INTRODUCTION

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The product of the ie2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is a ~49-kDa protein, IE2, which activates the transient expression of plasmid-borne reporter genes under the control of viral promoters in SF-21 cells. IE2 was originally identified by its ability to trans-activate expression from the early AcMNPV 39K promoter in the presence of another viral trans-regulator, IE1 (5, 16). The effect of IE2 on expression from the 39K promoter was traced to the ability of the IE2 gene, ie2, to trans-activate expression from the ie1 promoter (6) in transient expression assays. In SF-21 cells, ie2 is also required, along with 17 other viral genes including ie1, for optimal transient expression from reporter plasmids carrying a late viral promoter (26, 32). In these assays, efficient late expression requires the replication and stability of the reporter plasmid carrying a viral homologous repeat sequence which binds IE1 and is thought to serve as an origin of DNA replication (18, 21, 33). The requirement of IE2 in optimizing plasmid DNA replication and late gene expression is host specific, since it is required in SF-21 cells but not in TN-368 cells (25, 35). The host dependence of IE2 may explain the observation that ie2 is nonessential for replication of the closely related baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) in Bombyx mori cells (13).

IE2 is a nuclear protein which contains a RING finger motif at its center (23, 40). RING fingers, or C₃HC₄ motifs, form a cross-braced zinc coordinating structure (11, 12, 24) and are found in a number of proteins of diverse function. The AcMNPV genome alone contains a total of five genes encoding proteins with RING finger motifs: IE2, CG30 (41), PE-38 (22), IAP1 (3, 8), and IAP2 (1). Although the RING finger motifs of the IE2s of both AcMNPV and BmNPV differ slightly in the spacing of one cysteine pair from the canonical RING finger motif, a canonical RING finger motif is found in the ie2 homolog of Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) (40). Like the RING finger-containing mammalian oncogene PML, AcMNPV IE2 and PE38 localize to discrete structures within the nucleus (23).

While studying the effects of early AcMNPV genes on apoptosis in SF-21 cells (34), we noticed that transfection of plasmids expressing ie2 resulted in the enlargement of the cells. We now report that ie2 expression blocked cell cycle progression but did not block cellular DNA replication, resulting in an increase in the number of cells with an abnormal DNA content, greater than 4N. In addition, we found that mutants of IE2 containing either a deletion of the RING finger motif or a mutation of an individual conserved amino acid residue of the RING finger motif lacked the ability to block cell division but retained the ability to trans-activate the ie1 promoter.

	MATERIALS AND METHODS

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Cells. Spodoptera frugiperda IPBL-SF-21 (SF-21) (44), Trichoplusia ni BTI-TN-5B1-4 (46) and TN-368 (20), and Helicoverpa zea Hz-AM1 (28) cells were cultured at 27°C in TC-100 medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum and 0.26% tryptose broth, as described previously (31).

Reporter plasmids and plasmid constructs. The reporter plasmid phcIE1 (29) contains the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of the ie1 promoter (17) and a portion of the hr-5 (37) sequences of AcMNPV.

Transfection, transient expression assays, and CAT assays. SF-21 cells (2.0 × 10⁶ cells per 60-mm-diameter dish) were transfected with 2.0 µg of the reporter plasmid phcIE1 and 1.0 µg of each of the other plasmids by using Lipofectin (Gibco BRL). Transfected cells were incubated at 27°C and harvested at 24 h posttransfection. CAT assays (15, 37) were performed by using 1/50 of each cell lysate. In those experiments involving heat shock, the cells were heat shocked at 18 h posttransfection for 30 min at 42°C and then harvested 6 h after heat shock. The percentage of viable cells was determined at various times as described previously (7, 34).

