The adenovirus type 5 E1A enhancer contains two functionally distinct domains: One is specific for E1A and the other modulates all early units in cis

Cell, Vol. 45, 229-236,

April 25, 1986, Copyright

0 1986 by Cell Press

The Adenovirus Type 5 EIA Enhancer Contains Two Functionally Distinct Domains: One Is Specific for EIA and the Other Modulates All Early Units in Cis Patrick Hearing’ and Thomas Shenkt * Department of Microbiology Health Sciences Center State University of New York Stony Brook, New York 11794 t Department of Molecular Biology Princeton University Princeton, New Jersey 08544

Summary The adenovirus type 5 genome contains two distinct enhancer elements located at the left end of the viral chromosome. The first element is repeated and specifically regulates region ElA transcription within infected cells. One copy of this element is sufficient to fully activate ElA transcription in vivo. The second element is located between these repeated sequences and regulates transcription in cis of all early regions on the chromosome. These enhancer elements function independently of each other, and neither element is required for efficient viral DNA replication. Since mutations within the two ElA enhancer components generate different physiological responses, transcriptional enhancement can be achieved through multiple mechanisms. Introduction Enhancer sequences regulate a variety of viral and cellular genes (see Khoury and Gruss, 1983, for a review). By definition, enhancer elements augment transcription of a linked gene in a fashion relatively independent of orientation and location (Banerji et al., 1981; Moreau et al., 1981; Fromm and Berg, 1983). They have been shown to activate transcription over large distances (devilliers and Schaffner, 1981). Certain enhancers display tissue and species specificity (Laimins et al., 1982; devilliers et al., 1982; DesGroseillers et al., 1983). While enhancers can increase transcription of multiple linked genes, they often preferentially activate proximal, as compared with distal, promoters (Wasylyk et al., 1983). Cellular factors that recognize enhancer-specific sequences and/or structures have been identified in several systems (Payvar et al., 1983; Scholer and Gruss, 1984; Wildeman et al., 1985). The mechanisms by which enhancers activate transcription of linked genes, however, remain unclear. We previously described the sequences that regulate adenovirus type 5 (Ad5) region ElA transcription in vivo (Hearing and Shenk, 1983a). Our results demonstrated that ElA transcription is regulated within infected cells by an enhancer region located between sequence positions 194 and 358 (numbering according to Van Ormondt et al., 1978; -141 to -305 relative to the ElA cap site at +l).

This region activates ElA transcription when located at the 3’ end of the gene in both orientations and enhances the efficiency of thymidine kinase transformation in a long term transfection assay (Hearing and Shenk, 1983a). Here we describe a detailed mutational analysis of this region. Our results define two separate, functional enhancer elements located in the ElA 5’-flanking sequences. One element (repeated at sequence positions 200 and 300) specifically regulates ElA transcription in vivo. The second element (located between positions 250 and 280) regulates activity of all early transcription units on the viral chromosome. These enhancer elements function independently of each other, and neither is required for efficient viral DNA replication within infected cells. Results Construction and Propagation of Viral Mutants Small, random deletion mutations in the Ad5 enhancer region were initially constructed in a recombinant plasmid containing the left end of the Ad5 genome (pElA-WT) using a D-loop mutagenesis procedure (Hearing and Shenk, 1983b). The exact site of each deletion was determined by nucleotide sequence analysis, and a select group of mutations (Figure 1B) were rebuilt into intact viral chromosomes using the method of Stow (1981). Mutant viruses were propagated in 293 cells that complement adenovirus mutants defective for ElA and ElB function (Graham et al., 1977). We previously found that deletion of either the upstream half (sequence position 194 to 273, deletion A5 in Figure 1A) or the downstream half (sequence position 270 to 358, deletion Bl in Figure 1A) of the Ad5 enhancer region significantly reduced the ability of mutant viruses to package viral DNA into virions late in infection (Hearing and Shenk, 1983a). As a result, mutants A5, Bl, and 2 (Figure 1A) were propagated using a virus (in340) that contains the left-end packaging sequences and an enhancer region located at the right end of the viral chromosome (Hearing and Shenk, 1983a). None of the new mutations constructed for this study (Figure 1B) interfered with function of the left-end packaging sequences. They were all propagated in a wildtype (d/309) background with no right-end reiteration of the packaging signal. Since many of these mutants display transcriptional defects (see below), it is clear that the packaging and enhancer signals can be uncoupled by mutation. The Ad5 Enhancer Region Contains Two Functional Regulatory Sequences To study the effects of the mutations diagrammed in Figure 1B or ElA transcription, HeLa cells were infected with wild-type on mutant viruses, and cytoplasmic RNA was isolated early (6 hr) after infection. Steady state levels of ElA mRNAs were determined by Sl analysis using a uniformly 32P-labeled Ml3 probe DNA (Figure 2). Two mRNAs (13s and 12s) were synthesized from region ElA

Cell 230

A.

