genome organization of mouse adenovirus type 1 early region 1: a novel transcription map

VIROLOGY 170,523-536 (1989)

Genome Organization of Mouse Adenovirus Type 1 Early Region 1: A Novel Transcription Map

AMY OBERHAUSER BALL, CLAYTON W. BEARD, SAMBRA D. REDICK, AND KATHERINE R. SPINDLER’

Department of Genetics. University of Georgia, Athens, Georgia 30602

Received December 27, 1988; accepted Februav 23, 1989

Mouse adenovirus type 1 (MAV-1) genomic DNA from 8.9 to 13.7 map units was sequenced and the early region 1 (El) transcription map was determined by Sl nuclease, primer extension, and Northern analyses, and cDNA sequencing. The El transcription map of MAV-1 had marked dissimilarities from the conserved transcription maps of primate adenovirus El s. One major ElA and two El B mRNAs were identified in overlapping transcription units. The single ElA mRNA was composed of three exons; the last exon was coincident with the last exon of the El B mRNAs. While human adenovirus type 2 (Ad2) utilizes alternate splice donors for the first ElA mRNA exon, MAV-1 does not. Thus, no protein is predicted that would correspond to the Ad2 243 amino acid protein, although MAV-1 can encode a protein similar to the Ad2 289 amino acid protein (A. 0. Ball, M. E. Williams, and K. R. Spindler, 1988, J. Viral. 62, 3947-3957). Two spliced El B mRNAs differed from each other in an intron near the 5’end of the smaller El B mRNA. This smaller mRNA could encode only the 55K El B protein, while the larger mRNA could encode both the 21 K and 55K El B proteins. 0 1999 Academic Press, Inc.

INTRODUCTION

Much is known about the molecular biology of human adenoviruses, although molecular studies on in viva pathogenesis have been difficult because adenoviruses are generally species specific (Tooze, 1981). The mechanisms of adenovirus pathogenesis may be addressed using mouse adenovirus type 1 (MAV-1, also known as strain FL) as a model system. However, the molecular genetics of MAV-1 are not well characterized. Initial studies oriented the genome and showed regions of homology to human adenovirus type 2 (Ad2) (Antoine et al., 1982; Larsen et a/., 1979). MAV-1 early region 1 (El) was identified by functional and sequence homologies (Ball et a/., 1988).

Human adenovirus El has been well characterized in terms of transcription and protein structures and functions. ElA products regulate transcription from both cellular and viral promoters (for review, see Berk, 1986; Grand, 1987). The E 1 A gene encodes two major proteins of 243 and 289 amino acids (aa); 46 internal aa are unique to the larger E 1 A protein (Perricaudet et al., 1979). An El A transactivation function is localized to the unique region of the 289-aa protein (Glenn and Ricciardi, 1987; Lillie et a/., 1987; Monte11 et al., 1982; Ricciardi et al., 1981). The 243-aa protein functions as a transcriptional repressor (Lillie et al., 1986; Velcich and Ziff, 1985) and is necessary for viral DNA replica-

Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No. J03353.

’ To whom requests for reprints should be addressed,

tion in growth-arrested cells (Monte11 et al., 1984; Spin- dler et a/., 1985) and for growth factor induction in primary cells (Quinlan et a/., 1988). Both El A proteins are required for complete transformation by Ad2 (Hurwitz and Chinnadurai, 1985; Monte11 et al., 1984). The two E 1 B proteins, 2 1 K and 55K, are also required for cellular transformation (Graham, 1984). In addition, the El B proteins maintain the integrity of viral and cellular DNA, are involved in the selective transport of viral and cellular RNAs, and modulate viral replication and gene expression (Grand, 1987).

The El genomic sequence has been determined for many human adenovirus serotypes and other non-human adenoviruses (Ad2, Gingeras et a/., 1982; Ad4, Tokunaga et al., 1986; for a comparison of Ad5, Ad7, and Ad1 2, see van Ormondt and Hesper, 1983; van Ormondt et a/., 1980; Ad40, lshino et al., 1988; Ad41, Allard and Wadell, 1988; MAV-1, Ball et al., 1988; this paper; simian adenovirus 7 (SAV-7), Kimelman et al., 1985; tupaia adenovirus, Brinckmann et al., 1983; Flu- gel et a/., 1985). El transcripts were mapped for Ad2 and Ad5 (Baker and Ziff, 1981; Berk and Sharp, 1978; Bos et a/., 1981; Chow et al., 1979; Perricaudet et al., 1979, 1980); the transcription maps determined for Ad7,Ad12, andSAV--/areverysimilar(Boseta/., 1981; Dijkema et al., 1982, 1980; Kimelman et al., 1985). With other adenovirus El s, the conservation at the nucleotide and amino acid level was sufficient to predict transcription maps which were similar to that of Ad2 El. In Ad2, ElA and El B are discrete transcription units. The major early 12 and 13 S ElA mRNAs share

523 0042-6822189 $3.00 Copyright 0 1999 by Academic Press. Inc. All rights of reproduction in any form reserved.

