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Upregulation of the Golgi Protein GP73 by Adenovirus Infection Requires the E1A CtBP Interaction Domain
Virology 301, 236–246 (2002) doi:10.1006/viro.2002.1523
Upregulation of the Golgi Protein GP73 by Adenovirus Infection Requires the E1A CtBP Interaction Domain Raleigh D. Kladney,* Ann E. Tollefson,† William S. M. Wold,† and Claus J. Fimmel* ,‡ ,1 *GI Section, John Cochran Veterans Affairs Medical Center, St. Louis, Missouri 63106; †Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri 63104; ‡Division of Gastroenterology and Hepatology, Saint Louis University Medical School, St. Louis, Missouri 63104 Received March 15, 2002; returned to author for revision April 9, 2002; accepted April 25, 2002 GP73 is a novel type II Golgi transmembrane protein that is expressed at high levels in the hepatocytes of patients with viral hepatitis (R. D. Kladney, G. A. Bulla, L. Guo, A. L. Mason, A. E. Tollefson, D. J. Simon, Z. Koutoubi, and C. J. Fimmel, 2000, Gene 249, 53–65) and is induced in cultured cells by infection with viruses including adenoviruses. Its biological function and the mechanisms by which its expression may be regulated by viral infection are unknown. Here we report that GP73 is induced at the RNA and protein level in human Hep3B hepatoma cells infected by human Ad5 and Ad2. Hep3B cells were infected with wild-type or mutant adenoviruses. GP73 expression was measured by RNase protection assay, immunoblotting, or immunofluorescence microscopy. GP73 RNA and protein levels were strikingly induced following infection. The rise in GP73 expression coincided with the appearance of the adenovirus E1A and DBP proteins and preceded the expression of the fiber protein, a marker of the late phase of infection. Infection did not affect the expression of giantin, GPP130, or golgin-84, three integral Golgi membrane proteins with structural similarities to GP73. Mapping studies using a panel of mutant adenoviruses demonstrated that the E1A C-terminus, specifically its CtBP interaction domain (CID), is required for GP73 expression. Subsequently, Hep3B cells were transiently transfected with plasmids expressing wild-type or mutant E1A proteins. These studies confirmed that E1A induced GP73 expression via the CID. Our studies establish GP73 as a novel adenovirus-induced cellular protein whose expression is regulated through the CID of the E1A protein. © 2002 Elsevier Science (USA)
Key Words: GP73; Golgi protein; adenovirus; E1A; CtBP; transfection.
INTRODUCTION
Despite the cellular alterations brought about by Ad infection, the majority of host cell genes are not directly affected by the infection (Zantema and van der Eb, 1995). However, a few endogenous proteins have been shown to be expressed at high levels in response to infection. These include heat shock proteins (Glotzer et al., 2000; Nevins, 1982; Simon et al., 1987), cyclins (Spitkovsky et al., 1994), and -tubulin (Stein and Ziff, 1984). In most cases, the biological role for this upregulation is unclear. GP73 is a recently identified type II Golgi membrane protein that is expressed in hepatocytes of patients with adult giant-cell hepatitis, a severe form of hepatitis with a presumed viral etiology (Kladney et al., 2000), as well as in patients with liver disease due to chronic HBV- or HCV-infection (Kladney et al., 2002). The function of GP73 is unknown, but its expression pattern in human liver disease suggests that it might be involved in the cellular response to virus infection. In the present study, we report that GP73 is strikingly upregulated in response to Ad infection of cultured Hep3B hepatoma cells. GP73 induction is dependent upon the C-terminus of the E1A protein, and specifically, its C-terminal binding protein (CtBP) interaction domain (CID). To our knowledge, GP73 is the first cellular gene
Adenovirus (Ad) infection initiates a complex cascade of intracellular events in the host cell (Shenk, 1996). The early phase of infection is characterized by the expression of early Ad genes including the E1A immediate early transcriptional regulator. The late phase which follows the initiation of Ad DNA replication is characterized by the expression of Ad late genes required for assembly of new infectious virions and shutdown of host cell macromolecular synthesis. The molecular mechanisms by which early Ad genes subvert the host cell transcriptional machinery during the early stage of infection have been elucidated over the past decades. For E1A, specific domains have been identified that interact with cellular transcription factors to direct viral gene expression, deregulate the cell cycle, and modify critical intracellular signaling cascades, including interferon, STAT, NFB, IRF-3 and IRF-7, ATF2, c-jun, and other pathways (deGroot et al., 1995; Look et al., 1998; Wathelet et al., 1998).
1 To whom correspondence and reprint requests should be addressed at VA Medical Center, 915 North Grand Boulevard, Building 1, Room A901, St. Louis, MO 63106. Fax: (314) 289-7035. E-mail: [email protected].
0042-6822/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
shown to be upregulated through this particular domain of E1A. RESULTS Adenovirus infection induces GP73 expression in human Hep3B cells We had previously reported that Ad infection increases GP73 levels in HepG2 hepatoma cells (Kladney et al., 2000). GP73 levels were monitored at 24 h postinfection (p.i.), a period well into the late stages of infection. To determine whether an Ad early protein is responsible for induction of GP73, we have now examined GP73 in cells infected in the presence of Ara-C. Ara-C inhibits Ad DNA replication and therefore maintains the infection in the early phase. Hep3B cells were mock-infected or infected with the rec700 wild-type Ad in the presence of 20 g/ml Ara-C (Thomas and Matthews, 1980). At 24 h p.i., cellular RNA and proteins were separately extracted and analyzed for the GP73 mRNA and protein. Similar to our previous observation in HepG2 cells (Kladney et al., 2000), uninfected Hep3B cells expressed barely detectable levels of GP73 (Fig. 1A). In contrast, rec700 infection was associated with a marked upregulation of GP73 expression, both at the mRNA and at the protein level. The expression of GP73 in the presence of Ara-C suggests that this effect occurs during the early phase of infection, independent of Ad DNA replication. Immunofluorescence studies demonstrated specific GP73 reactivity in rare, mock-infected cells (Fig. 1B). Approximately 5% of cells spontaneously expressed GP73 (data not shown). In comparison, essentially all rec700-infected cells displayed GP73 fluorescence at 24 h p.i. Furthermore, the signal intensity in rec700-infected cells was strikingly increased as compared to the mock-infected cells. The pattern of GP73 immunoreactivity was punctate with a perinuclear accentuation, typical of the Golgi pattern in cultured hepatoma cells. Coexpression experiments using giantin, a Golgi marker protein, confirmed that the GP73 immunoreactivity was entirely localized to the Golgi compartment (data not shown). Omission of Ara-C in mock infections did not affect the GP73 fluorescence signals (data not shown). GP73 expression is a feature of early adenovirus infection The finding that GP73 expression could be induced by rec700 infection in the presence of Ara-C suggests that GP73 is induced during the early stages of infection. To relate the expression of GP73 to the time course of Ad infection, we compared GP73 expression in rec700-infected cells with that of the E1A, DNA-binding protein (DBP), and fiber proteins. E1A and DBP are first produced in early infection, whereas fiber is expressed during the late stage of infection after the initiation of Ad
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DNA replication. This experiment was performed in the absence of Ara-C to allow the infection to progress into the late stage. GP73 and viral proteins were detected by immunofluorescence microscopy; the number of Hep3B cells expressing the respective proteins was calculated as a function of time postinfection. As demonstrated in Fig. 2, E1A was present at 5 h p.i. and reached a plateau of ⬎90% of nuclei E1A-positive by 14 h p.i. DBP was first detected at 9 h p.i. and rose steadily until 24 h p.i. when ⬎90% of all nuclei were positive. GP73 expression was present in a small percentage (⬍5%) of cells at the earliest time points following infection; this was comparable to the background expression seen in mock-infected cells (see above). A definitive increase in the percentage of GP73-positive cells was noted at 14 h p.i. The percentage of GP73-expressing cells continued to rise steadily until 24 h p.i., when approximately 90% of the cells stained positive. In comparison, a definitive increase in the percentage of fiber-positive cells became evident only at 20–24 h p.i. These data suggest that the increased GP73 expression is a feature of early Ad infection. Adenovirus infection does not affect the expression of other Golgi membrane proteins To determine whether the effect of rec700 infection was specific to GP73 or instead representative of a generalized upregulation of Golgi membrane proteins, the expression of three resident Golgi membrane proteins, giantin, GPP130, and golgin-84, was examined. Giantin is a C-terminally anchored Golgi transmembrane protein (Linstedt and Hauri, 1993) that was previously found to colocalize with GP73 in 293 cells (Kladney et al., 2000). GPP130 is a type II membrane protein with general structural similarities to GP73, including the presence of a large, acidic C-terminal domain with two coiled-coil motifs and a short cytoplasmic N-terminal domain (Linstedt et al., 1997). Golgin-84 is a type I integral membrane protein with a large cytoplasmic N-terminus containing a coiled-coil domain and a small lumenal C-terminal domain (Bascom et al., 1999). The expression of the Golgi marker proteins was determined by Western blotting (Fig. 3A) and immunofluorescence microscopy (Fig. 3B). As demonstrated in Fig. 3A, giantin, GPP130, and golgin-84 signals were readily apparent in mock-infected Hep3B cells. No changes in expression levels were evident in rec700-infected cells. The immunofluorescence images (Fig. 3B) demonstrated similar Golgi expression patterns and signal intensities for the three proteins in mock-infected and rec700-infected cells. These data suggest that the changes observed with GP73 are not due to a general effect of Ad infection on the expression of Golgi membrane proteins.