499

loo I

TERMINAL REPEAT 340-A6

Is4

?I3

340-w 3$0-2

B.

*PO

2!0 220 230 240 260 270 260 ACACAGGAAGWACAATTTTCGCGC6GTTTTAGGC6GATGTTGTAkTAAATTTGGbCGT&#XGAbTAAGATTTG6CCA~JTC I IL -11 *g A A

B

c

309-97

204-?4B Q 9

280 ~,~AT~~*~AT~~~ATAAT~~ II I B

-I

309-141 309 -64 251

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252

Jos- IO 309-21

282,

P 3ol

9 9

Figure

1. ElA Transcriptional

Control

Region

(A) Schematic view of the left end of the Ad5 genome including the ElA transcription unit with its 5’-flanking sequences. The inverted terminal repeat, enhancer/packaging region, and CAAT and TATA homologies are identified by open rectangles. Large deletions that originally defined the enhancer/packaging domain are designated by solid rectangles. Nucleotide sequence numbers (Van Ormondt et al., 1976) designate the last base pair present on either side of deletions. (B) Nucleotide sequence of the ElA enhancer domain and mutations carried by viral variants. Arrows designate repeated constituents of enhancer elements (I and II) as well as repeats that play a role in packaging (A and B).

early after infection. The protected fragments in Figure 2 correspond to the ElA 13s and 12s 5’exons (613 and 475 nucleotides, respectively) and to the common 13s and 12s 3’exon (345 nucleotides). The first two lanes in Figure 2 (d/309 and in340) show the levels of ElA mRNAs observed in wild-type virus infections. As previously reported (Hearing and Shenk, 1963a), deletion of the upstream half of the Ad5 enhancer region (d/340-A6, Figure 1A) or of the downstream half of this region (d/340-61, Figure 1A) resulted in a 4 to 5 fold decrease in cytoplasmic ElA mRNAs (Figure 2). Deletion of the entire enhancer region (d/340-2, Figure 1A) resulted in a 20-fold decrease in ElA mRNAs. The decrease in the level of steady state cytoplasmic ElA mRNAs with mutant d/340-2 reflects a similar decrease in the rate of nuclear ElA transcription when analyzed by in vivo pulse-labeling experiments (Hearing and Shenk, 1983a). A repeated sequence (element I in Figure lB), located in the Ad5 enhancer region at sequence positions 200 and 300, closely resembles the core sequence of several eukaryotic enhancer elements (Hearing and Shenk, 1983a). As shown in Figure 2, deletion of either the upstream copy of this repeat (d/309-RH) or the downstream copy of the repeat (d/309-67) had no effect on ElA transcription within infected cells. Deletion of both copies (d/309-RH/87), however, resulted in a 4 to 5 fold decrease in levels of ElA transcripts. Sequences upstream of the element I repeat at position 200 or downstream of the repeat at position 300 are not essential for optimal ElA enhancement (d/309-3 and mutations described in Hearing and Shenk, 1983b). This refines our original estimate of the enhancer domain from that defined by in340-2 to the region between sequence positions 194 and 308.

,613n

,475n

,345n

Figure 2. Si Endonuclease HeLa Cells after Infection Mutants

Analysis

of ElA mRNAs

Synthesized

in

with Wild-Type Virus or Enhancer Region

Total cytoplasmic RNA was isolated 6 hr after infection and analyzed using a uniformly labeled ElA-Ml3 probe corresponding to the left 1574 nucleotides of the Ad5 genome. Bands corresponding to the ElA 1% (613 nucleotides) and 125 (475 nucleotides) 5’exons and their common 3’ exon (345 nucleotides) are designated.

