, the orf3 gene was essential for enhanced replication of the E4 orf6-mutant viruses in S-phase-infected cells (Table 2 and Fig. 2). Growth of the E4 orf3-orf6 double-mutant viruses in cells infected during G₁ was restricted to a fraction of infected cells, similar to that of the E4 orf6-mutant viruses (Table 2). However, when cells were infected during S phase with the E4 orf3-orf6-double-mutant viruses the fraction of infected cells producing virus did not increase (Table 2) and these cells did not produce greater yields of virus than did cells infected during G₁ (Fig. 2). These results indicate that E4 orf3-orf6 double-mutant viruses may be restricted for growth by mechanisms other than the cell cycle.

It is not clear what mechanism other than the cell cycle restriction may limit virus production to a fraction of infected cells. Perhaps there is a limited probability (20 to 30%) that a cell infected with the E1B 55-kDa- or E4 orf6-mutant virus will produce progeny virus. This probability can be modulated by a number of factors such as the stage of the cell cycle at the time of infection, the temperature at which the infection is maintained, and expression of the E4 orf3 gene. Although the specific function of the E4 orf3 gene product in promoting cell cycle-independent growth is not understood, these results demonstrate that, in the absence of other E4 proteins, the E4 orf3 protein acts in the S-phase-infected cell to overcome restrictions on virus growth. In this regard, it is interesting to note that expression of an unidentified E4 gene product(s) in recombinant, replication-defective Ad vectors other than the orf6 protein resulted in dysregulation of the cell cycle and inappropriate cyclin expression (73). Perhaps the E4 orf3 protein contributes to such an effect.

Since the restriction for the E1B 55-kDa-mutant viruses is similar to that for the E4 orf6-mutant viruses, we speculate that the E1B 55-kDa-mutant viruses may also rely on the presence of the E4 orf3 gene for enhanced growth in cells infected during S phase. The interaction of these two viral proteins is suggested by the findings that the E1B 55-kDa protein is physically associated with the E4 orf3 protein (49) and colocalizes with the E4 orf3 protein in the absence of other E4 proteins (39). Preliminary results suggest that enhanced growth of the E1B 55-kDa-mutant virus requires an intact E4 orf3 gene (25). Such a finding would imply that the E4 orf3 protein is not necessary for cell cycle-independent replication in the presence of the E1B 55-kDa and E4 orf6 proteins but is necessary to establish the permissive state in S-phase cells infected with the E1B 55-kDa or E4 orf6-mutant virus. If substantiated, these results have implications for the development of oncolytic Ad vectors for the treatment of cancer. These results would predict that the E4 orf3 gene may be essential for the selective replication of oncolytic Ad vectors that lack the E1B 55-kDa gene in tumor cells (7, 31).

The contribution of the E4 orf3 protein to enhanced growth of E4 orf6-mutant viruses in S-phase cells may be linked to its role in disrupting nuclear domains called ND10s or promyelocytic leukemia oncogenic domains. Redistribution of proteins sequestered within ND10s, such as PML and Sp100, may trigger cell cycle dysregulation and oncogenesis, as in acute promyelocytic leukemia (reviewed in reference 17). The functions of ND10-associated proteins are not well understood, but the proteins have been hypothesized to promote cellular events analogous to the activation of a viral program of replication and may function in cell cycle regulation (2, 17-19). The morphology of ND10s changes with cell cycle progression, becoming more amorphous and diffuse, perhaps reflecting an active state, when cells enter S phase (19). Furthermore, localization of PML and Sp100 to interchromatin granules suggests that ND10-associated proteins may function in mRNA metabolism (56). The E1A and E1B 55-kDa proteins localize to ND10s early in infection (13, 18). Late in infection, some ND10-associated proteins are translocated to the viral inclusions (18, 56). Perhaps the E4 orf3 protein disrupts these domains to make ND10-associated proteins accessible to E1B 55-kDa and E4 orf6 proteins for the promotion of cell cycle-independent replication or virus-mediated mRNA transport. In the absence of the E1B 55-kDa or E4 orf6 proteins during Ad infection, ND10-associated proteins may contribute to the virus infection when cells are infected during S phase with a virus that expresses the E4 orf3 protein. Intriguingly, the ICP0 protein of herpes simplex virus type 1 (20, 40, 44, 59) and the IE1 and IE2 proteins of human cytomegalovirus (1, 38) also induce redistribution of ND10-associated proteins to viral inclusions, strongly suggesting a role for these proteins in viral replication.

In summary, we have demonstrated a role for the E4 orf6 protein in overcoming cell cycle restrictions imposed on virus growth. This role is similar to that described for the E1B 55-kDa protein (26). Our results indicate that the lytic infection of Ad is differentially affected by the stage of the cell cycle at the time of infection with respect to virus-mediated mRNA transport, late gene expression, and virus growth. Interestingly, the E4 orf3 protein was required for enhanced replication of E4 orf6-mutant viruses in cells infected during S phase. The mechanisms employed by Ad to promote cell cycle-independent virus growth and their relationship to mRNA biogenesis warrant continued investigation.