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FIG. 1. Adenovirus infection elicits GP73 expression in human Hep3B hepatoma cells. Cells were mock-infected or infected with rec700 wild-type Ad in the presence of Ara-C (20 g/ml) for 24 h. GP73 mRNA and protein levels were measured by ribonuclease protection assay (RPA) and Western blotting, respectively. GP73 immunofluorescence was detected using a rabbit polyclonal primary antibody directed against GP73 and a goat anti-rabbit IgG/Alexa Fluor 488 secondary antibody (Molecular Probes). (A) Top: Representative Western blot of GP73. The GP73 signal is apparent as a broad band with an approximate molecular size of 73 kDa. Middle: GP73 mRNA levels as measured by RPA. A single protected fragment represents the GP73 mRNA. Bottom: RPA for GAPDH mRNA (loading control). Equal RNA loading is evident in both samples. (B) Immunofluorescence detection of GP73 (green fluorescence signal) and corresponding nuclear DNA stain (blue fluorescence signal) in mock-infected and rec700-infected Hep3B cells. The lens magnification was 100-fold. In mock-infected cells, weak GP73 expression can only be detected in occasional cells. Intense GP73 fluorescence is uniformly present in rec700-infected cells. GP73 immunoreactivity is predominantly found in a punctate, perinuclear, and cytoplasmic pattern, typical of Golgi distribution in Hep3B cells. FIG. 3. Ad infection does not affect the expression of giantin, GPP130, or golgin-84. Cells were mock-infected or infected with rec700 in the presence of Ara-C (20 g/ml). The expression of giantin, GPP130, and golgin-84 was measured 24 h p.i. by Western blotting or immunofluorescence microscopy.
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
FIG. 2. GP73 expression is induced during the early stages of Ad infection. Hep3B cells were infected with rec700 and fixed after varying lengths of time ranging from 5 to 24 h. The expression of GP73 (䡺), E1A (F), DBP (Œ), and fiber protein (f) was assessed by indirect immunofluorescence. The percentage of cells staining positively for each of the respective proteins is shown for each time point.
GP73 upregulation requires the E1A CtBP interacting domain The data shown in Figs. 1 and 2 indicate that GP73 expression is upregulated during the early phase of infection, indicating that early viral gene functions might be responsible for this effect. We used a series of mutants defective in specific Ad early genes to determine whether or not these mutants could elicit GP73 expression. When necessary, infections were allowed to proceed for up to 48 h to compensate for variability in the time courses of infection in certain E1A mutants. As shown in Fig. 4A, a robust GP73 signal was detected in Hep3B cells infected with the wild-type strains rec700 and Ad2, respectively, whereas mock-infected cells demonstrated only a minimal signal. Cells infected with the 201.2 mutant (deletion of E1A and E1B) did not express GP73. In contrast, mutants containing deletions that were limited to the E1B genes (dl110 [55K ⫺] and dl111[19K ⫺]) elicited a robust GP73 response, suggesting that E1A but not E1B functions were required. Any major role for the E2 gene was unlikely, given the observation that GP73 induction in cells infected with the wild-type virus was not diminished by Ara-C. A nonreplicating Ad vector lacking the E1A and E1B regions and expressing
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only the E3 proteins (Ad/E3) did not induce GP73. Infection with dl7001 (Ranheim et al., 1993), a mutant with a complete deletion of E3, was associated with a strong GP73 signal (data not shown), establishing that E3 function is dispensable for GP73 expression. Infection with dl808, a mutant containing a large E4 deletion, did not diminish GP73 expression, suggesting that E4 gene function was not required (data not shown). Taken together, these preliminary mapping studies suggested that E1A function alone might be necessary for GP73 expression. We therefore performed more detailed mapping studies focusing on specific domains within the E1A gene. Ad2/ 5pm975 or 12S infections resulted in GP73 levels that were comparable to wild-type infections (Figs. 4A and 4B), suggesting that conserved region 3 (CR3) function was not required. A robust GP73 signal was present in dl1101/1107infected cells, suggesting that functions of the conserved regions 1 and 2 (CR1, CR2) were not required. GP73 expression was not induced in cells infected with dl313, a mutant with a deletion extending into C-terminus of E1A, or in cells infected with the 177-9 mutant containing a large C-terminal deletion in E1A. These data suggest that the E1A C-terminus is required for GP73 expression. This interpretation is supported by a direct comparison of the E1A mutant viruses (Fig. 4B). In this experiment, we measured E1A levels to document that the viral mutants were functioning as expected and at comparable levels. rec700, pm975, and 12S infections elicited robust GP73 and E1A signals (Fig. 4B). GP73 expression was strikingly diminished in 177-9- and dl1135-infected cells, even though E1A expression in dl1135-infected cells was similar to that in 12S-infected cells. The molecular size and signal intensity of E1A in 177-9-infected cells were reduced, most likely due to the loss of antigenic C-terminal epitopes recognized by the M73 monoclonal antibody in the E1A antibody mixture (Arsenault and Webber, 1993). However, the reduced E1A signal was not due to a poorer infection, since 177-9 infection elicited a strong DBP signal (data not shown). Mutant 177-9 lacks aa 178–238 of the Ad2 243R E1A protein, and dl1135 lacks aa 225–238 of the Ad5 243R protein; these deleted regions include the CtBP interacting domain (CID). Therefore, these data suggest that the C-terminal domain of E1A, and specifically its CID, was required for GP73 expression.
(A) Representative results of an immunoblot experiment. Giantin (top), GPP130 (middle), and golgin-84 (bottom) are present in extracts of mock-infected cells. No changes in expression levels are evident following rec700 infection. The exposure times of the immunoblots for the individual proteins varied, and the signal intensities do not reflect the absolute protein levels. (B) Immunofluorescence images. Cells were immunostained for giantin or GPP130 using mouse monoclonal primary antibodies and a goat anti-mouse IgG/Alexa Fluor 549 secondary antibody. Golgin-84 staining was performed using a rabbit polyclonal primary antibody and goat anti-rabbit IgG/Alexa Fluor 488 secondary antibody (Molecular Probes). Immunofluorescence signals for both mock- and rec700-infections display an apparent Golgi distribution. The overall signal intensities are not affected by Ad infection.
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FIG. 4. Expression of GP73 following Ad infection requires the CID of E1A. Hep3B cells were mock-infected or infected with wild-type or mutant Ad viruses for 24 or 48 h in the presence of Ara-C (20 g/ml). GP73 expression was measured by Western blotting. (A) Gross mapping of GP73 expression to E1A. The figure is representative of three independent experiments. (B) Detailed mapping GP73 expression to the C-terminus of E1A. In addition to GP73, E1A protein was measured by Western blotting using a mixture of monoclonal antibodies (Harlow et al., 1985). The molecular size of E1A is reduced in cells infected with 177-9 virus carrying a large C-terminal deletion mutation (arrow).
