Vol. 64, No. 6
JOURNAL OF VIROLOGY, June 1990, p. 2780-2787
0022-538X/90/062780-08$02.00/0 Copyright C 1990, American Society for Microbiology
cis-Dominant Defect in Activation of Adenovirus Type. 5 Elb Early RNA Synthesis CHRISTOPHER L. PARKS,t AND DAVID J. SPECTOR* Department of Microbiology and Immunology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Received 27 November 1989/Accepted 7 March 1990
A cis-dominant mutation in the adjacent Ela gene disrupted the accumulation of adenovirus type 5 Elb mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear Elb RNAs in cells infected with d1312, a strain that lacks the Ela TATA box, cap site, and much of the coding sequence, were reduced 5- to 10-fold even when the Ela activator was provided in trans. The strain was defective for early Elb RNA synthesis but not for Elb RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in Elb RNA synthesis could not be attributed to the Ela promoter sequences missing in d1312 DNA. If the Ela protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped Elb transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient Elb expression during the early phase of infection.
lished data). Removing sequences 5' of position -100 reduces the expression of the related adenovirus 12 Elb gene (6). However, there is no direct evidence implicating distal sequences as major contributors to adenovirus 5 or 2 Elb transcription during productive infection. We initiated studies to analyze further the contributions of both proximal and distal sequences to Elb transcriptional control. In the process we observed that removing sequences 5' of the previously studied regions resulted in dramatically reduced expression of Elb mRNA.
Elb RNA and proteins of the group C adenoviruses (prototypes 5 and 2) are synthesized during the early phase of infection and continue to accumulate at high levels during the late phase after the onset of viral DNA replication (17, 48). A single primary Elb transcript structure is processed to one of five known species of mRNA (17, 53). At least two of these mRNAs encode polypeptides that have critical roles in both the lytic cycle of the virus and transformation (17, 48, 58). The frequency of transcription initiation by RNA polymerase II determines the overall activity of the Elb transcription unit (37), whereas the rates of accumulation of individual Elb gene products are regulated posttranscriptionally (36, 61). Both viral and cellular proteins govern the synthesis of early Elb RNA. These include Ela 289R, the 289-aminoacid residue primary translation product of the viral immediate-early gene Ela (4, 11, 37), and two cellular sequencespecific transcription factors, Spl and TFIID. The Spl recognition sequence (GC box) is located between 48 and 39 base pairs (bp) 5' of the initiation (cap) site (positions -48 to -39), whereas the TFIID-binding site (TATA box) is located between positions -29 and -23 (43, 65). Mutation of the GC sequence reduces early transcription by at least 60% in vivo and in vitro, whereas TATA box mutations reduce transcription to barely detectable levels (39, 43, 63, 65). Ela 289R stimulates basal transcription (4, 11). Transactivation requires an intact TATA sequence, a result that implicates TFIID in the process (40, 64, 65). These findings have suggested that, unlike the other early adenovirus genes (11, 26), Elb has a relatively simple promoter located within 50 bp of the cap site (65). No definitive contributions to Elb transcription have been assigned to other sequences, although there are hints of a more complex control region. Deletion between positions -449 and -127 reduces transcription by 50% in vivo (64). This region includes sequences that stimulate transcription (39) and interact specifically with nuclear proteins in vitro (39, 43; C. L. Parks, D. S. Cohen, and D. J. Spector, unpub-
MATERIALS AND METHODS Cells and viruses. Monolayers of KB cells and 293 cells (15) were maintained in Dulbecco modified Eagle medium (KC Biologicals) supplemented with 2 mM glutamine, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml (DMEM). For 293 cells, DMEM was supplemented with 0.07% sodium bicarbonate and 10% fetal bovine serum (KC Biologicals or Gibco Laboratories) or with a combination of 5% fetal bovine serum and 5% bovine calf serum (Hyclone Laboratories, Inc.). For KB cells, 0.23% sodium bicarbonate and 5% fetal bovine serum were added. The virus strains used in these studies (see Fig. 1) included adenovirus type 5 wild-type (wt) strain dl309 (27), Ela mutant strains dl347 and dl348 (62), hrl (19), dl312 (27), dl340-13 (20), and NTdllOlO and CTdl1009 (59) and adenovirus type 2 Ela mutant strain d11500 (35). The Elb mutant strain dlll8/E3+, containing the dlll8 mutation (2) in a wt E3 genetic background and constructed by Lee Babiss, was generously provided by C. S. H. Young (Columbia University). The construction of the other adenovirus strains is described below. For preparation of virus stocks, infected 293 cell monolayers were incubated for 36 to 48 h. Viruses in cell lysates were quantified by plaque titration on 293 cell monolayers. Recombinant DNA. Adenovirus type 2 and 5 region El DNA sequences are very similar (50). Recombinant DNAs contained type 5 sequences unless indicated otherwise; sequence numbers are as described previously (50). All recombinant DNA procedures were performed as described previously (39). Plasmid pXCl, derived from pXC7 (39), contains adenovirus 2 DNA sequences from
* Corresponding author. t Present address: Department of Biology, Princeton University, Princeton, NJ 08544.