Flow cytometry. For flow cytometry, the medium was removed at the indicated times posttransfection and the cells were fixed and stained with DAPI (4',6-diamidino-2-phenylindole; Sigma, St. Louis, Mo.). The cells were harvested and washed once with ice-cold phosphate-buffered saline (PBS), pH 6.2. After fixation in 80% ethanol for 30 min on ice, the cells were washed again with ice-cold PBS and stained with a solution containing 1 µg of DAPI per ml, 0.1 mM EDTA, and 1 µg of RNase A per ml in PBS. DAPI-stained cells were analyzed with a Coulter EPIC 753 flow cytometer (Coulter Electronics, Hialeah, Fla.), as described previously (7).

Microphotography and immunofluorescence. SF-21 cells were seeded on glass coverslips in 35-mm-diameter culture dishes and transfected with the indicated plasmids, as described above. At 48 h posttransfection, growth medium was aspirated and the cells were rinsed with PBS (pH 6.2) and fixed in methanol at 10°C for 20 min, followed by two washes in PBS. To detect the microtubule network, cells were incubated with a 1:500 dilution of DM1 anti-alpha tubulin (Sigma) in PBS containing 1 µg of DAPI per ml for 30 min at room temperature. The cells were washed in PBS and incubated with a 1:50 dilution of lissamine rhodamine-conjugated goat anti-mouse immunoglobulin G (IgG) and IgM (Jackson Immunoresearch Laboratories) in PBS. Cells were washed twice in PBS and mounted on glass slides in Gelmount.

Immunoblot analysis. Transfected cells were heat shocked at 18 h posttransfection and harvested 3 h after heat shock. Cells were lysed in SDS buffer (4% sodium dodecyl sulfate [SDS], 125 mm Tris-HCl [pH 6.7], 30% [vol/vol] glycerol, 0.002% [wt/vol] bromphenol blue, 2% [vol/vol] -mercaptoethanol). Proteins from lysates of the transfected cells were separated on SDS-12% polyacrylamide gels and transferred to Immobilon P membranes (Millipore). FLAG-tagged proteins were detected with a 1:10,000 dilution of anti-FLAG M2 monoclonal antibody (Eastman Kodak Co., New Haven, Conn.) followed by a 1:10,000 dilution of rabbit anti-mouse IgG-horseradish peroxidase conjugate (Amersham). Immunoblots were visualized by the chemiluminescence method (Amersham).

	RESULTS

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Expression of AcMNPV ie2 blocks cell division. By light microscopy, we observed that SF-21 cells transfected with a plasmid containing the PstI-N fragment of the AcMNPV genome, pBs-PstN, became enlarged by 48 h posttransfection (Fig. 1A). The pBs-PstN-transfected cells continued to increase in size until 72 h and existed in this enlarged state for more than 96 h after transfection without obvious signs of stress (data not shown). This unusual morphology was not detected in SF-21 cells transfected with the vector plasmid pBluescript KS⁺ (pBs) (Fig. 1B).

View larger version (93K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   FIG. 1.   Expression of the AcMNPV PstI-N fragment blocks cell division. SF-21 cells (0.5 × 106 per 35-mm-diameter dish) were transfected either with 0.5 µg of plasmid pBs-PstN (A, C, and E) or with the same amount of the control vector plasmid, pBs (B, D, and F). At 48 h posttransfection, the cells were fixed in methanol and then double stained with DAPI (C and D) and DM1 anti-alpha tubulin (E and F), and the same field of cells was examined by phase-contrast (A and B) and immunofluorescence (C to F) microscopy. All cells were photographed at the same magnification; note the larger sizes of the cells transfected with pBs-PstN. Also, one of the four cells in panels A, C, and E does not appear to have been transfected with pBs-PstN. In panels B and F, mitotic spindles can be observed in the cell on the right. Bar, 25 µm. (G) SF-21 cells were transfected with either pBs or pBs-PstN and harvested after 24, 48, 72, and 96 h, and the numbers of viable cells were counted in the presence of trypan blue.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Physical map of the 96.9- to 0.0-m.u. region of AcMNPV with a partial restriction map. Locations and directions of ORFs between 96.9 and 0.0 m.u. are based on the sequence determined by Ayres et al. (1). Schematic representations of wild-type and mutant forms of ie2 are shown below the lines representing each plasmid. The RING finger motif is indicated by the shaded R box. Restriction sites are abbreviated as follows: HIII, HindIII; PI, PstI; HI, HpaI; B, BglII; S, SnaBI; D, DraI; HI*, HpaI-like site (see Materials and Methods).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Expression of ie2 arrests the cell cycle. SF-21 cells were transfected with either pHSP70FLAG-IE2 or control pHSP70PLVI+CAT and heat shocked at 18 h posttransfection. At 48, 72, and 96 h posttransfection, the cells were harvested and counted as described in Materials and Methods.