E1A is sufficient for induction of GP73 and requires the CID To unequivocally establish a role of the E1A CID for GP73 induction, we introduced mutated forms of E1A into Hep3B cells by transient transfection of plasmid constructs and followed GP73 and E1A expression by immunofluorescence microscopy. As shown in Fig. 5A, GP73 (and E1A) expression was negligible in the pUC18 vector-transfected cells. With the 13S- and 12S-transfected cells most cells that expressed E1A also expressed GP73. In contrast, a minority of 177-9- or dl1135transfected cells expressing E1A was positive for GP73. A quantitative analysis demonstrated that approximately 90% of 13S- or 12S-transfected, E1A-positive cells expressed GP73, compared to 29 and 26% of cells transfected with plasmids carrying the C-terminal mutations (Fig. 5B). As mentioned under Materials and Methods, cells were scored positive even if the fluorescence was weak, as was the case with the cells transfected with 177-9 and dl1135 plasmids (Fig. 5A). Therefore, the percentage values do not reflect the true magnitude of the difference in the 13S and 12S cells vs the 177-9 and dl1135 cells. This point is illustrated by the dramatic difference in GP73 levels observed in the corresponding infection studies (Fig. 4). The results of this experiment
indicate that E1A is sufficient for GP73 induction and that the CtBP-interacting domain of E1A is necessary for this function. DISCUSSION Our data indicate that GP73 is a Golgi membrane protein that is upregulated at the RNA and protein levels in response to Ad infection. GP73 joins a small group of cellular proteins that are regulated in a similar fashion. This group includes certain heat shock proteins, cyclins, and -tubulin. Until recently, it was unclear whether the upregulation of these proteins was biologically important for the virus or the host cell or whether it was simply a bystander consequence of the viral need to activate transcription. A recent study by Glotzer (2000) in the avian Ad model of CELO virus infection provided the first direct evidence that heat shock protein expression may be required for productive infection. The authors demonstrated that the antiapoptotic function of Gam1, a CELO protein required for viral replication, is dependent on the expression of hsp70 and hsp40. To our knowledge, no comparable studies have been carried out for any of the other Ad-induced cellular genes. GP73 was first identified in a genetic screen for cellular proteins involved in the development of adult giant-
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
cell hepatitis, a rare form of hepatitis with a postulated viral etiology (Fimmel et al., 1998). Subsequently, we observed that GP73 expression by hepatocytes was strikingly induced in chronic hepatitis B virus and hepatitis C virus infection (Kladney et al., 2002). The absence of suitable in vitro models of productive HBV and HCV infection prompted us to explore alternative in vitro models to study the effect of viral infection on GP73 expression. Initial studies in human HepG2 hepatoma cells suggested that Ad infection provided such a model (Kladney et al., 2000). The present study demonstrates that GP73 expression is strikingly increased in Hep3B cells following infection with Ad. Three distinct but structurally related Golgi membrane proteins were not increased in infection, indicating that the upregulation of GP73 is unlikely to be due to a general effect of Ad infection on Golgi protein expression. Time-course studies suggested that GP73 expression requires the function of one or more early Ad genes. This hypothesis was tested by a series of infections with Ad viruses containing deletion mutations in early genes. These studies demonstrated that GP73 expression required E1A function. Further mapping experiments revealed that the C-terminus of E1A was critical for this effect, in particular the CID. Some functions of conserved regions 1 (CR1), 2 (CR2), and 3 (CR3) did not play a major role, although the slight stimulatory effect of CID-deleted E1A proteins observed in the transfection studies (Fig. 5) leaves open the possibility that alternate domains of E1A might compensate, albeit inefficiently, for the loss of CID. The E1A C-terminus has been identified as an important regulator of Ad gene expression in a rat tumor model. Initial studies with viral mutants carrying deletions in this domain demonstrated a dramatic increase in cell transformation, tumorigenesis, and tumor progression (Subramanian et al., 1989). A novel cellular protein, named CtBP, was shown to bind to a highly conserved amino acid motif (PLDLS) of E1A, the CtBP-interacting domain (Schaeper et al., 1995). Recent studies have demonstrated that CtBP is a member of a novel family of transcriptional co-repressors that recognize PXDLS motifs in a variety of DNA-binding proteins and that function as transcriptional co-repressors in Drosophila, Xenopus, and mammals (Turner and Crossley, 2001). CtBP proteins bind to a variety of transcription factors, including Drosophila Hairy, Snail, and Kru¨ppel, and the SP1-related zinc finger protein, mammalian Basic Kru¨ppel-like Factor/Kru¨ppel-like Factor 3 (BLKF/KLF3). CtBP/E1A interactions result in the expression of epithelial genes such as desmoglein-2, plakoglobin, and E-cadherin (Fischer and Quinlan, 1998; Grooteclaes and Frisch, 2000). E1A-mediated de-repression by sequestration of CtBP may have wide-range effect on cellular genes, as recently demonstrated by Sundqvist et al. (2001). These authors performed functional knock-out studies in cell lines that
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allowed the inducible expression of the CID of E1A and identified differentially expressed genes by gene array analysis. They found that the expression of approximately 7% of the analyzed genes was regulated in a CID-dependent fashion. Interestingly, the most pronounced effects were observed on genes involved in cell-cycle regulation, apoptosis, and intracellular communication, including cyclin-dependent kinase 5 activator, MEKK, PKC-␥, TRAIL receptor, death domain-containing protein CRADD, caspase 9-precursor, death-associated protein kinase 1, and RATS1 (Sundqvist et al., 2001). We hypothesize that Ad infection, through binding of the E1A CID to CtBP, results in GP73 expression by a derepression mechanism at the GP73 promoter. A potential additional function for CtBP has recently been revealed by the work of Corda and colleagues (Spano et al., 1999; Weigert et al., 1999). These authors found that CtBP/BARS, a rat homolog of human CtBP1, has a direct effect on Golgi lipid metabolism. CtBP/BARS catalyzed the acylation of lysophosphatidic acid to phosphatidic acid in pure lipid systems and in Golgi membranes. Ribosylation of CtBP/BARS by Brefeldin A was associated with the disassembly of the Golgi complex. These data suggest that CtBP might play a direct, enzymatic role in the formation and maintenance of the Golgi complex and raises the hypothesis that a CID-mediated interaction between Ad and CtBP at the Golgi apparatus somehow results in the increased expression of GP73. To our knowledge, GP73 is the first cellular Golgi protein shown to be upregulated by Ad infection. Our studies to date have not demonstrated an obvious function for GP73, and its structure provides no specific clues. In principle, Golgi membrane proteins may have the following four basic functions: (1) posttranslational modification of proteins, including glycosylation; (2) transport and sorting of secretory and resident proteins; (3) establishment and regulation of the Golgi structure; and (4) specific roles in cell signaling and apoptosis regulation. The absence of significant homologies or shared motifs in GP73 with the known Golgi glycosidases argues against the first possibility. The lack of a significant cytoplasmic domain argues against a role in vesicle trafficking and tethering (“Velcro function”). Consequently, we are considering the possibility that GP73 functions by interacting with viral or cellular proteins undergoing modification within the Golgi lumen. The Ad model established in this article may provide us with a suitable experimental model to elucidate the role(s) of GP73 in viral infection. Our study suggests that the Golgi apparatus might be a specific target in Ad infection. Precedents for viral interference with Golgi structure or functions have been established in studies of HSV 1 (Campadelli et al., 1993; Ward et al., 1998), poliovirus (Sandoval and Carrasco, 1997), and corona mouse hepatitis virus (Lavi et al., 1996). Interestingly, early electron microscopic studies
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have hinted at similar morphological alterations in the Golgi apparatus of Ad-infected cells (Lenk et al., 1980). In summary, our study establishes GP73 as a novel protein with upregulated expression in Ad infection. The upregulation appears to be specific to this particular Golgi protein and requires the function of the E1A Cterminus and, specifically, its CID. Its biochemical function and role in the cellular response to Ad infection remain to be elucidated. MATERIALS AND METHODS Cell lines Human Hep3B (Hep 3B2.1–7) cells were obtained from the American Type Culture Collection (ATCC). This cell line is derived from a hepatocellular cancer and contains an integrated copy of the hepatitis B virus genome (Knowles et al., 1980). Hep3B cells resemble normal hepatocytes by expressing a number of hepatocyte marker proteins, including alpha fetoprotein, albumin, ␣-2-macroglobulin, and transferrin (Knowles et al., 1980). They are considered to be highly differentiated (Kawai et al., 2001). Hep3B cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin and cultured at 37°C in 95% air and 5% CO 2. Viruses and infection The Ad2 wild-type (wt) virus was obtained from ATCC. rec700 is a Ad5-Ad2-Ad5 recombinant that has a wt E1A region and is used as a wt control (Wold et al., 1986). The mutant E1A viruses (see Table 1) are named Ad2/ 5pm975 (13S only; carrying a deletion in the 12S donor splice site) (Montell et al., 1982), 12S [Ad5 derivative; 12S only, conserved region (CR3) transactivation defective] (Stephens and Harlow, 1987), dl313 (Ad5 derivative; 2350-bp deletion of map units 3.5–10.6, deleting the second exon of E1A and E1B) (Jones and Shenk, 1979), dl201.2 (Ad2 derivative; deletion of map units ⬃2–6, E1A-, E1B-) (Brusca and Ehinnadurai, 1983), dl1101/1107 [Ad5 derivative containing exon 1 deletion of aa 4–25 (dl1101) and aa 111–163(dl1107), resulting in the inability of E1A proteins to bind p300 and p105-Rb] (Howe et al., 1990), dl110 (Ad5 derivative, deletion of bp 2333–2804, resulting
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Mutant Viruses Used in This Study a Virus rec700 dl201.2 Ad2/5 pm975 12S d/313 dl1101/1107 177-9 dl1135
dl110 dl111 Ad/E3 dl808
Phenotype [reference] Wild-type E1A [Wold et al., 1986] Lacks E1A and E1B [Brusca and Chinnadurai, 1983] 289R only [Montell et al., 1982] 243R only [Stephens and Harlow, 1987] Lacks aa 174–243 of the EIA c-terminus (including CID) [Jones and Shenk, 1979] Defective in CR1 and CR2 [Howe et al., 1990] Lacks aa 178–238 of the E1A C-terminus (including CID) b [Subramanian et al., 1989] Lacks aa 225–238 of the E1A C-terminus (including CID) b [Arsenault and Webber, 1993], [Boyd et al., 1993] Lacks E1B 55K [Babiss and Ginsberg, 1984] Lacks E1B 19K [Babiss et al., 1984] E3 proteins only (except ADP) [Tollefson et al., 2001] Lacks E4 [Challberg and Ketner, 1981]
a The following mutants were derived from the parental virus dl309: Ad2/5 pm975, 12S, dl313, dl1101/1107, d/1135, dl110, dl111. b 12S background.