2780
cis ACTIVATION OF ADENOVIRUS Elb EARLY RNA
VOL. 64, 1990
nucleotides 1 to 1336 (XbaI site) and adenovirus 5 DNA sequences from nucleotides 1338 (XbaI) to 5792 (XhoI). Plasmid pXC3, used to prepare strain d12002, was constructed from pXCi by removing Elb nucleotides 1771 (Sacl) to 3328 (BglII). Plasmid pXCiOi, used to construct strain d12004, was derived from pXCl and has the mutated Elb sequences from dlii2 (2). The construction was facilitated by a gift from L. Babiss of plasmid pBi, which has an XbaI-KpnI DNA fragment with the dlii2 Elb mutation. Plasmid pXC201, used to construct strain d12005, has Elb sequences from pXCi0i and Ela sequences from pXC2 (a gift from S. Banerjee), a pXCi derivative with a d1312 Ela
2781
enzyme produced uniquely end-labeled fragments that were gel purified. Viral RNA was quantified by hybridization in solution to end-labeled DNA probes and electrophoresis (32) of hybrids protected from nuclease Si digestion (5, 57, 65) as described by Parks et al. (39), except that hybrids were digested with 830 units of nuclease Si (Boehringer Mann-
heim Biochemicals) per ml for 1 h at 37°C. Probe DNA was used at a concentration of no less than 0.9 nM; a twofold increase in probe concentration did not increase the intensities of the hybridization signals. The relative intensities of the autoradiographic bands were determined by scanning preflashed films (30) as described previously (39).
region.
Plasmid pXC7 was the source of Elb hybridization probes. Plasmid pGemE3 was constructed to provide E3 probe DNA. An EcoRI-KpnI DNA fragment from map units 75.9 to 81.0 of adenovirus type 5 DNA was isolated from a recombinant M13 phage (21; provided by P. Hearing). Bacteriophage replicative-form DNA was prepared as described by Messing (34). The E3 EcoRI-KpnI DNA fragment was cloned between the EcoRI and KpnI sites in the polylinker of plasmid pGem4Z (Promega Corp.). Recombinant viruses. Recombinant adenoviruses were constructed by overlap recombination into the d1309 genome (7, 49). Viral DNA digested with ClaI and XbaI was used directly without further processing. Plasmid DNAs were digested within vector sequences and purified by extraction with phenol-chloroform and ethanol precipitation. Two micrograms of d1309 DNA and 2 to 4 ,ug of plasmid DNA were used to transfect two 60-mm dishes of 293 cells by calcium phosphate coprecipitation (16, 60) as described by Spector and Tevethia (47). The uptake of DNA was stimulated 4 h after transfection by incubation of cells in DMEM containing 20% glycerol for exactly 1 min (12). Viral plaques appeared 5 to 6 days posttransfection. To determine the structures of recombinant genomes,
we
prepared viral DNA from cells harvested 24 h after infection by the modified Hirt procedure described by Volkert and Young (54) and analyzed it by restriction enzyme digestion and gel electrophoresis. Viral isolates of the desired genotype were plaque purified twice. All recombinants had adenovirus 2 DNA sequences to the left of the XbaI site at about 4 map units. Infections and RNA extractions. For viral RNA analysis, KB cell monolayers, growing on 150-mm dishes at approximately 75% confluence, were infected with 20 PFU of virus from a lysate stock per cell. Coinfected cells received 20 PFU of each virus per cell. For early RNA, cultures were incubated for 5 to 6 h in the presence or absence of 20 ,ug of cytosine arabinoside (araC) per ml. Late RNA was isolated 16 to 24 h after infection. RNA also was prepared from cells infected for 30 to 36 h in the presence of 20 ,ug of araC per ml; fresh araC was added every 8 to 12 h.