IE2 arrested the cell cycle in the S phase. To determine whether IE2 prevented cell division by arresting cells at a specific point in the cell cycle, we analyzed the DNA content of ie2-transfected cells by flow cytometry (Fig. 4). Cells transfected with the control plasmid pBs had similar DNA profiles at 24, 48, and 72 h posttransfection (Fig. 4), correlating with stable 4N:2N DNA content ratios of 0.93, 0.92, and 0.94, respectively (Table 1). In contrast, significant changes occurred in the cell cycle distribution of SF-21 cells transfected with pBs-PstN expressing ie-2 (Fig. 4). At 24 h posttransfection, a lower proportion of pBs-PstN-transfected cells appeared to have a 4N complement of DNA, and greater proportions were found in the G₁ and S phases (Fig. 4; Table 1). At 48 h posttransfection, a large proportion of ie2-expressing cells contained a 4N or higher amount of DNA compared with cells transfected with the control, pBs (Fig. 4; Table 1). By 48 h, the 4N:2N DNA content ratio had increased to 5.4 in pBs-PstN-transfected cells, compared to 0.92 in control cells (Table 1). By 72 h posttransfection, more than 50% of the pBs-PstN-transfected cells had abnormal DNA contents of greater than 4N (Fig. 4; Table 1), indicating that expression of ie2 blocks cell division and that cellular DNA replication continues. On the basis of these data and the lack of mitotic spindle formation, it appears that ie2 arrested SF-21 cells in the S phase of the cell cycle and that the normal control of cellular DNA replication was altered.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   DNA content analysis in ie2-transfected cells. SF-21 cells were transfected with either pBs-PstN expressing ie2 or the vector alone (pBs). The cells were washed, fixed, and stained with DAPI at the indicated times. Histograms show the results of flow cytometric analysis of DAPI staining intensity. The abscissas show relative amounts of DAPI fluorescence, reflecting DNA content, and the ordinates show cell numbers. The results shown are representative of at least two independent experiments.

Deletion of the RING finger region blocked the ability of IE2 to arrest the cell cycle. To determine if a specific region of IE2 was important for its ability to arrest the cell cycle, we constructed two deletion mutants of IE2 and tested their transactivation capabilities and their effects on cell cycle progression in transient assays. Plasmid pBs-IE2(215-274) has an in-frame deletion of the DraI-HpaI* fragment resulting in the deletion of amino acids 215 to 274, thereby eliminating the RING finger motif from IE2 (Fig. 2). In pBs-IE2(94-173), the HpaI-SnaBI fragment of PstI-N was deleted, leaving the RING finger domain intact but deleting amino acids 94 to 173 (Fig. 2), including several arginine, proline, and serine residues that are conserved between the AcMNPV and OpMNPV ie2 genes. To ensure that the ie2 deletion mutants could be expressed in SF-21 cells, we also constructed plasmids containing FLAG-tagged coding sequences of the ie2 mutants under hsp70 promoter control. These constructs, pHSP70FLAG-IE2(94-173) and pHSP70FLAG-IE2(215-274), as well as pHSP70FLAG-IE2, were tested for expression in SF-21 cells (Fig. 5). Plasmids pHSP70FLAG-IE2(94-173), pHSP70FLAG-IE2(215-274), and pHSP70FLAG-IE2 were transfected into SF-21 cells, followed by heat shock treatment. FLAG-IE2 and IE2(94-173) showed comparable levels of expression (Fig. 5; compare lanes 1 and 2). Slightly higher levels of expression were observed for the IE2(215-274) mutant protein (Fig. 5, lane 3).