A second control sequence (element II in Figure 1B) is nested between the element I repeats. Mutant d/309-64 and an overlapping mutant, d/309-141, both lack portions of this second element and showed a 4 to 5 fold decrease in cytoplasmic ElA mRNAs (Figure 2). Viruses carrying deletions that approach the endpoints of these two mutations, from either the upstream side (d/309-109 and d/30955) or the downstream side (d/309-21), also show reduced levels of ElA mRNAs. Mutants d/309-97 and di309-10 both

Ad5 ElA Enhancer 231

TR

ENH

Domains

499

A. -ENH-SR -2

EIA

1573

Table 1. Both ElA Enhancer Transformation by the Herpes

G?’

3’-WT 3’-643’-RH/87-

B.

Elements Function in Cis to Enhance Simplex Virus Thymidine Kinase Gene Coloniesl3ug

DNA

Plasmid

Expt. 1 (FoldEnhancement)

Expt. 2 (FoldEnhancement)

pHSV106 pHSV106-Ad5-WT pHSV106-Ad5-64 pHSVlO6-Ad5RH187 pHSV106SV40

a 54 31 20 166

5 30 18 13 79

(1 .O) (6.7) (3.9) (2.5) (20.8)

(1 .O) (6.0) (3.6) (2.6) (15.8)

Enhancer sequences were inserted at sequence position - 200 relative to the thymidine kinase mRNA cap site at + 1. Inserts maintained the normal orientation of enhancer segment relative to augmented gene. pHSV106SV40 contains the SV40 enhancer domain (sequence position 5235 to 270) inserted in the same location and orientation as the Ad5 elements. Human 143 thymidine kin-minus cells were transfected with 1 ug plasmid DNA per culture on a 100 mm plate (about 1 x lo6 cells) according to the procedure of Wigler et al. (1977). Thymidine kinase-positive colonies were quantitated IO days after transfection.

Figure 3. Analysis hancer Segments

of Ad5 Variants Containing Wild-Type at the 3’ End of Region ElA

or Mutant En-

(A) Schematic view of variants. Enhancer segments were inserted at sequence position 1573 in an in340-2 background. Also shown is a variant (dBO%ENH-SR) that contains the enhancer region inverted in its normal location. (6) RNA blot analysis. RNA was isolated 6 hr after infection, poly(A)+selected, and analyzed using an EIA-specific probe DNA.

synthesized wild-type levels of ElA transcripts and define maximal boundaries of this second regulatory region between sequence positions 248 and 282 (Figure 1B). Both Regulatory Sequences Act as Enhancer Elements for ElA Transcription Viruses carrying the alterations diagrammed in Figure 3A were constructed to test whether the regulatory elements described above could augment EIA transcription independently of location and orientation. The variants lack the ElA enhancer region at its normal location (deletion 2, Figure 1A) and carry an insertion of the same region from the wild-type or the enhancer region mutants near the S’end of the ElA transcription unit. All of these viruses were constructed in an in340 background (packaging region at the right end of the viral chromosome) because deletion 2 disrupts the Ad5 packaging signals (Hearing and Shenk, 1983a). In the first variant (in340-2/3’ENH-WT), a wild-type enhancer region was introduced at the 3’ end of region ElA. The other two variants (in340-2/3’ENH-84 and in340-2/3’ENHRH/87) carry enhancer fragments containing deletions 64 and RH187 (Figure lB), respectively. A final variant containing the Ad5 enhancer region in an inverted orientation at its normal location (dl-309-ENH-SR) was also constructed. These viruses were used to infect HeLa cells, and cytoplasmic RNA was isolated early (6 hr) after infection. Polyadenylated RNA was selected and levels of ElA mRNAs were measured by RNA blot analysis using an ElA-specific probe DNA (Figure 38). The 13s and 12s ElA mRNAs migrate as a single species in the gel system