Cytoplasmic RNA
was isolated
by phenol-chloroform
extraction of cytoplasm fractions prepared in an isotonic buffer (33) as described previously (51). Nuclear RNA was isolated by centrifugation of lysed nuclei, prepared in an isotonic buffer (46), through a cushion of 9.5 molal cesium chloride (67). The pelleted RNA was suspended in 0.01 M Tris hydrochloride (pH 7.5) and extracted once with chloroform. RNA concentrations were determined spectrophoto-
metrically. Probes 5' end labeled with 32p were prepared as described previously (39), except that the polynucleotide kinase reaction was modified as described by Harrison and Zimmerman (18). After labeling, digestion with an appropriate restriction
RESULTS Accumulation of Elb mRNA in mutants with Ela sequence alterations. A genetic scheme was devised to facilitate an analysis of Elb transcriptional activity in the presence and absence of virus-encoded trans activators and repressors. Despite having sequences that should have included a functional Elb promoter, a test virus strain with a large Ela substitution (nucleotides 454 to 1338 replaced) and a large Elb deletion was defective for Elb RNA accumulation even when Ela 289R was provided in trans (data not shown). To test whether the Ela mutation produced a cis-acting defect in Elb RNA accumulation, we carried out further studies with dl312, whose DNA contains a wt Elb coding region and lacks Ela sequences between nucleotide positions 447 and 1350 (25, 27, 66). Cells were coinfected with dl2002 to or another provide Ela gene products in trans and with dl312 Ela deletion mutant (Fig. 1). Three of these strains do not make a functional trans activator. Strain dlSO00 has an 8-bp deletion that prevents the splicing of Ela 289R mRNA (36), a 1-bp deletion in hrl DNA results in a prematurely terminated protein (42), and the deletion in d1347 DNA removes sequences encoding the activating domain (62). Strain dl348, used as a positive control, has a functional Ela 289R gene, although it lacks the 289R mRNA intron (62). The amount of Elb RNA was about 15 to 25% the amounts dl312 early obtained with the other strains, which all produced about the same
quantities (data not shown).
Specfficity and cis dominance of the defect in d1312 Elb mRNA accumulation. To determine whether the effect of the deletion on mRNA accumulation was specific for dl312 Elawhether the mutation was cis dominant, we used a and Elb
different complementation strategy. The helper strain, wt adenovirus 5, provides normal early mRNAs and proteins, including transacting viral proteins required for early viral mRNA accumulation. The test strain, d12005, has the dl312 Ela mutation in a background of Elb and E3 genes that are Elb region of genetically distinct from the wt genes. The d12005 DNA has a small deletion and linker substitution derived from dlii2 (2), whereas the E3 region (as with most of the viruses used in this study) is derived from d1309, a strain that makes altered E3 mRNAs. To test for cis dominance, we found it important to establish that the Elb and E3 mutations in the test strain DNA were phenotypically silent with respect to early mRNA accumulation. Strain d12004 has the same Elb and E3 alleles as d12005 but is nondefective because of an intact, functional Ela region. Early RNA expression could be analyzed in d12004-infected cells in the absence of helper virus. Separate cultures were infected either with wt adenovirus 5 or with dl2004. There were no significant differences in the amounts of either Elb or E3 mRNAs made from d12004 and wt adenovirus 5 DNAs respectively (Fig. 2).