View larger version (37K): [in this window] [in a new window]   FIG. 5.   Expression of FLAG-IE2 and FLAG-IE2 mutants. SF-21 cells were transfected with 2.5 µg of pHSP70FLAG-IE2 (lane 1), pHSP70FLAG-IE2(94-173) (lane 2), pHSP70FLAG-IE2(215-274) (lane 3), or pHSP70FLAG-IE2C251A (lane 4), heat shocked at 18 h posttransfection, and harvested 3 h after heat shock. Equal amounts of cell lysates were analyzed by SDS-12% polyacrylamide gel electrophoresis followed by Western blotting with anti-FLAG monoclonal antibody.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   DNA content analysis of SF-21 cells transfected with deletion mutants of ie2. SF-21 cells were transfected with pBs-IE2(94-173), pBs-IE2(215-274), or the control vector plasmid, pBs. Transfected cells were harvested at 24 or 48 h posttransfection. After being washed, fixed, and stained with DAPI, the cells were analyzed by flow cytometry. DAPI fluorescence is plotted on the x axes and is proportional to DNA content. Cell numbers are shown on the y axes.

Transactivation properties of IE2 mutants. To determine if the IE2 deletion mutants retained transregulatory activity, we analyzed their abilities to activate expression from phcIE1, a reporter plasmid containing the CAT gene under ie1 promoter control. When SF-21 cells were cotransfected with phcIE1 and pBs-PstN or pHSP70FLAG-IE2 expressing ie2 under the control of either its own promoter or the hsp70 promoter, the levels of CAT activity increased approximately threefold compared with that in cells transfected with the reporter plasmid alone (Fig. 7; compare lane 1 with lanes 2 and 3). Higher levels of CAT activity were also observed for cells cotransfected with phcIE1 and plasmids expressing ie2(215-274) (Fig. 7, lanes 6 and 7). Thus, deletion of the RING finger motif did not impair the ability of IE2 to activate the ie1 promoter. Plasmids pBs-IE2(94-173) and pHSP70FLAG-IE2(94-173) were not able to transactivate the CAT gene under ie1 promoter control (Fig. 7, lanes 4 and 5). Moreover, the levels of CAT activity in these cells were approximately twofold lower than in cells transfected with the reporter plasmid alone, suggesting interference with factors responsible for ie1 promoter activation or direct repression of the ie1 promoter.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   Transactivation of the ie1 promoter by IE2 deletion mutants. SF-21 cells were transfected with the reporter plasmid phcIE1 alone or cotransfected with phcIE1 and pBs-PstN, pBs-IE2(94-173), pBs-IE2(215-274), pHSP70FLAG-IE2, pHSP70FLAG-IE2(94-173), pHSP70FLAG-IE2(215-274), or pHSP70FLAG-IE2C251A. CAT activity relative to that from phcIE1 cotransfected with wild-type ie2 under its own promoter (lane 2, 100%) was determined. The data are averages of two or more experiments, and standard errors are indicated.

Site-specific mutation of the IE2 RING finger. To determine if the RING finger was specifically involved in blocking cell cycle progression, we constructed pHSP70FLAG-IE2C251A, a plasmid containing FLAG-tagged IE2 with an alanine in place of one of the seven conserved cysteine residues of the IE2 RING finger motif. Transient expression of pHSP70FLAG-IE2C251A in SF-21 cells did not result in an enlargement of the cell size (Fig. 8). Analysis of the trans-regulatory activity of FLAG-IE2C251A showed that this mutant was able to stimulate levels of CAT activity from the ie1 promoter comparable to that determined for wild-type IE2 (Fig. 7; compare lanes 3 and 8). Thus, modification of a single conserved residue of the RING finger motif of IE2 abolished its ability to arrest the cell cycle but did not affect its trans-regulatory activity.