in a truncated form of the 55-kDa protein of E1B) (Babiss and Ginsberg, 1984), dl111 (derived from the Ad5 mutant dl309, E1B ⫺19K ⫺, RID ⫺, 14.7K ⫺) (Babiss et al., 1984), 177-9 (Ad2 derivative, 12S E1A background, deletion of aa 178–238 of E1A, defective in CtBP binding) (Subramanian et al., 1989), and dl1135 (Ad5 derivative in a 12S background; deletion of aa 225–238 of E1A, lacking the CtBP binding domain) (Arsenault and Webber, 1993; Boyd et al., 1993). dl808 is an Ad5 derivative that lacks E4 function (1030-bp deletion of map units 92.0–97.1, defective in E4 subregions 1 or 2 through 7) (Challberg and Ketner, 1981). All mutants except rec700, dl201.2, 177-9, and dl808 were derived from the parental virus dl309 which has a deletion in the E3 region that removes the genes for E3-RID and E3-14.7K. Ad/E3 is an E1-minus replication-deficient Ad vector with an expression cassette consisting of the E3 genes driven off the CMV promoter inserted into the E1 region (Tollefson et al., 2001). The E1A deletion mutants, Ad/E3 and dl808, were grown in monolayers of human 293 cells (Green and
FIG. 5. GP73 expression is induced by transfection with E1A expression plasmids; induction requires the CID of E1A. Hep3B cells were transfected with a control plasmid (pUC18) or with plasmids carrying E1A wild-type 13S or 12S genes, the large C-terminal deletion mutation (177-9) of E1A in a 12S background, or a deletion of the CtBP-binding domain (dl1135) in a 12S E1A background. GP73 protein expression was assessed by immunofluorescence (green fluorescence signal) using a GP73-specific polyclonal antibody. E1A coexpression was detected using the pooled mouse monoclonal antibody mix (red fluorescence). Nuclear DNA was visualized using DAPI (blue fluorescence). (A) Representative fluorescence images from transiently transfected cells. E1A and GP73 signals are absent in pUC18-transfected cells. 13S- and 12S-transfected cells exhibit a marked increase in GP73 signal, and GP73 and E1A are coexpressed extensively. In comparison, a minority of E1A-expressing cells transfected with the 177-9 or dl1135 plasmids expressed GP73. (B) Quantitation of GP73/E1A coexpression. Immunofluorescence data were quantitated by cell counting and expressed as the percentage of E1A-positive cells with an unequivocal GP73 signal. Results are shown as means ⫾ SEM. Extensive coexpression of GP73 and E1A was found in 13S- and 12S-transfected cells. In contrast, a minority of cells transfected with 177-9 or dl1135 plasmids coexpressed GP73.
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Wold, 1979). Hep3B cells were infected with 100 to 300 PFU of the appropriate virus, and the infection was allowed to proceed for 24 to 48 h prior to harvest. In some experiments, 1--D-arabinofuranosylcytosine (Ara-C) (20 g/ml) was added to the culture media to prevent adenoviral DNA replication (Thomas and Matthews, 1980). Transfections Hep3B cells were transiently transfected with pUC18 vector (Life Technologies, Gaithersburg, MD) or with pUC18-derived plasmids carrying Ad wt E1A 13S or 12S genes, or E1A with mutations within the C-terminus (177-9, dl1135). The terms 13S and 12S refer to the sizes of E1A-derived mRNAs that encode the 289R and 243R proteins, respectively. The Hep3B cells were grown to approximately 75% confluency on six-well plates containing coverslips. Transfections were carried out using Lipofectamine 2000, following the manufacturer’s protocol (Life Technologies). Aliquots of transfection-grade plasmid DNA were diluted in Opti-MEM I Reduced Serum (Life Technologies). Lipofectamine 2000 was suspended in Opti-MEM I (1:20 v/v) for 5 min. Aliquots of diluted plasmid DNA and Lipofectamine 2000 suspension were combined in microcentrifuge tubes and incubated at room temperature (RT) for 20 min. Immediately prior to transfection, culture media was removed from the Hep3B cells and replaced with Opti-MEM I. Aliquots of plasmid DNA/Lipofectamine 2000 complex (corresponding to 5 g plasmid DNA and 12.5 l Lipofectamine 2000 per well) were added to the cells, followed by a 2.5 h incubation at 37°C. The medium was removed and replaced with DMEM/10% FBS. Cells were incubated for 48–72 h and then fixed for immunofluorescence microscopy (see below). RNase protection assays Total cellular RNA was isolated from cultured cells using a rapid, guanidine-based method (RNA Stat 60, Tel-Test, Friendswood, TX). A cRNA antisense probe directed against the GP73 cDNA was prepared as previously described (Kladney et al., 2000). Twenty microgram aliquots of RNA were hybridized to the probe for the ribonuclease protection assay (RPA) using a commercial kit (RPAIII, Ambion, Austin, TX). The RPA control probe was directed against human GAPDH. Antibodies GP73 was measured using a polyclonal rabbit antiserum raised against recombinant human GP73 (Kladney et al., 2000). Giantin was detected using a rabbit polyclonal antiserum directed against the N-terminal moiety of the protein for immunoblotting assays or a mouse monoclonal antibody for immunofluorescence experiments (Linstedt and Hauri, 1993). GPP130 and golgin-84 were detected using a mouse monoclonal anti-
body or a rabbit polyclonal antiserum against the human proteins, respectively (Linstedt et al., 1997; Bascom et al., 1999). E1A was detected with a monoclonal antibody mixture consisting of pooled supernatants of Harlow M1, M2, M37, M58, and M73 hybridomas (Harlow et al., 1985). Adenoviral DNA binding protein (DBP) was detected with a rabbit polyclonal antibody (Lillie et al., 1987), and fiber protein was detected with a mouse monoclonal antibody (generously provided by Jeff Engler). Western blotting Hep3B cells were washed three times with PBS followed by the addition of lysis buffer (62.5 mM Tris–Cl pH 6.8, 20 mM DTT, 10% glycerol, 1% SDS). Adherent cells were removed with plastic scrapers, and the lysates transferred to microcentrifuge tubes. Samples were boiled for 3 min, sonicated for 30 s, and centrifuged at 14,000 rpm at 4°C for 20 min. The cleared lysates were stored at ⫺80°C and analyzed in batch fashion. Protein concentrations of the lysates were determined using a modification of the micro-Lowry method with bovine serum albumin as a standard (Protein Assay Kit, Sigma, St. Louis, MO). Aliquots corresponding to 15 g of total cellular protein were subjected to SDS–PAGE using precast 10% Tris–HCl gels (Ready Gels, Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris–Cl, 192 mM glycine, and 0.1% SDS. Gels were run at 100 V for 8 min and then at 200 V for 30 min. A control sample consisting of 7.5 g of total cellular protein from HEK293 cells (ATCC) was included in each gel run. 293 cells were found to give a robust GP73 protein signal, which was used as a standard to facilitate quantitative comparisons between gels. Separated proteins were transferred to PVDF membranes (PVDF-Plus, Micron Separations, Westborough, MA) with a semidry transfer cell (TransBlot SD, Bio-Rad), using an optimized buffer (48 mM Tris–Cl, 39 mM glycine, 0.005% SDS, 20% methanol) and standardized transfer conditions (10 V, 30 min). Membranes were kept overnight in blocking buffer [10% nonfat dry milk in Tris-buffered saline (pH 7.6) containing 0.5% Tween 20 (TBST)] at 4°C. They were then washed three times in wash buffer (0.5% nonfat dry milk in TBST) and incubated for 1 h at RT in 15 ml of wash buffer containing primary antibodies. Several washes with high-salt buffer (0.5% nonfat dry milk in TBST containing 0.5 M NaCl) and wash buffer were then performed. This was followed by an incubation for 30 min in washing buffer with 1:1000 v/v preadsorbed anti-rabbit or antimouse IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA). After repeat washes with high-salt and wash buffer, GP73 or E1A immunoreactivity was visualized by enhanced chemiluminescence using a commercial kit (ECL, Amersham Pharmacia Biotech, Buckinghamshire, U.K.).
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
Immunofluorescence microscopy Hep3B cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 10 min at RT, permeabilized with methanol containing 4,6-diamidino-2-phenylindole (DAPI) for 6 min at ⫺20°C, and washed with ⫺20°C methanol without DAPI for 2 min at RT. For GPP130 and golgin-84 staining, the paraformaldehyde step above was omitted. After PBS washes, coverslips were incubated with a blocking solution containing 4% (v/v) goat serum in PBS for 20 min. This was followed by a 1 h incubation with primary antibodies at RT. After two PBS washes, coverslips were incubated with secondary antibodies in PBS containing 4% goat serum. Secondary antibodies were goat anti-mouse IgG/Alexa Fluor 594 or goat anti-rabbit IgG/Alexa Fluor 488 conjugates (1:400 v/v) (Molecular Probes, Eugene, OR). Following two PBS washes, coverslips were mounted in elvanol-containing p-phenylenediamine (Heimer and Taylor, 1974). The images shown in Fig. 5A were viewed using a Nikon Optiphot-2 microscope equipped with epifluorescence and photographed using Kodak Elite CHROME 400 film. All other fluorescent images were captured using a Nikon DXM1200 digital still camera with ACT-1 software (Nikon, Tokyo, Japan). The final images were processed using Photoshop. Quantitation of immunofluorescent signals The cellular expression of marker proteins by immunofluorescence microscopy was quantitated by cell counting. All measurements were performed by one investigator (C.J.F.). Whenever possible, counting was performed without the investigator’s knowledge of the experimental condition. All cells in a visual field (counted by DAPI fluorescence) that were positive for a chosen marker were included in the counting, regardless of the signal strength. Typically, 500 to 2000 cells (corresponding to 10 to 40 randomly selected fields, respectively) were assessed for each data point. ACKNOWLEDGMENTS This work was supported by a Veterans Affairs Merit Review grant to C.J.F. and by NIH Grants CA58538 and CA71704 to W.S.M.W. We thank T. Subramanian and G. Chinnadurai for providing 177-9, dl1135, and for the E1A plasmids, Jeff Engler for the fiber antibody, Maurice Green for the DBP antibody, Robert Nussbaum for the golgin-84 antibody, and Adam Linstedt for the giantin and GPP130 antibodies.