J. VIROL.
PARKS AND SPECTOR
2782
A 3
2
I
0
KB
pIX
Elb
Ela
4
I
_
13S (E1 a 289R) 1112
499
22S (El b 496R, 176R)
4070
3510
1632
13S (Elb 176R)
12S (E1 a 243R) 3595
2255
1702
974 1229
9S (pIX 140R) 3580
maim1
1920
probe 2
2210 0 2BO
probe 3
B d1312
447
1350
dl347 974 1229
d1348
1112 1229
-4
dli 500 hrl
d12002
d12004 d12005 d1340-13
NTdIlOlO CTdl1 009 dll 18
3329
1770
1885 1955 1350 1885 1955
447 454 550 563
1010
1009
1338
2049
2591
FIG. 1. Map of region El of adenovirus 5 DNA and structures of viral genomes. (A) The left end of the adenovirus 5 genome from nucleotides 1 to 4200 is represented as a bar showing the locations of the Ela, Elb, and pIX genes. The exon-intron structures of the major El mRNAs are shown (nucleotide numbers indicate the exon ends) and designated by mRNA size (S value), gene region (Ela, Elb, or pIX), and polypeptide size (in number of amino acid residues [R]). The open reading frames are indicated as open (frame 1), hatched (frame 2), and shaded (frame 3) boxes. Hybridization probes 1 to 3 were prepared from BstEII, NcoI, and Hindlll DNA fragments, respectively, of plasmid DNA containing wt Elb sequences. The 32P-labeled termini are indicated by asterisks. KB, kilobase pairs. (B) Structures of viral El DNA sequences. Deletions are indicated by open bars (the numbers indicate the endpoints of the DNA sequences present). Arrows above the genomes of d11500 and hrl designate the positions of a 9-bp deletion mutation and a l-bp deletion mutation, respectively.
co
cis ACTIVATION OF ADENOVIRUS Elb EARLY RNA
VOL. 64, 1990
2783
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o M
JOURNAL OF VIROLOGY, June 1990, p. 2780-2787
0022-538X/90/062780-08$02.00/0 Copyright C 1990, American Society for Microbiology
cis-Dominant Defect in Activation of Adenovirus Type. 5 Elb Early RNA Synthesis CHRISTOPHER L. PARKS,t AND DAVID J. SPECTOR* Department of Microbiology and Immunology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Received 27 November 1989/Accepted 7 March 1990
A cis-dominant mutation in the adjacent Ela gene disrupted the accumulation of adenovirus type 5 Elb mRNA during the early phase of infection. Steady-state levels of cytoplasmic and nuclear Elb RNAs in cells infected with d1312, a strain that lacks the Ela TATA box, cap site, and much of the coding sequence, were reduced 5- to 10-fold even when the Ela activator was provided in trans. The strain was defective for early Elb RNA synthesis but not for Elb RNA made late or during prolonged incubation in the presence of an inhibitor of DNA replication. The defect in Elb RNA synthesis could not be attributed to the Ela promoter sequences missing in d1312 DNA. If the Ela protein-coding region contains cis-acting regulatory sequences, they are not part of the previously mapped Elb transcriptional control region and may represent additional regulatory elements that ensure prompt and efficient Elb expression during the early phase of infection.
lished data). Removing sequences 5' of position -100 reduces the expression of the related adenovirus 12 Elb gene (6). However, there is no direct evidence implicating distal sequences as major contributors to adenovirus 5 or 2 Elb transcription during productive infection. We initiated studies to analyze further the contributions of both proximal and distal sequences to Elb transcriptional control. In the process we observed that removing sequences 5' of the previously studied regions resulted in dramatically reduced expression of Elb mRNA.