View larger version (56K): [in this window] [in a new window]   FIG. 8.   Effects of FLAG-IE2C251A on cell size and growth. SF-21 cells were transfected with plasmids expressing the FLAG-tagged version of wild-type (pHSP70FLAG-IE2) or RING finger mutant (pHSP70FLAG-IE2C251A) IE2 or with the control CAT gene (pHSP70PLVI+CAT) and examined by light microscopy at 72 h posttransfection. Bar, 50 µm.

Effects of ie2 expression in different cell lines. Since the requirement for IE2 in transient expression assays is cell line specific, we analyzed the ability of IE2 to arrest cell division in cell lines derived from other insect species. SF-21, TN-368, BTI-TN-5B1-4, and Hz-AM1 cells were transfected with pHSP70FLAG-IE2 and heat shocked at 18 h posttransfection. At 72 h posttransfection, approximately 50% of the normal numbers of viable cells were obtained in the pHSP70FLAG-IE2-transfected TN-368, BTI-TN-5B1-4, and Hz-AM1 cells at 72 h posttransfection (Fig. 9A), similar to the reduction in cell numbers observed in the pHSP70FLAG-IE2-transfected SF-21 cell culture. There was no evidence of apoptosis or necrotic cell death in any of the transfected cultures. Thus, IE2 expression affected the ability of cells to divide, and this resulted in decreases in the numbers of viable cells. This result was confirmed by light microscopic analysis of pHSP70FLAG-IE2-transfected cells (Fig. 9B). No enlarged cells were observed in control pHSP70PLVI⁺CAT-transfected cells. In contrast, many of the TN-368, BTI-TN-5B1-4, and Hz-AM1 cells transfected with ie2 exhibited altered cellular morphologies at 72 h posttransfection. A dramatic enlargement of cells was observed in pHSP70FLAG-IE2-transfected BTI-TN-5B1-4 cells. A less dramatic but nevertheless noticeable increase in cell size was observed in TN-368 cells transfected with pHSP70FLAG-IE2, whereas Hz-AM1 cells expressing IE2 became elongated and had long protrusions at 72 h posttransfection (Fig. 9B). We conclude that AcMNPV ie2 was able to affect the cell division of cell lines derived from other species.

View larger version (44K): [in this window] [in a new window]   View larger version (149K): [in this window] [in a new window]   FIG. 9.   Effects of ie2 expression in different cell lines. (A) Percentage of viable cells in pHSP70FLAG-IE2-transfected cultures relative to that observed in control cells transfected with pHSP70PLVI+CAT in each cell line. The cells (0.5 × 106 cells per 35-mm-diameter dish) were transfected with 0.5 µg of pHSP70FLAG-IE2 and heat shocked at 18 h posttransfection. The cells were harvested, stained, and counted at 72 h posttransfection. (B) Morphology of TN-368, BTI-TN-5B1-4, and Hz-AM1 cells transfected with pHSP70FLAG-IE2 or control plasmid. Transfected cells were heat shocked at 18 h posttransfection and photographed at the same magnification at 72 h posttransfection. Cells transfected with pHSP70PLVI+CAT served as controls for normal cell growth and regular morphology. Bar, 50 µm.

	DISCUSSION

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We have identified a new activity of IE2: cell cycle arrest. SF-21 cells transfected with ie2 cease dividing and eventually contain a single enlarged nucleus with a greater than 4N complement of DNA. Cell cycle arrest appears to occur in the S phase, based on several observations. During the first 24 h following transfection, cells were able to progress from G₂ through M to G₁ and S, based on the fact that the proportion of cells with a 4N complement of DNA decreased during this period while the proportion of cells in the G₁ and S phase (2N and 2N to 4N) increased (Table 1). By 48 h, however, cells with a 4N or greater complement of DNA predominated, indicating that DNA synthesis continued in these cells in an unregulated fashion which precluded transition into G₂ and M. There were also no signs of mitotic spindle formation in the ie2-enlarged cells.