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Upregulation of the Golgi Protein GP73 by Adenovirus Infection Requires the E1A CtBP Interaction Domain Raleigh D. Kladney,* Ann E. Tollefson,† William S. M. Wold,† and Claus J. Fimmel* ,‡ ,1 *GI Section, John Cochran Veterans Affairs Medical Center, St. Louis, Missouri 63106; †Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri 63104; ‡Division of Gastroenterology and Hepatology, Saint Louis University Medical School, St. Louis, Missouri 63104 Received March 15, 2002; returned to author for revision April 9, 2002; accepted April 25, 2002 GP73 is a novel type II Golgi transmembrane protein that is expressed at high levels in the hepatocytes of patients with viral hepatitis (R. D. Kladney, G. A. Bulla, L. Guo, A. L. Mason, A. E. Tollefson, D. J. Simon, Z. Koutoubi, and C. J. Fimmel, 2000, Gene 249, 53–65) and is induced in cultured cells by infection with viruses including adenoviruses. Its biological function and the mechanisms by which its expression may be regulated by viral infection are unknown. Here we report that GP73 is induced at the RNA and protein level in human Hep3B hepatoma cells infected by human Ad5 and Ad2. Hep3B cells were infected with wild-type or mutant adenoviruses. GP73 expression was measured by RNase protection assay, immunoblotting, or immunofluorescence microscopy. GP73 RNA and protein levels were strikingly induced following infection. The rise in GP73 expression coincided with the appearance of the adenovirus E1A and DBP proteins and preceded the expression of the fiber protein, a marker of the late phase of infection. Infection did not affect the expression of giantin, GPP130, or golgin-84, three integral Golgi membrane proteins with structural similarities to GP73. Mapping studies using a panel of mutant adenoviruses demonstrated that the E1A C-terminus, specifically its CtBP interaction domain (CID), is required for GP73 expression. Subsequently, Hep3B cells were transiently transfected with plasmids expressing wild-type or mutant E1A proteins. These studies confirmed that E1A induced GP73 expression via the CID. Our studies establish GP73 as a novel adenovirus-induced cellular protein whose expression is regulated through the CID of the E1A protein. © 2002 Elsevier Science (USA)
Key Words: GP73; Golgi protein; adenovirus; E1A; CtBP; transfection.
INTRODUCTION
Despite the cellular alterations brought about by Ad infection, the majority of host cell genes are not directly affected by the infection (Zantema and van der Eb, 1995). However, a few endogenous proteins have been shown to be expressed at high levels in response to infection. These include heat shock proteins (Glotzer et al., 2000; Nevins, 1982; Simon et al., 1987), cyclins (Spitkovsky et al., 1994), and -tubulin (Stein and Ziff, 1984). In most cases, the biological role for this upregulation is unclear. GP73 is a recently identified type II Golgi membrane protein that is expressed in hepatocytes of patients with adult giant-cell hepatitis, a severe form of hepatitis with a presumed viral etiology (Kladney et al., 2000), as well as in patients with liver disease due to chronic HBV- or HCV-infection (Kladney et al., 2002). The function of GP73 is unknown, but its expression pattern in human liver disease suggests that it might be involved in the cellular response to virus infection. In the present study, we report that GP73 is strikingly upregulated in response to Ad infection of cultured Hep3B hepatoma cells. GP73 induction is dependent upon the C-terminus of the E1A protein, and specifically, its C-terminal binding protein (CtBP) interaction domain (CID). To our knowledge, GP73 is the first cellular gene
Adenovirus (Ad) infection initiates a complex cascade of intracellular events in the host cell (Shenk, 1996). The early phase of infection is characterized by the expression of early Ad genes including the E1A immediate early transcriptional regulator. The late phase which follows the initiation of Ad DNA replication is characterized by the expression of Ad late genes required for assembly of new infectious virions and shutdown of host cell macromolecular synthesis. The molecular mechanisms by which early Ad genes subvert the host cell transcriptional machinery during the early stage of infection have been elucidated over the past decades. For E1A, specific domains have been identified that interact with cellular transcription factors to direct viral gene expression, deregulate the cell cycle, and modify critical intracellular signaling cascades, including interferon, STAT, NFB, IRF-3 and IRF-7, ATF2, c-jun, and other pathways (deGroot et al., 1995; Look et al., 1998; Wathelet et al., 1998).
1 To whom correspondence and reprint requests should be addressed at VA Medical Center, 915 North Grand Boulevard, Building 1, Room A901, St. Louis, MO 63106. Fax: (314) 289-7035. E-mail: [email protected].
0042-6822/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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shown to be upregulated through this particular domain of E1A. RESULTS Adenovirus infection induces GP73 expression in human Hep3B cells We had previously reported that Ad infection increases GP73 levels in HepG2 hepatoma cells (Kladney et al., 2000). GP73 levels were monitored at 24 h postinfection (p.i.), a period well into the late stages of infection. To determine whether an Ad early protein is responsible for induction of GP73, we have now examined GP73 in cells infected in the presence of Ara-C. Ara-C inhibits Ad DNA replication and therefore maintains the infection in the early phase. Hep3B cells were mock-infected or infected with the rec700 wild-type Ad in the presence of 20 g/ml Ara-C (Thomas and Matthews, 1980). At 24 h p.i., cellular RNA and proteins were separately extracted and analyzed for the GP73 mRNA and protein. Similar to our previous observation in HepG2 cells (Kladney et al., 2000), uninfected Hep3B cells expressed barely detectable levels of GP73 (Fig. 1A). In contrast, rec700 infection was associated with a marked upregulation of GP73 expression, both at the mRNA and at the protein level. The expression of GP73 in the presence of Ara-C suggests that this effect occurs during the early phase of infection, independent of Ad DNA replication. Immunofluorescence studies demonstrated specific GP73 reactivity in rare, mock-infected cells (Fig. 1B). Approximately 5% of cells spontaneously expressed GP73 (data not shown). In comparison, essentially all rec700-infected cells displayed GP73 fluorescence at 24 h p.i. Furthermore, the signal intensity in rec700-infected cells was strikingly increased as compared to the mock-infected cells. The pattern of GP73 immunoreactivity was punctate with a perinuclear accentuation, typical of the Golgi pattern in cultured hepatoma cells. Coexpression experiments using giantin, a Golgi marker protein, confirmed that the GP73 immunoreactivity was entirely localized to the Golgi compartment (data not shown). Omission of Ara-C in mock infections did not affect the GP73 fluorescence signals (data not shown). GP73 expression is a feature of early adenovirus infection The finding that GP73 expression could be induced by rec700 infection in the presence of Ara-C suggests that GP73 is induced during the early stages of infection. To relate the expression of GP73 to the time course of Ad infection, we compared GP73 expression in rec700-infected cells with that of the E1A, DNA-binding protein (DBP), and fiber proteins. E1A and DBP are first produced in early infection, whereas fiber is expressed during the late stage of infection after the initiation of Ad
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DNA replication. This experiment was performed in the absence of Ara-C to allow the infection to progress into the late stage. GP73 and viral proteins were detected by immunofluorescence microscopy; the number of Hep3B cells expressing the respective proteins was calculated as a function of time postinfection. As demonstrated in Fig. 2, E1A was present at 5 h p.i. and reached a plateau of ⬎90% of nuclei E1A-positive by 14 h p.i. DBP was first detected at 9 h p.i. and rose steadily until 24 h p.i. when ⬎90% of all nuclei were positive. GP73 expression was present in a small percentage (⬍5%) of cells at the earliest time points following infection; this was comparable to the background expression seen in mock-infected cells (see above). A definitive increase in the percentage of GP73-positive cells was noted at 14 h p.i. The percentage of GP73-expressing cells continued to rise steadily until 24 h p.i., when approximately 90% of the cells stained positive. In comparison, a definitive increase in the percentage of fiber-positive cells became evident only at 20–24 h p.i. These data suggest that the increased GP73 expression is a feature of early Ad infection. Adenovirus infection does not affect the expression of other Golgi membrane proteins To determine whether the effect of rec700 infection was specific to GP73 or instead representative of a generalized upregulation of Golgi membrane proteins, the expression of three resident Golgi membrane proteins, giantin, GPP130, and golgin-84, was examined. Giantin is a C-terminally anchored Golgi transmembrane protein (Linstedt and Hauri, 1993) that was previously found to colocalize with GP73 in 293 cells (Kladney et al., 2000). GPP130 is a type II membrane protein with general structural similarities to GP73, including the presence of a large, acidic C-terminal domain with two coiled-coil motifs and a short cytoplasmic N-terminal domain (Linstedt et al., 1997). Golgin-84 is a type I integral membrane protein with a large cytoplasmic N-terminus containing a coiled-coil domain and a small lumenal C-terminal domain (Bascom et al., 1999). The expression of the Golgi marker proteins was determined by Western blotting (Fig. 3A) and immunofluorescence microscopy (Fig. 3B). As demonstrated in Fig. 3A, giantin, GPP130, and golgin-84 signals were readily apparent in mock-infected Hep3B cells. No changes in expression levels were evident in rec700-infected cells. The immunofluorescence images (Fig. 3B) demonstrated similar Golgi expression patterns and signal intensities for the three proteins in mock-infected and rec700-infected cells. These data suggest that the changes observed with GP73 are not due to a general effect of Ad infection on the expression of Golgi membrane proteins.