Elb RNA and proteins of the group C adenoviruses (prototypes 5 and 2) are synthesized during the early phase of infection and continue to accumulate at high levels during the late phase after the onset of viral DNA replication (17, 48). A single primary Elb transcript structure is processed to one of five known species of mRNA (17, 53). At least two of these mRNAs encode polypeptides that have critical roles in both the lytic cycle of the virus and transformation (17, 48, 58). The frequency of transcription initiation by RNA polymerase II determines the overall activity of the Elb transcription unit (37), whereas the rates of accumulation of individual Elb gene products are regulated posttranscriptionally (36, 61). Both viral and cellular proteins govern the synthesis of early Elb RNA. These include Ela 289R, the 289-aminoacid residue primary translation product of the viral immediate-early gene Ela (4, 11, 37), and two cellular sequencespecific transcription factors, Spl and TFIID. The Spl recognition sequence (GC box) is located between 48 and 39 base pairs (bp) 5' of the initiation (cap) site (positions -48 to -39), whereas the TFIID-binding site (TATA box) is located between positions -29 and -23 (43, 65). Mutation of the GC sequence reduces early transcription by at least 60% in vivo and in vitro, whereas TATA box mutations reduce transcription to barely detectable levels (39, 43, 63, 65). Ela 289R stimulates basal transcription (4, 11). Transactivation requires an intact TATA sequence, a result that implicates TFIID in the process (40, 64, 65). These findings have suggested that, unlike the other early adenovirus genes (11, 26), Elb has a relatively simple promoter located within 50 bp of the cap site (65). No definitive contributions to Elb transcription have been assigned to other sequences, although there are hints of a more complex control region. Deletion between positions -449 and -127 reduces transcription by 50% in vivo (64). This region includes sequences that stimulate transcription (39) and interact specifically with nuclear proteins in vitro (39, 43; C. L. Parks, D. S. Cohen, and D. J. Spector, unpub-
MATERIALS AND METHODS Cells and viruses. Monolayers of KB cells and 293 cells (15) were maintained in Dulbecco modified Eagle medium (KC Biologicals) supplemented with 2 mM glutamine, 100 U of penicillin per ml, and 100 ,ug of streptomycin per ml (DMEM). For 293 cells, DMEM was supplemented with 0.07% sodium bicarbonate and 10% fetal bovine serum (KC Biologicals or Gibco Laboratories) or with a combination of 5% fetal bovine serum and 5% bovine calf serum (Hyclone Laboratories, Inc.). For KB cells, 0.23% sodium bicarbonate and 5% fetal bovine serum were added. The virus strains used in these studies (see Fig. 1) included adenovirus type 5 wild-type (wt) strain dl309 (27), Ela mutant strains dl347 and dl348 (62), hrl (19), dl312 (27), dl340-13 (20), and NTdllOlO and CTdl1009 (59) and adenovirus type 2 Ela mutant strain d11500 (35). The Elb mutant strain dlll8/E3+, containing the dlll8 mutation (2) in a wt E3 genetic background and constructed by Lee Babiss, was generously provided by C. S. H. Young (Columbia University). The construction of the other adenovirus strains is described below. For preparation of virus stocks, infected 293 cell monolayers were incubated for 36 to 48 h. Viruses in cell lysates were quantified by plaque titration on 293 cell monolayers. Recombinant DNA. Adenovirus type 2 and 5 region El DNA sequences are very similar (50). Recombinant DNAs contained type 5 sequences unless indicated otherwise; sequence numbers are as described previously (50). All recombinant DNA procedures were performed as described previously (39). Plasmid pXCl, derived from pXC7 (39), contains adenovirus 2 DNA sequences from
* Corresponding author. t Present address: Department of Biology, Princeton University, Princeton, NJ 08544.
2780
cis ACTIVATION OF ADENOVIRUS Elb EARLY RNA
VOL. 64, 1990
nucleotides 1 to 1336 (XbaI site) and adenovirus 5 DNA sequences from nucleotides 1338 (XbaI) to 5792 (XhoI). Plasmid pXC3, used to prepare strain d12002, was constructed from pXCi by removing Elb nucleotides 1771 (Sacl) to 3328 (BglII). Plasmid pXCiOi, used to construct strain d12004, was derived from pXCl and has the mutated Elb sequences from dlii2 (2). The construction was facilitated by a gift from L. Babiss of plasmid pBi, which has an XbaI-KpnI DNA fragment with the dlii2 Elb mutation. Plasmid pXC201, used to construct strain d12005, has Elb sequences from pXCi0i and Ela sequences from pXC2 (a gift from S. Banerjee), a pXCi derivative with a d1312 Ela
2781
enzyme produced uniquely end-labeled fragments that were gel purified. Viral RNA was quantified by hybridization in solution to end-labeled DNA probes and electrophoresis (32) of hybrids protected from nuclease Si digestion (5, 57, 65) as described by Parks et al. (39), except that hybrids were digested with 830 units of nuclease Si (Boehringer Mann-
heim Biochemicals) per ml for 1 h at 37°C. Probe DNA was used at a concentration of no less than 0.9 nM; a twofold increase in probe concentration did not increase the intensities of the hybridization signals. The relative intensities of the autoradiographic bands were determined by scanning preflashed films (30) as described previously (39).