used for this analysis. As expected, deletion of the entire enhancer region (d/340-2) resulted in a 20-fold decrease in ElA mRNAs relative to the wild-type viruses (d/309 and in340). Insertion of a wild-type enhancer region at the 3’ end of the ElA transcription unit (in340-2/3’ENH-WT) resulted in a 4 to 5 fold increase in ElA mRNA. Insertion of the same region from mutants 84 and RH/87 also resulted in increased ElA transcription relative to d/340-2, but the increase with each variant was about half of that observed using the wild-type enhancer region. These results mimic the effect of the mutations when present in the ElA 5’-flanking sequences (Figure 2) and demonstrate that both elements I and II are functional components of the wild-type enhancer region. Inversion of the enhancer region at its normal location (in340-ENH-SR) also resulted in increased ElA transcription relative to a mutant with no enhancer, but clearly this region functions most efficiently when positioned in its normal location and orientation. As a second test of element I and element II enhancer properties, the wild-type and mutant enhancer regions were inserted upstream of the herpes simplex virus thymidine kinase gene (-200 relative to the cap site at +l) in plasmid pHSV106 (Colbere-Garapin et al., 1979). These plasmids were used to transform human 143 thymidine kinase-minus cells, and positive colonies were selected in HAT medium (Table 1). Insertion of the wild-type Ad5 enhancer region upstream of the thymidine kinase gene enhanced the efficiency of transformation in human cells about 6-fold compared with pHSV106 containing no exogenous enhancer sequences. Enhancer regions carrying deletion RH/87 (pHSV106Ad5-RH/87) or deletion 64 (pHSV106-Ad564) also enhanced the efficiency of transformation but at levels that were intermediate between plasmids containing no enhancer (pHSV106) and those containing the Ad5 wild-type enhancer region (pHSVlOGAd5-WT). We conclude that elements I and II can function independently to enhance transcription as judged by their activity near the 3’ end of the ElA unit or by their ability to augment thymidine kinase transformation. Furthermore, their effects appear to be additive since neither

Cell 232

functions tions.

alone as efficiently

as the intact domain

func-

Element II Regulates Transcription of All Early Regions in Cis Since enhancers activate transcription of linked genes over considerable distances, we tested the effect of EIA enhancer mutations on transcription from the other adenovirus early regions. Activation of adenovirus early transcription is dependent on the ElA gene products (Jones and Shenk, 1979b; Berk et al., 1979). Therefore, 293 cells were used in this experiment because they contain and express the adenovirus ElA and ElB genes (Graham et al., 1977) and complement viruses expressing reduced levels of the ElA products. Total cytoplasmic RNA was isolated early (5 hr) after infection, and polyadenylated RNA was quantitated by RNA blot analysis using E2A-, E3-, and ECspecific probe DNAs (Figure 4). Mutant d/343 contains a deletion in the ElA coding region. It is defective for ElA function (Hearing and Shenk, 1985) and serves as a control for the ability of 293 cells to fully complement ElA defects. mRNAs accumulated to wild-type (d/309 and in340) levels in cells infected with d/343 for each of the regions tested. Enhancer element II mutants (in34OA5, in340-Bl, and d/309-64) generated reduced steady state mRNA levels for each of the early regions tested. The mRNA levels of regions E2A and E3 were reduced 5 to 7 fold, and E4-specific mRNAs were reduced about 3-fold. In contrast, element I mutant-infected cells contained normal levels of mRNAs encoded by these early transcription units. Transcription rates were measured for the E2A and E4 units (data not shown) and found to be normal in element I and reduced in element II mutant-infected cells. These analyses strongly suggest that element II modulates transcription in cis across the entire viral chromosome. To confirm this surprising result, an internally controlled coinfection experiment was performed. All of the enhancer mutants were constructed in a d/309 or an in340 background. Although these parental viruses are phenotypically wild-type, they carry a substitution mutation at 64 map units (Jones and Shenk, 1979a). The mutation generates altered E3 mRNAs and removes an EcoRl cleavage site. As a result, it is possible to distinguish the origin of both viral DNA and E3 mRNAs in cells coinfected with genotypically wild-type virus (wf300) and derivatives of di309 or in340.