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FIG. 1. Adenovirus infection elicits GP73 expression in human Hep3B hepatoma cells. Cells were mock-infected or infected with rec700 wild-type Ad in the presence of Ara-C (20 g/ml) for 24 h. GP73 mRNA and protein levels were measured by ribonuclease protection assay (RPA) and Western blotting, respectively. GP73 immunofluorescence was detected using a rabbit polyclonal primary antibody directed against GP73 and a goat anti-rabbit IgG/Alexa Fluor 488 secondary antibody (Molecular Probes). (A) Top: Representative Western blot of GP73. The GP73 signal is apparent as a broad band with an approximate molecular size of 73 kDa. Middle: GP73 mRNA levels as measured by RPA. A single protected fragment represents the GP73 mRNA. Bottom: RPA for GAPDH mRNA (loading control). Equal RNA loading is evident in both samples. (B) Immunofluorescence detection of GP73 (green fluorescence signal) and corresponding nuclear DNA stain (blue fluorescence signal) in mock-infected and rec700-infected Hep3B cells. The lens magnification was 100-fold. In mock-infected cells, weak GP73 expression can only be detected in occasional cells. Intense GP73 fluorescence is uniformly present in rec700-infected cells. GP73 immunoreactivity is predominantly found in a punctate, perinuclear, and cytoplasmic pattern, typical of Golgi distribution in Hep3B cells. FIG. 3. Ad infection does not affect the expression of giantin, GPP130, or golgin-84. Cells were mock-infected or infected with rec700 in the presence of Ara-C (20 g/ml). The expression of giantin, GPP130, and golgin-84 was measured 24 h p.i. by Western blotting or immunofluorescence microscopy.
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
FIG. 2. GP73 expression is induced during the early stages of Ad infection. Hep3B cells were infected with rec700 and fixed after varying lengths of time ranging from 5 to 24 h. The expression of GP73 (䡺), E1A (F), DBP (Œ), and fiber protein (f) was assessed by indirect immunofluorescence. The percentage of cells staining positively for each of the respective proteins is shown for each time point.
GP73 upregulation requires the E1A CtBP interacting domain The data shown in Figs. 1 and 2 indicate that GP73 expression is upregulated during the early phase of infection, indicating that early viral gene functions might be responsible for this effect. We used a series of mutants defective in specific Ad early genes to determine whether or not these mutants could elicit GP73 expression. When necessary, infections were allowed to proceed for up to 48 h to compensate for variability in the time courses of infection in certain E1A mutants. As shown in Fig. 4A, a robust GP73 signal was detected in Hep3B cells infected with the wild-type strains rec700 and Ad2, respectively, whereas mock-infected cells demonstrated only a minimal signal. Cells infected with the 201.2 mutant (deletion of E1A and E1B) did not express GP73. In contrast, mutants containing deletions that were limited to the E1B genes (dl110 [55K ⫺] and dl111[19K ⫺]) elicited a robust GP73 response, suggesting that E1A but not E1B functions were required. Any major role for the E2 gene was unlikely, given the observation that GP73 induction in cells infected with the wild-type virus was not diminished by Ara-C. A nonreplicating Ad vector lacking the E1A and E1B regions and expressing
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only the E3 proteins (Ad/E3) did not induce GP73. Infection with dl7001 (Ranheim et al., 1993), a mutant with a complete deletion of E3, was associated with a strong GP73 signal (data not shown), establishing that E3 function is dispensable for GP73 expression. Infection with dl808, a mutant containing a large E4 deletion, did not diminish GP73 expression, suggesting that E4 gene function was not required (data not shown). Taken together, these preliminary mapping studies suggested that E1A function alone might be necessary for GP73 expression. We therefore performed more detailed mapping studies focusing on specific domains within the E1A gene. Ad2/ 5pm975 or 12S infections resulted in GP73 levels that were comparable to wild-type infections (Figs. 4A and 4B), suggesting that conserved region 3 (CR3) function was not required. A robust GP73 signal was present in dl1101/1107infected cells, suggesting that functions of the conserved regions 1 and 2 (CR1, CR2) were not required. GP73 expression was not induced in cells infected with dl313, a mutant with a deletion extending into C-terminus of E1A, or in cells infected with the 177-9 mutant containing a large C-terminal deletion in E1A. These data suggest that the E1A C-terminus is required for GP73 expression. This interpretation is supported by a direct comparison of the E1A mutant viruses (Fig. 4B). In this experiment, we measured E1A levels to document that the viral mutants were functioning as expected and at comparable levels. rec700, pm975, and 12S infections elicited robust GP73 and E1A signals (Fig. 4B). GP73 expression was strikingly diminished in 177-9- and dl1135-infected cells, even though E1A expression in dl1135-infected cells was similar to that in 12S-infected cells. The molecular size and signal intensity of E1A in 177-9-infected cells were reduced, most likely due to the loss of antigenic C-terminal epitopes recognized by the M73 monoclonal antibody in the E1A antibody mixture (Arsenault and Webber, 1993). However, the reduced E1A signal was not due to a poorer infection, since 177-9 infection elicited a strong DBP signal (data not shown). Mutant 177-9 lacks aa 178–238 of the Ad2 243R E1A protein, and dl1135 lacks aa 225–238 of the Ad5 243R protein; these deleted regions include the CtBP interacting domain (CID). Therefore, these data suggest that the C-terminal domain of E1A, and specifically its CID, was required for GP73 expression.
(A) Representative results of an immunoblot experiment. Giantin (top), GPP130 (middle), and golgin-84 (bottom) are present in extracts of mock-infected cells. No changes in expression levels are evident following rec700 infection. The exposure times of the immunoblots for the individual proteins varied, and the signal intensities do not reflect the absolute protein levels. (B) Immunofluorescence images. Cells were immunostained for giantin or GPP130 using mouse monoclonal primary antibodies and a goat anti-mouse IgG/Alexa Fluor 549 secondary antibody. Golgin-84 staining was performed using a rabbit polyclonal primary antibody and goat anti-rabbit IgG/Alexa Fluor 488 secondary antibody (Molecular Probes). Immunofluorescence signals for both mock- and rec700-infections display an apparent Golgi distribution. The overall signal intensities are not affected by Ad infection.
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FIG. 4. Expression of GP73 following Ad infection requires the CID of E1A. Hep3B cells were mock-infected or infected with wild-type or mutant Ad viruses for 24 or 48 h in the presence of Ara-C (20 g/ml). GP73 expression was measured by Western blotting. (A) Gross mapping of GP73 expression to E1A. The figure is representative of three independent experiments. (B) Detailed mapping GP73 expression to the C-terminus of E1A. In addition to GP73, E1A protein was measured by Western blotting using a mixture of monoclonal antibodies (Harlow et al., 1985). The molecular size of E1A is reduced in cells infected with 177-9 virus carrying a large C-terminal deletion mutation (arrow).