region.
Plasmid pXC7 was the source of Elb hybridization probes. Plasmid pGemE3 was constructed to provide E3 probe DNA. An EcoRI-KpnI DNA fragment from map units 75.9 to 81.0 of adenovirus type 5 DNA was isolated from a recombinant M13 phage (21; provided by P. Hearing). Bacteriophage replicative-form DNA was prepared as described by Messing (34). The E3 EcoRI-KpnI DNA fragment was cloned between the EcoRI and KpnI sites in the polylinker of plasmid pGem4Z (Promega Corp.). Recombinant viruses. Recombinant adenoviruses were constructed by overlap recombination into the d1309 genome (7, 49). Viral DNA digested with ClaI and XbaI was used directly without further processing. Plasmid DNAs were digested within vector sequences and purified by extraction with phenol-chloroform and ethanol precipitation. Two micrograms of d1309 DNA and 2 to 4 ,ug of plasmid DNA were used to transfect two 60-mm dishes of 293 cells by calcium phosphate coprecipitation (16, 60) as described by Spector and Tevethia (47). The uptake of DNA was stimulated 4 h after transfection by incubation of cells in DMEM containing 20% glycerol for exactly 1 min (12). Viral plaques appeared 5 to 6 days posttransfection. To determine the structures of recombinant genomes,
we
prepared viral DNA from cells harvested 24 h after infection by the modified Hirt procedure described by Volkert and Young (54) and analyzed it by restriction enzyme digestion and gel electrophoresis. Viral isolates of the desired genotype were plaque purified twice. All recombinants had adenovirus 2 DNA sequences to the left of the XbaI site at about 4 map units. Infections and RNA extractions. For viral RNA analysis, KB cell monolayers, growing on 150-mm dishes at approximately 75% confluence, were infected with 20 PFU of virus from a lysate stock per cell. Coinfected cells received 20 PFU of each virus per cell. For early RNA, cultures were incubated for 5 to 6 h in the presence or absence of 20 ,ug of cytosine arabinoside (araC) per ml. Late RNA was isolated 16 to 24 h after infection. RNA also was prepared from cells infected for 30 to 36 h in the presence of 20 ,ug of araC per ml; fresh araC was added every 8 to 12 h.
Cytoplasmic RNA
was isolated
by phenol-chloroform
extraction of cytoplasm fractions prepared in an isotonic buffer (33) as described previously (51). Nuclear RNA was isolated by centrifugation of lysed nuclei, prepared in an isotonic buffer (46), through a cushion of 9.5 molal cesium chloride (67). The pelleted RNA was suspended in 0.01 M Tris hydrochloride (pH 7.5) and extracted once with chloroform. RNA concentrations were determined spectrophoto-
metrically. Probes 5' end labeled with 32p were prepared as described previously (39), except that the polynucleotide kinase reaction was modified as described by Harrison and Zimmerman (18). After labeling, digestion with an appropriate restriction
RESULTS Accumulation of Elb mRNA in mutants with Ela sequence alterations. A genetic scheme was devised to facilitate an analysis of Elb transcriptional activity in the presence and absence of virus-encoded trans activators and repressors. Despite having sequences that should have included a functional Elb promoter, a test virus strain with a large Ela substitution (nucleotides 454 to 1338 replaced) and a large Elb deletion was defective for Elb RNA accumulation even when Ela 289R was provided in trans (data not shown). To test whether the Ela mutation produced a cis-acting defect in Elb RNA accumulation, we carried out further studies with dl312, whose DNA contains a wt Elb coding region and lacks Ela sequences between nucleotide positions 447 and 1350 (25, 27, 66). Cells were coinfected with dl2002 to or another provide Ela gene products in trans and with dl312 Ela deletion mutant (Fig. 1). Three of these strains do not make a functional trans activator. Strain dlSO00 has an 8-bp deletion that prevents the splicing of Ela 289R mRNA (36), a 1-bp deletion in hrl DNA results in a prematurely terminated protein (42), and the deletion in d1347 DNA removes sequences encoding the activating domain (62). Strain dl348, used as a positive control, has a functional Ela 289R gene, although it lacks the 289R mRNA intron (62). The amount of Elb RNA was about 15 to 25% the amounts dl312 early obtained with the other strains, which all produced about the same
quantities (data not shown).