Accordingly, 293 cells were coinfected with wt300 and either d/309 or in340, or their enhancer mutant derivatives. Each virus was used at a multiplicity of 20 pfulcell to ensure that all cells received both viruses in coinfections. Both cytoplasmic RNA and nuclear DNA were isolated early (5 hr) after infection. ESspecific mRNA was quantitated by Sl analysis using wild-type E3 DNA as probe (Figure 5A). The E9specific probe protected fragments of 490 and 375 nucleotides with RNA from cells infected with wt300, and fragments of 375, 300, and 190 nucleotides with RNA from cells infected with viruses of a d/309 or an in340 background. Cells infected with each of the phenotypically wild-type viruses (wt300, d/309, and in340) contained equivalent levels of E3-specific mRNAs. The element II mutants (d/340-A6, d/340-Bl, and d/309-64) all showed 5-fold reduced levels of E3 mRNAs, while the element I mutant (d/309-RH187) synthesized wild-type quantities of the RNAs. In mixed infections with wt300, each of the element II mutants still displayed reduced levels of E3 mRNAs relative to the coinfecting wild-type virus, confirming that reduced E3 mRNA accumulation within mutant-infected cells is not due to lack of a frans-acting factor. In contrast, wild-type E3 mRNA levels were detected in mixed infections with each of the other viruses tested (wt300 and d/309, in340, and d/309-RH/87). High molecular weight nuclear DNA from mixed infections was digested with EcoRl and analyzed by DNA blot analysis (Figure 58). Ad5 wt300 genome contains two EcoRl sites at 76 and 64 map units, generating fragments corresponding to 76,8, and 16 map units in length. The 8 and 16 map unit fragments are fused in viruses derived from d/309 or in340. Infected cell nuclei from each of the mixed infections contained equivalent levels of the two input genomes. Even though nuclei from cells infected with both viruses contained equal numbers of wf300 and mutant DNA molecules, 5-fold less E8specific mRNA was encoded by the enhancer element II mutant templates than by the wild-type templates. Similar results were obtained for ElBspecific mRNAs (data not shown) in mixed infections of HeLa cells with enhancer mutants and d/338, which carries a deletion within the ElB region (Pilder et al., 1986). We conclude that element I provides enhancer function specifically for region ElA while element II has a global effect, enhancing transcription across the entire viral chromosome.

Ad5 EIA Enhancer 233

Domains

300/309/340

,O-76mu *76-IOOmu +84-IOOmu

300

Figure

5. Viral mRNA

Production

and DNA Replication

within 293 Ceils Coinfected

with Wild-Type

.76-84mu

Virus

and Enhancer

Mutants

(A) Analysis of E3 mRNAs in single and mixed infections. RNA was isolated 5 hr after infection of 293 cells and analyzed using a wild-type ES-specific Ml3 probe DNA. The sizes in nucleotides (n) as well as the origins (~1300, d/309, or in340) of protected fragments are indicated. Mixed infections are designated as virus 1 x virus 2. (B) Analysis of viral DNA within mixedly infected 293 cells. High molecular weight DNA was isolated from the same cells described in A, digested with EcoRI, and analyzed by blot hybridization using an AdSspecific probe DNA. The positions in map units (mu) as well as the origins (wf300, d/309, or in340) of DNA bands are kdicated.

The Ad5 Enhancer Elements Are Not Required for Efficient Viral DNA Replication It was recently shown that the polyoma virus enhancer region is required not only for polyoma transcription but also for efficient viral DNA replication (devilliers et al., 1984). The polyoma equivalent of Ad5 element I (Figure 16) constitutes the minimal sequence required for this function (Veldman et al., 1985). We wanted to determine, therefore, if either element I or element II is required for efficient adenovirus DNA replication. To test this point, mixed infections were again performed in 293 cells. wt300 was used as the second virus in mixed infections because it could supply normal levels of early viral functions and, as described above, its DNA could be distinguished from mutant DNAs. High molecular weight DNA was isolated at various times after infection (9,13, and 17 hr) and digested with EcoRI. Viral DNAs were then quantitated by blot analysis (Figure 8). In each of the mixed infections, waO0 and mutant DNA accumulated at the same rate. We conclude that neither enhancer domain is required for efficient viral DNA replication in vivo.

Discussion We have defined two separate enhancer elements that regulate Ad5 transcription within infected cells. The two elements are functionally distinct in that one enhances transcription of the ElA unit alone while the other augments transcriptional activity of all early viral units. Thus, the two elements must mediate enhancement by different mechanisms. The first element (element I, Figure 1B) is repeated and specifically regulates ElA transcription. It resembles sequences present in polyomavirus, in a variety of retroviral control regions (Hearing and Shenk, 1983a), and in the transcriptional control regions of mouse and human a- and p-interferon genes (Goodbourn et al., 1985). The polyomavirus enhancer domain contains a perfect (10110 base pairs) element I homology (Herbomel et al., 1984; Mueller et al., 1984; Veldman et al., 1985). The sequence is duplicated in a number of naturally occurring polyomavirus strains (Ruley and Fried, 1983); and it plays a key role in the cell-type specificity displayed by polyomavirus vari-

Cell 234

Figure 6. Analysis of Viral DNA Replication in 293 Cells Infected with Enhancer Element I and Element II Mutants