E1A is sufficient for induction of GP73 and requires the CID To unequivocally establish a role of the E1A CID for GP73 induction, we introduced mutated forms of E1A into Hep3B cells by transient transfection of plasmid constructs and followed GP73 and E1A expression by immunofluorescence microscopy. As shown in Fig. 5A, GP73 (and E1A) expression was negligible in the pUC18 vector-transfected cells. With the 13S- and 12S-transfected cells most cells that expressed E1A also expressed GP73. In contrast, a minority of 177-9- or dl1135transfected cells expressing E1A was positive for GP73. A quantitative analysis demonstrated that approximately 90% of 13S- or 12S-transfected, E1A-positive cells expressed GP73, compared to 29 and 26% of cells transfected with plasmids carrying the C-terminal mutations (Fig. 5B). As mentioned under Materials and Methods, cells were scored positive even if the fluorescence was weak, as was the case with the cells transfected with 177-9 and dl1135 plasmids (Fig. 5A). Therefore, the percentage values do not reflect the true magnitude of the difference in the 13S and 12S cells vs the 177-9 and dl1135 cells. This point is illustrated by the dramatic difference in GP73 levels observed in the corresponding infection studies (Fig. 4). The results of this experiment
indicate that E1A is sufficient for GP73 induction and that the CtBP-interacting domain of E1A is necessary for this function. DISCUSSION Our data indicate that GP73 is a Golgi membrane protein that is upregulated at the RNA and protein levels in response to Ad infection. GP73 joins a small group of cellular proteins that are regulated in a similar fashion. This group includes certain heat shock proteins, cyclins, and -tubulin. Until recently, it was unclear whether the upregulation of these proteins was biologically important for the virus or the host cell or whether it was simply a bystander consequence of the viral need to activate transcription. A recent study by Glotzer (2000) in the avian Ad model of CELO virus infection provided the first direct evidence that heat shock protein expression may be required for productive infection. The authors demonstrated that the antiapoptotic function of Gam1, a CELO protein required for viral replication, is dependent on the expression of hsp70 and hsp40. To our knowledge, no comparable studies have been carried out for any of the other Ad-induced cellular genes. GP73 was first identified in a genetic screen for cellular proteins involved in the development of adult giant-
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
cell hepatitis, a rare form of hepatitis with a postulated viral etiology (Fimmel et al., 1998). Subsequently, we observed that GP73 expression by hepatocytes was strikingly induced in chronic hepatitis B virus and hepatitis C virus infection (Kladney et al., 2002). The absence of suitable in vitro models of productive HBV and HCV infection prompted us to explore alternative in vitro models to study the effect of viral infection on GP73 expression. Initial studies in human HepG2 hepatoma cells suggested that Ad infection provided such a model (Kladney et al., 2000). The present study demonstrates that GP73 expression is strikingly increased in Hep3B cells following infection with Ad. Three distinct but structurally related Golgi membrane proteins were not increased in infection, indicating that the upregulation of GP73 is unlikely to be due to a general effect of Ad infection on Golgi protein expression. Time-course studies suggested that GP73 expression requires the function of one or more early Ad genes. This hypothesis was tested by a series of infections with Ad viruses containing deletion mutations in early genes. These studies demonstrated that GP73 expression required E1A function. Further mapping experiments revealed that the C-terminus of E1A was critical for this effect, in particular the CID. Some functions of conserved regions 1 (CR1), 2 (CR2), and 3 (CR3) did not play a major role, although the slight stimulatory effect of CID-deleted E1A proteins observed in the transfection studies (Fig. 5) leaves open the possibility that alternate domains of E1A might compensate, albeit inefficiently, for the loss of CID. The E1A C-terminus has been identified as an important regulator of Ad gene expression in a rat tumor model. Initial studies with viral mutants carrying deletions in this domain demonstrated a dramatic increase in cell transformation, tumorigenesis, and tumor progression (Subramanian et al., 1989). A novel cellular protein, named CtBP, was shown to bind to a highly conserved amino acid motif (PLDLS) of E1A, the CtBP-interacting domain (Schaeper et al., 1995). Recent studies have demonstrated that CtBP is a member of a novel family of transcriptional co-repressors that recognize PXDLS motifs in a variety of DNA-binding proteins and that function as transcriptional co-repressors in Drosophila, Xenopus, and mammals (Turner and Crossley, 2001). CtBP proteins bind to a variety of transcription factors, including Drosophila Hairy, Snail, and Kru¨ppel, and the SP1-related zinc finger protein, mammalian Basic Kru¨ppel-like Factor/Kru¨ppel-like Factor 3 (BLKF/KLF3). CtBP/E1A interactions result in the expression of epithelial genes such as desmoglein-2, plakoglobin, and E-cadherin (Fischer and Quinlan, 1998; Grooteclaes and Frisch, 2000). E1A-mediated de-repression by sequestration of CtBP may have wide-range effect on cellular genes, as recently demonstrated by Sundqvist et al. (2001). These authors performed functional knock-out studies in cell lines that
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allowed the inducible expression of the CID of E1A and identified differentially expressed genes by gene array analysis. They found that the expression of approximately 7% of the analyzed genes was regulated in a CID-dependent fashion. Interestingly, the most pronounced effects were observed on genes involved in cell-cycle regulation, apoptosis, and intracellular communication, including cyclin-dependent kinase 5 activator, MEKK, PKC-␥, TRAIL receptor, death domain-containing protein CRADD, caspase 9-precursor, death-associated protein kinase 1, and RATS1 (Sundqvist et al., 2001). We hypothesize that Ad infection, through binding of the E1A CID to CtBP, results in GP73 expression by a derepression mechanism at the GP73 promoter. A potential additional function for CtBP has recently been revealed by the work of Corda and colleagues (Spano et al., 1999; Weigert et al., 1999). These authors found that CtBP/BARS, a rat homolog of human CtBP1, has a direct effect on Golgi lipid metabolism. CtBP/BARS catalyzed the acylation of lysophosphatidic acid to phosphatidic acid in pure lipid systems and in Golgi membranes. Ribosylation of CtBP/BARS by Brefeldin A was associated with the disassembly of the Golgi complex. These data suggest that CtBP might play a direct, enzymatic role in the formation and maintenance of the Golgi complex and raises the hypothesis that a CID-mediated interaction between Ad and CtBP at the Golgi apparatus somehow results in the increased expression of GP73. To our knowledge, GP73 is the first cellular Golgi protein shown to be upregulated by Ad infection. Our studies to date have not demonstrated an obvious function for GP73, and its structure provides no specific clues. In principle, Golgi membrane proteins may have the following four basic functions: (1) posttranslational modification of proteins, including glycosylation; (2) transport and sorting of secretory and resident proteins; (3) establishment and regulation of the Golgi structure; and (4) specific roles in cell signaling and apoptosis regulation. The absence of significant homologies or shared motifs in GP73 with the known Golgi glycosidases argues against the first possibility. The lack of a significant cytoplasmic domain argues against a role in vesicle trafficking and tethering (“Velcro function”). Consequently, we are considering the possibility that GP73 functions by interacting with viral or cellular proteins undergoing modification within the Golgi lumen. The Ad model established in this article may provide us with a suitable experimental model to elucidate the role(s) of GP73 in viral infection. Our study suggests that the Golgi apparatus might be a specific target in Ad infection. Precedents for viral interference with Golgi structure or functions have been established in studies of HSV 1 (Campadelli et al., 1993; Ward et al., 1998), poliovirus (Sandoval and Carrasco, 1997), and corona mouse hepatitis virus (Lavi et al., 1996). Interestingly, early electron microscopic studies
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ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
have hinted at similar morphological alterations in the Golgi apparatus of Ad-infected cells (Lenk et al., 1980). In summary, our study establishes GP73 as a novel protein with upregulated expression in Ad infection. The upregulation appears to be specific to this particular Golgi protein and requires the function of the E1A Cterminus and, specifically, its CID. Its biochemical function and role in the cellular response to Ad infection remain to be elucidated. MATERIALS AND METHODS Cell lines Human Hep3B (Hep 3B2.1–7) cells were obtained from the American Type Culture Collection (ATCC). This cell line is derived from a hepatocellular cancer and contains an integrated copy of the hepatitis B virus genome (Knowles et al., 1980). Hep3B cells resemble normal hepatocytes by expressing a number of hepatocyte marker proteins, including alpha fetoprotein, albumin, ␣-2-macroglobulin, and transferrin (Knowles et al., 1980). They are considered to be highly differentiated (Kawai et al., 2001). Hep3B cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin and cultured at 37°C in 95% air and 5% CO 2. Viruses and infection The Ad2 wild-type (wt) virus was obtained from ATCC. rec700 is a Ad5-Ad2-Ad5 recombinant that has a wt E1A region and is used as a wt control (Wold et al., 1986). The mutant E1A viruses (see Table 1) are named Ad2/ 5pm975 (13S only; carrying a deletion in the 12S donor splice site) (Montell et al., 1982), 12S [Ad5 derivative; 12S only, conserved region (CR3) transactivation defective] (Stephens and Harlow, 1987), dl313 (Ad5 derivative; 2350-bp deletion of map units 3.5–10.6, deleting the second exon of E1A and E1B) (Jones and Shenk, 1979), dl201.2 (Ad2 derivative; deletion of map units ⬃2–6, E1A-, E1B-) (Brusca and Ehinnadurai, 1983), dl1101/1107 [Ad5 derivative containing exon 1 deletion of aa 4–25 (dl1101) and aa 111–163(dl1107), resulting in the inability of E1A proteins to bind p300 and p105-Rb] (Howe et al., 1990), dl110 (Ad5 derivative, deletion of bp 2333–2804, resulting
243 TABLE 1
Mutant Viruses Used in This Study a Virus rec700 dl201.2 Ad2/5 pm975 12S d/313 dl1101/1107 177-9 dl1135
dl110 dl111 Ad/E3 dl808
Phenotype [reference] Wild-type E1A [Wold et al., 1986] Lacks E1A and E1B [Brusca and Chinnadurai, 1983] 289R only [Montell et al., 1982] 243R only [Stephens and Harlow, 1987] Lacks aa 174–243 of the EIA c-terminus (including CID) [Jones and Shenk, 1979] Defective in CR1 and CR2 [Howe et al., 1990] Lacks aa 178–238 of the E1A C-terminus (including CID) b [Subramanian et al., 1989] Lacks aa 225–238 of the E1A C-terminus (including CID) b [Arsenault and Webber, 1993], [Boyd et al., 1993] Lacks E1B 55K [Babiss and Ginsberg, 1984] Lacks E1B 19K [Babiss et al., 1984] E3 proteins only (except ADP) [Tollefson et al., 2001] Lacks E4 [Challberg and Ketner, 1981]
a The following mutants were derived from the parental virus dl309: Ad2/5 pm975, 12S, dl313, dl1101/1107, d/1135, dl110, dl111. b 12S background.