Specfficity and cis dominance of the defect in d1312 Elb mRNA accumulation. To determine whether the effect of the deletion on mRNA accumulation was specific for dl312 Elawhether the mutation was cis dominant, we used a and Elb
different complementation strategy. The helper strain, wt adenovirus 5, provides normal early mRNAs and proteins, including transacting viral proteins required for early viral mRNA accumulation. The test strain, d12005, has the dl312 Ela mutation in a background of Elb and E3 genes that are Elb region of genetically distinct from the wt genes. The d12005 DNA has a small deletion and linker substitution derived from dlii2 (2), whereas the E3 region (as with most of the viruses used in this study) is derived from d1309, a strain that makes altered E3 mRNAs. To test for cis dominance, we found it important to establish that the Elb and E3 mutations in the test strain DNA were phenotypically silent with respect to early mRNA accumulation. Strain d12004 has the same Elb and E3 alleles as d12005 but is nondefective because of an intact, functional Ela region. Early RNA expression could be analyzed in d12004-infected cells in the absence of helper virus. Separate cultures were infected either with wt adenovirus 5 or with dl2004. There were no significant differences in the amounts of either Elb or E3 mRNAs made from d12004 and wt adenovirus 5 DNAs respectively (Fig. 2).
J. VIROL.
PARKS AND SPECTOR
2782
A 3
2
I
0
KB
pIX
Elb
Ela
4
I
_
13S (E1 a 289R) 1112
499
22S (El b 496R, 176R)
4070
3510
1632
13S (Elb 176R)
12S (E1 a 243R) 3595
2255
1702
974 1229
9S (pIX 140R) 3580
maim1
1920
probe 2
2210 0 2BO
probe 3
B d1312
447
1350
dl347 974 1229
d1348
1112 1229
-4
dli 500 hrl
d12002
d12004 d12005 d1340-13
NTdIlOlO CTdl1 009 dll 18
3329
1770
1885 1955 1350 1885 1955
447 454 550 563
1010
1009
1338
2049
2591
FIG. 1. Map of region El of adenovirus 5 DNA and structures of viral genomes. (A) The left end of the adenovirus 5 genome from nucleotides 1 to 4200 is represented as a bar showing the locations of the Ela, Elb, and pIX genes. The exon-intron structures of the major El mRNAs are shown (nucleotide numbers indicate the exon ends) and designated by mRNA size (S value), gene region (Ela, Elb, or pIX), and polypeptide size (in number of amino acid residues [R]). The open reading frames are indicated as open (frame 1), hatched (frame 2), and shaded (frame 3) boxes. Hybridization probes 1 to 3 were prepared from BstEII, NcoI, and Hindlll DNA fragments, respectively, of plasmid DNA containing wt Elb sequences. The 32P-labeled termini are indicated by asterisks. KB, kilobase pairs. (B) Structures of viral El DNA sequences. Deletions are indicated by open bars (the numbers indicate the endpoints of the DNA sequences present). Arrows above the genomes of d11500 and hrl designate the positions of a 9-bp deletion mutation and a l-bp deletion mutation, respectively.
co
cis ACTIVATION OF ADENOVIRUS Elb EARLY RNA
VOL. 64, 1990
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