O-76rnu 76-lOOmu 84-lOOmu

300

ants for differentiated, as compared to undifferentiated, murine cells (Herbomel et al., 1984). It also comprises a key domain required for efficient polyoma DNA replication (Veldman et al., 1985). In contrast to the polyomavirus case, adenovirus DNA replication proceeded at wildtype levels in the absence of both copies of element I (Figure 8). The second enhancer element identified in this study (element II, Figure 16) is located between the repeated sequences. It regulates transcription from the entire adenoviral chromosome (Figures 4 and 5) and, again, is not required for efficient adenovirus DNA replication (Figure 8). This element is delimited by deletions 97 and 10 in Figure 1B and spans a region of about 30 base pairs. It contains several interesting features. The first is one copy of a repeated sequence (element A in Figure 1B) that is required for efficient packaging of viral DNA late in infection (Hearing and Shenk, unpublished data). The second copy of element A is located immediately upstream of the boundaries of element II. Quite interestingly, a nearly identical copy of element A (lo/12 base pairs) is found at an equivalent position relative to the right end of the adenovirus genome (Steenberg and Sussenbach, 1979). However, this sequence does not appear to be the functional enhancer component of element II for several reasons. First, most element II mutants leave the upstream copy of this repeated sequence intact (Figure la), suggesting the somewhat unlikely possibility that only the downstream copy of this element can provide enhancer function. Second, the copy of this repeat located at the right end of the viral genome is not capable of replacing element II when placed at the left end of the viral chromosome (Leza and Hearing, unpublished data). A second sequence motif is repeated four times within the boundaries of enhancer element II (arrows below the sequence in Figure 1B) and is arranged as two sets of direct repeats that are inverted relative to each other. The Enconsensus for this repeated sequence is gGCG:AA. hancer mutants having the most dramatic effect on element II function delete part or all of two or more copies of this repeated sequence (Figure lB, deletions 84, 141, A5, and Bl; and Figure 2). Mutants having a less dramatic effect on element II function delete only one copy of this se-

High molecular weight DNA was isolated at 9, 13, and 17 hr after infection, digested with EcoRI, and analyzed by blot hybridization using an Ad&specific probe. Mixed infections are designated virus 1 x virus 2, and the positions in map units (mu) as well as the origins (wf300, d/309, or in340) of DNA bands are indicated.

76-84mu

quence (Figure 16, deletions 55, 109, and 21; and Figure 2). The structure of these repeated sequences strongly resemble binding site II for the SV40 large T antigen (DeLucia et al., 1983) and suggests that four binding proteins might conceivably interact with this region. A homology search of the entire Ad2 genome (approximately 38,000 base pairs; Gingeras et al., 1982) revealed that this consensus sequence is present 22 times. It is noteworthy that six of these homologies (27% of the total) are present within the enhancer region defined by deletion 2, between sequence positions 194 and 358 (0.4% of the viral genome). Because of the distinct left-end-specific polarity observed for the function of element II (discussed below), it appears unlikely that these other homologies represent functional enhancer sequences. It is possible, however, that the remainder of these repeats act as a sink, directing transcription factors to the adenovirus genome in a mechanism similar to that proposed for the Xenopus ribosomal RNA gene promoter repeated elements (Reeder, 1984). How does the left-end element II augment transcription across the entire chromosome? As yet, one can only speculate. Perhaps the sequence helps to position the viral chromosome into a transcriptionally active compartment within the nucleus. Conceivably, it could mediate attachment of the viral genome to the nuclear matrix and thereby facilitate transcription. Alternatively, the element might serve as a highly efficient entry site for a transcription factor, which could then scan downstream to function at one or another viral transcription unit. Because enhancer element II lies in close proximity to sequences required for efficient packaging of Ad5 DNA into virions, an argument could be made that element II affects transcription from the early regions in the viral genome by affecting an unpackaging event early after infection. Genetic data argue strongly that packaging and enhancer sequences are functionally distinct. The packaging sequences do not function when located at the 3’ end of the ElA transcription unit (Samulski, Hearing, and Shenk, unpublished data) while enhancer element II clearly functions at this location (Figure 3). Conversely, the packaging sequences function perfectly well at the right end of the viral chromosome (Hammarskjbld and Wliberg, 1980; Hearing and Shenk, 1983a), yet both mu-