in a truncated form of the 55-kDa protein of E1B) (Babiss and Ginsberg, 1984), dl111 (derived from the Ad5 mutant dl309, E1B ⫺19K ⫺, RID ⫺, 14.7K ⫺) (Babiss et al., 1984), 177-9 (Ad2 derivative, 12S E1A background, deletion of aa 178–238 of E1A, defective in CtBP binding) (Subramanian et al., 1989), and dl1135 (Ad5 derivative in a 12S background; deletion of aa 225–238 of E1A, lacking the CtBP binding domain) (Arsenault and Webber, 1993; Boyd et al., 1993). dl808 is an Ad5 derivative that lacks E4 function (1030-bp deletion of map units 92.0–97.1, defective in E4 subregions 1 or 2 through 7) (Challberg and Ketner, 1981). All mutants except rec700, dl201.2, 177-9, and dl808 were derived from the parental virus dl309 which has a deletion in the E3 region that removes the genes for E3-RID and E3-14.7K. Ad/E3 is an E1-minus replication-deficient Ad vector with an expression cassette consisting of the E3 genes driven off the CMV promoter inserted into the E1 region (Tollefson et al., 2001). The E1A deletion mutants, Ad/E3 and dl808, were grown in monolayers of human 293 cells (Green and
FIG. 5. GP73 expression is induced by transfection with E1A expression plasmids; induction requires the CID of E1A. Hep3B cells were transfected with a control plasmid (pUC18) or with plasmids carrying E1A wild-type 13S or 12S genes, the large C-terminal deletion mutation (177-9) of E1A in a 12S background, or a deletion of the CtBP-binding domain (dl1135) in a 12S E1A background. GP73 protein expression was assessed by immunofluorescence (green fluorescence signal) using a GP73-specific polyclonal antibody. E1A coexpression was detected using the pooled mouse monoclonal antibody mix (red fluorescence). Nuclear DNA was visualized using DAPI (blue fluorescence). (A) Representative fluorescence images from transiently transfected cells. E1A and GP73 signals are absent in pUC18-transfected cells. 13S- and 12S-transfected cells exhibit a marked increase in GP73 signal, and GP73 and E1A are coexpressed extensively. In comparison, a minority of E1A-expressing cells transfected with the 177-9 or dl1135 plasmids expressed GP73. (B) Quantitation of GP73/E1A coexpression. Immunofluorescence data were quantitated by cell counting and expressed as the percentage of E1A-positive cells with an unequivocal GP73 signal. Results are shown as means ⫾ SEM. Extensive coexpression of GP73 and E1A was found in 13S- and 12S-transfected cells. In contrast, a minority of cells transfected with 177-9 or dl1135 plasmids coexpressed GP73.
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Wold, 1979). Hep3B cells were infected with 100 to 300 PFU of the appropriate virus, and the infection was allowed to proceed for 24 to 48 h prior to harvest. In some experiments, 1--D-arabinofuranosylcytosine (Ara-C) (20 g/ml) was added to the culture media to prevent adenoviral DNA replication (Thomas and Matthews, 1980). Transfections Hep3B cells were transiently transfected with pUC18 vector (Life Technologies, Gaithersburg, MD) or with pUC18-derived plasmids carrying Ad wt E1A 13S or 12S genes, or E1A with mutations within the C-terminus (177-9, dl1135). The terms 13S and 12S refer to the sizes of E1A-derived mRNAs that encode the 289R and 243R proteins, respectively. The Hep3B cells were grown to approximately 75% confluency on six-well plates containing coverslips. Transfections were carried out using Lipofectamine 2000, following the manufacturer’s protocol (Life Technologies). Aliquots of transfection-grade plasmid DNA were diluted in Opti-MEM I Reduced Serum (Life Technologies). Lipofectamine 2000 was suspended in Opti-MEM I (1:20 v/v) for 5 min. Aliquots of diluted plasmid DNA and Lipofectamine 2000 suspension were combined in microcentrifuge tubes and incubated at room temperature (RT) for 20 min. Immediately prior to transfection, culture media was removed from the Hep3B cells and replaced with Opti-MEM I. Aliquots of plasmid DNA/Lipofectamine 2000 complex (corresponding to 5 g plasmid DNA and 12.5 l Lipofectamine 2000 per well) were added to the cells, followed by a 2.5 h incubation at 37°C. The medium was removed and replaced with DMEM/10% FBS. Cells were incubated for 48–72 h and then fixed for immunofluorescence microscopy (see below). RNase protection assays Total cellular RNA was isolated from cultured cells using a rapid, guanidine-based method (RNA Stat 60, Tel-Test, Friendswood, TX). A cRNA antisense probe directed against the GP73 cDNA was prepared as previously described (Kladney et al., 2000). Twenty microgram aliquots of RNA were hybridized to the probe for the ribonuclease protection assay (RPA) using a commercial kit (RPAIII, Ambion, Austin, TX). The RPA control probe was directed against human GAPDH. Antibodies GP73 was measured using a polyclonal rabbit antiserum raised against recombinant human GP73 (Kladney et al., 2000). Giantin was detected using a rabbit polyclonal antiserum directed against the N-terminal moiety of the protein for immunoblotting assays or a mouse monoclonal antibody for immunofluorescence experiments (Linstedt and Hauri, 1993). GPP130 and golgin-84 were detected using a mouse monoclonal anti-
body or a rabbit polyclonal antiserum against the human proteins, respectively (Linstedt et al., 1997; Bascom et al., 1999). E1A was detected with a monoclonal antibody mixture consisting of pooled supernatants of Harlow M1, M2, M37, M58, and M73 hybridomas (Harlow et al., 1985). Adenoviral DNA binding protein (DBP) was detected with a rabbit polyclonal antibody (Lillie et al., 1987), and fiber protein was detected with a mouse monoclonal antibody (generously provided by Jeff Engler). Western blotting Hep3B cells were washed three times with PBS followed by the addition of lysis buffer (62.5 mM Tris–Cl pH 6.8, 20 mM DTT, 10% glycerol, 1% SDS). Adherent cells were removed with plastic scrapers, and the lysates transferred to microcentrifuge tubes. Samples were boiled for 3 min, sonicated for 30 s, and centrifuged at 14,000 rpm at 4°C for 20 min. The cleared lysates were stored at ⫺80°C and analyzed in batch fashion. Protein concentrations of the lysates were determined using a modification of the micro-Lowry method with bovine serum albumin as a standard (Protein Assay Kit, Sigma, St. Louis, MO). Aliquots corresponding to 15 g of total cellular protein were subjected to SDS–PAGE using precast 10% Tris–HCl gels (Ready Gels, Bio-Rad, Hercules, CA) in a running buffer containing 25 mM Tris–Cl, 192 mM glycine, and 0.1% SDS. Gels were run at 100 V for 8 min and then at 200 V for 30 min. A control sample consisting of 7.5 g of total cellular protein from HEK293 cells (ATCC) was included in each gel run. 293 cells were found to give a robust GP73 protein signal, which was used as a standard to facilitate quantitative comparisons between gels. Separated proteins were transferred to PVDF membranes (PVDF-Plus, Micron Separations, Westborough, MA) with a semidry transfer cell (TransBlot SD, Bio-Rad), using an optimized buffer (48 mM Tris–Cl, 39 mM glycine, 0.005% SDS, 20% methanol) and standardized transfer conditions (10 V, 30 min). Membranes were kept overnight in blocking buffer [10% nonfat dry milk in Tris-buffered saline (pH 7.6) containing 0.5% Tween 20 (TBST)] at 4°C. They were then washed three times in wash buffer (0.5% nonfat dry milk in TBST) and incubated for 1 h at RT in 15 ml of wash buffer containing primary antibodies. Several washes with high-salt buffer (0.5% nonfat dry milk in TBST containing 0.5 M NaCl) and wash buffer were then performed. This was followed by an incubation for 30 min in washing buffer with 1:1000 v/v preadsorbed anti-rabbit or antimouse IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA). After repeat washes with high-salt and wash buffer, GP73 or E1A immunoreactivity was visualized by enhanced chemiluminescence using a commercial kit (ECL, Amersham Pharmacia Biotech, Buckinghamshire, U.K.).
ADENOVIRUS INDUCES GOLGI PROTEIN GP73 EXPRESSION
Immunofluorescence microscopy Hep3B cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 10 min at RT, permeabilized with methanol containing 4,6-diamidino-2-phenylindole (DAPI) for 6 min at ⫺20°C, and washed with ⫺20°C methanol without DAPI for 2 min at RT. For GPP130 and golgin-84 staining, the paraformaldehyde step above was omitted. After PBS washes, coverslips were incubated with a blocking solution containing 4% (v/v) goat serum in PBS for 20 min. This was followed by a 1 h incubation with primary antibodies at RT. After two PBS washes, coverslips were incubated with secondary antibodies in PBS containing 4% goat serum. Secondary antibodies were goat anti-mouse IgG/Alexa Fluor 594 or goat anti-rabbit IgG/Alexa Fluor 488 conjugates (1:400 v/v) (Molecular Probes, Eugene, OR). Following two PBS washes, coverslips were mounted in elvanol-containing p-phenylenediamine (Heimer and Taylor, 1974). The images shown in Fig. 5A were viewed using a Nikon Optiphot-2 microscope equipped with epifluorescence and photographed using Kodak Elite CHROME 400 film. All other fluorescent images were captured using a Nikon DXM1200 digital still camera with ACT-1 software (Nikon, Tokyo, Japan). The final images were processed using Photoshop. Quantitation of immunofluorescent signals The cellular expression of marker proteins by immunofluorescence microscopy was quantitated by cell counting. All measurements were performed by one investigator (C.J.F.). Whenever possible, counting was performed without the investigator’s knowledge of the experimental condition. All cells in a visual field (counted by DAPI fluorescence) that were positive for a chosen marker were included in the counting, regardless of the signal strength. Typically, 500 to 2000 cells (corresponding to 10 to 40 randomly selected fields, respectively) were assessed for each data point. ACKNOWLEDGMENTS This work was supported by a Veterans Affairs Merit Review grant to C.J.F. and by NIH Grants CA58538 and CA71704 to W.S.M.W. We thank T. Subramanian and G. Chinnadurai for providing 177-9, dl1135, and for the E1A plasmids, Jeff Engler for the fiber antibody, Maurice Green for the DBP antibody, Robert Nussbaum for the golgin-84 antibody, and Adam Linstedt for the giantin and GPP130 antibodies.
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