The purpose of arresting the cell cycle in S phase remains unclear, although the most obvious possibility would be to provide a nuclear environment which is more conducive to viral DNA replication. However, ie2 is a dispensable gene for both AcMNPV and BmNPV replication in at least some cell lines (13, 35), so cell cycle arrest does not appear to be essential for virus replication in those cell lines. IE2 of BmNPV appears to accelerate viral DNA replication slightly but reproducibly (13), although it is not known whether this effect is due to the transactivation or cell cycle arrest properties of IE2.

The ability of IE2 to arrest the cell cycle requires the presence of the RING finger but appears to be independent of the ability of IE2 to trans-activate gene expression. Deletion of residues 215 to 274, which encompass the majority of the RING finger motif of IE2 (only the first two cysteines of the motif remain), eliminated the ability of IE2 to block the cell cycle but did not eliminate its ability to trans-activate expression from the ie1 promoter. A similar effect was observed for an IE2 mutant with an alteration in a single conserved residue of the RING finger motif. Our observation that modification or deletion of the RING finger motif did not affect the ability of IE2 to trans-activate the ie1 promoter contrasts with the properties of an IE2 mutant described by Yoo and Guarino (48) which precisely eliminated the RING finger motif (residues 207 to 254). This 207-to-254 mutant was found to be severely impaired in its ability to transactivate the ie1 promoter (48). Since a mutant lacking the nine residues just N-terminal of the RING finger (i.e., a mutant deleted between residues 198 and 206) also lacked the ability to trans-activate the ie1 promoter (48), it is possible that residues 207 to 215 are crucial for IE2 trans-activation potential in our 215-to-274 mutant.

The IE2 mutant lacking residues 94 to 173 was unable to trans-activate the ie1 promoter but retained the ability to block the cell cycle, reinforcing the view that the trans-activation activity of IE2 may be unrelated to its ability to influence the cell cycle. Interestingly, the IE2(94-173) mutant not only failed to trans-activate the ie1 promoter but also had a negative effect on its expression. It is possible that this negative effect on ie1-promoted gene expression is due to the arrest of the cells in S phase, in which case IE2 would require a transactivation activity in order to compensate for this negative regulatory effect. However, it is also possible that the mutant form of IE2 interacts with factors affecting ie1 expression in a negative fashion. It is clear that gene regulation by IE2 is far more complex than originally envisioned.

A number of other viruses are known to affect the cell cycle during infection. Smaller DNA viruses such as simian virus 40, papillomaviruses, and adenoviruses depend on many cellular enzymes for viral DNA replication and induce cellular replication factors (reviewed in references 30 and 43). Simian virus 40 stimulates cells to synthesize DNA, and the majority of cells acquire a greater-than-G₂ tetraploid DNA content. Human adenovirus E1A proteins induce quiescent cells to enter S phase of the cell cycle by deregulating checkpoint controls, while the E1B proteins block apoptosis and promote a favorable cellular environment for viral replication (10, 14, 36, 39, 45). Large mammalian DNA viruses also arrest the cell cycle during infection. Human cytomegalovirus, for example, seems to inhibit cell cycle progression at multiple points, including the transition from G₁ to S (4, 9, 27). The Vpr protein of human immunodeficiency virus type 1 causes the accumulation of cells in the G₂ phase of the cell cycle and stimulates replication (19, 38). In contrast to human immunodeficiency virus type 1, the oncoviruses require host cell proliferation for productive infection (2).

Baculovirus infection has long been known to induce significant changes in infected cells during the first 6 h of infection, including cytoskeletal rearrangements and host chromatin dispersal within the nucleus, which enlarges during this period (47). The ability of baculoviruses to arrest the cell cycle has not been previously reported, and the involvement of a RING finger protein in this process is especially intriguing. Understanding the interaction between baculoviruses and the cell cycle should lead to new insights into viral pathogenesis. We intend to investigate the basis of the host range dependence on IE2 in the context of virus infections.