Ad5 EIA 235

Enhancer

Domains

tams d/340& and d/340-61 contain a duplicated copy of the enhancer/packaging region at their right terminus and still display an element II mutant phenotype (Figures 2,4, and 5). Element II, therefore, functions only when located at the left end of the viral genome. Furthermore, if the transcriptional defect displayed by element II mutants (Figures 2, 4, and 5) were due to an unpackaging defect, the variants would be expected to display a lag in DNA replication. This is not the case (Figure 6). Finally, element II functions as an enhancer in long term thymidine kinase gene transformation assays (Table 1). In sum, these observations lend strong support to the conclusion that element II plays a direct role in transcription. We have no clear explanation for the inability of element II to function at the right end of the viral chromosome. This sequence is present at the right end of the d/340-A6 and 81 genomes, yet these mutants display the same transcriptional phenotype as d/309-64 or 141, which lack the right-end insert (Figures 2,4, and 5). Possibly, asequence at the right end of the viral genome blocks its function. Alternatively, element II might function with an additional sequence at the left end that lies outside the domain transposed to the right end in d/340& and Bl. A number of enhancer elements have been mapped in the adenovirus ElA Y-flanking sequences using a variety of assays. The enhancer described by Weeks and Jones (1983) most likely corresponds to element I defined above. The minor enhancer element defined by Sassone-Corsi et al. (1983) is probably equivalent to element II. The major enhancer sequence defined by Sassone-Corsi et al. (1983) and the element described by lmperiale et al. (1983) map in regions that we have found dispensable for efficient ElA transcription within infected cells (Hearing and Shenk, 1983a). The functional role of these other sequences in regulation of viral transcription is not clear. Experimental

Procedures

Viruses and Cells Growth of virus stocks and preparation of viral DNA have been described (Jones and Shenk, 1978). Mutant d/309 was selected as an Ad5 variant that contained only one Xbal cleavage site at 3.8 map units (Jones and Shenk, 1979a). d/309 grows like wild-type Ad5 by all criteria tested. Mutant in340 is identical to d/309 except that it also contains the cis-acting Ad5 packaging sequence (left-end 353 base pairs) at the right terminus of the viral genome (Hearing and Shenk, 1983a). d/343 contains a two base pair deletion early in the EIA coding sequences and is defective for EIA function (Hearing and Shenk, 1985). 293 cells, which contain and express the left end of the Ad5 genome (Graham et al., 1977) were grown in medium containing 10% calf serum. Spinner cultures of HeLa cells were maintained in medium containing 5% calf serum. Construction and Propagation of Viral Mutants Deletion mutations in the Ad5 enhancer region were initially constructed using a D-loop mutagenesis procedure as previously described (Hearing and Shenk, 1983b) in plasmid pEiA-WT (Hearing and Shenk, 1983a). A fragment containing the left 1% (353 bp) of the viral genome was used in the mutagenesis procedure. Deletion RH was constructed by deleting the sequences between an Rsal site at nucleotide position 194 and an Hhal site at position 218. The exact endpoint of each deletion was determined by nucleotide sequence analysis using the procedure of Sanger et al. (1977). The mutations were then rebuilt into intact viral chromosomes using the method of Stow (1981). Viruses were propagated using 293 cells.

Variants containing the Ad5 enhancer region inserted at the 3’ end of the EIA coding sequences or in an inverted orientation were constructed as described previously (Hearing and Shenk, 1983a) except the enhancer fragments (Hpall at nucleotide position 188 to Sstll at position 353) were prepared from the wild-type and mutant plasmids described in the text. RNA and DNA Preparatlon and Analysis RNAs for Northern and Sl analysis were prepared as described (McGrogan et al., 1979). RNAs were harvested 8 hr after infection of HeLa cells and five hours after infection of 293 cells. Polyadenylated RNA was selected and analyzed by Northern analysis as described (Hearing and Shenk, 198%). Sl analysis was performed as described by Osborne et al. (1982) using a uniformly 32P-labeled Ml3 probe that spanned the region from Ad5 nucleotides 1 to 1574 for region EIA and map units 75.9 to 81.0 for early region 3. High molecular weight DNA was isolated and analyzed by blot hybridixation analysis as described by Maniatis et al. (1982). Probes were labeled to high specific activity by nick translation (Rigby et al., 1977). Acknowledgments This work was supported by a grant from the American Cancer Society (MV-45) to T S. and grants from the National Institutes of Health (Al 20412) and American Cancer Society (JFRA-91) to f? H.; T S. is an American Cancer Society Research Professor. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adveffisefnent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

27, 1985.

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