524 BALL ET AL.

5’ and 3’termini and differ only in the position of an internal donor splice site. Two major Ad2 El B mRNAs, 13 and 22 S, also share 5’ and 3’termini. The 13 S El B mRNA can only encode the 21 K protein, since a splice removes much of the coding sequence of the 55K El B protein. The 22 S El B mRNA can encode both the 21 K and 55K El B proteins.

The genomic and predicted protein sequences of MAV-1 El were distinct enough from those of Ad2 El that, although open reading frames (orfs) for El proteins could be identified, the mRNA structures could not be easily deduced (Ball et a/., 1988). In the present study, early transcripts for MAV-1 El A and El B were mapped by a combination of Northern blot analyses, nuclease protection and primer extension assays, and genomic and cDNA sequencing. The data indicated that MAV-1 ElA and ElB mRNAs had structures different from those of other adenoviruses. The most notable difference is that MAV-1 transcribed only a single major ElA mRNA which overlapped the El B transcripts.

MATERIALS AND METHODS

Plasmids and genomic sequencing

The genomic DNA of MAV-1 is about 31,500 bp; the genome is divided into 100 map units (m.u.), with 0 m.u. at the left end of the genome (Ball et al., 1988). The construction of plasmids containing MAV-1 genomic fragments used in this work was described previously (Ball et al., 1988). The insert of pMSCL consists of nucleotides (nt) 90 to 2556; pMBE contains the BarnHI-E fragment, nt 359 to 1846; pMPl includes the Pstl-I fragment, nt 2556 to 4333; prK5 contains nt 1 to 828; pMXD contains the Xbal-D fragment, 0 to 17 m.u.; pMBA includes the BarnHI-A fragment, 30 to 64 m.u. For sequencing genomic DNA, the Pstl-I fragment was subcloned into M 13mplO in both orientations and a series of deletion clones was obtained by the Bal 31 exonuclease method (Poncz et al., 1982). Clones were sequenced by the dideoxy-chain termination method (Sanger et al., 1977), by using either the large fragment of DNA polymerase I or Sequenase enzyme (United States Biochemical Corp.). Computer analysis was done using lntelligenetics and International Biotechnol- ogies, Inc./Pustell software.

Cells, virus, and RNA preparation

Mouse L cells were grown and MAV-1 infections were carried out as described (Ball eta/., 1988). For the isolation of RNA, L cells were infected with MAV-1 at an m.o.i. of 1 to 5 PFU/cell and incubated for 8 to 48 hr. Total cytoplasmic RNA was isolated from the mock- infected or MAV-1 -infected cells by Nonidet P-40 lysis

and phenol/chloroform extraction as described (Berk et al., 1979). Polyadenylated RNA was selected by two passes over oligothymidylic acid-cellulose columns (Aviv and Leder, 1972).

Northern blots

For Northern blots, 2 pg per lane of poly(A)+ RNA were separated on 1.2% agarose/formaldehyde gels and transferred to nitrocellulose in 20X SSC (Maniatis et a/., 1982). Blots were air-dried, baked for 30 min in a vacuum oven at 80“, and prehybridized, hybridized, and washed as described (Thomas, 1980). Probes were labeled to a specific activity of 1 X 1 O8 cpm/pg or greater by the random primer method (Feinberg and Vogelstein, 1983). For the hybridizations, 1 X 1 O7 cpm of denatured probe were used. Molecular size standards were end-labeled HindIll fragments of Ad5 DNA.

Nuclease protection and primer extension assays

For probes and primers, plasmids or isolated fragments were first digested with the appropriate enzyme, and then either 5’ end-labeled with [T-~~P]ATP, using polynucleotide kinase, or 3’ end-labeled with [a-32P]dATP, using the large fragment of DNA polymerase I (Maniatis et a/., 1982). The labeled DNA was digested with a second enzyme to remove the label at one end and the desired fragment was isolated from a gel. Sl nuclease protection assays were performed using the method of Berk and Sharp (1977) as modified by Weaver and Weissman (1979). Primer extension reactions were hybridized and then extended with avian myeloblastosis virus reverse transcriptase (Promega Corp.) as described (Treisman et al., 1982). For the Sl nuclease and primer extension reactions, 25 to 100 pg of RNA were used. Hybridization temperatures were experimentally determined for each probe. Overdiges- tion by the Sl nuclease was a problem because some hybrids, being AT-rich, were unstable (Martin and Ti- noco, 1980) and so were subject to digestion when reactions were incubated at 26” or 37”. Therefore, the hybridized samples were chilled by addition of 200 ~1 of ice-cold Sl buffer followed by 250 @I of ice-cold Sl buffer containing 100 units of Sl nuclease; the digestions were incubated on ice except as noted in the figure legends. Sl nuclease and primer extension reactions were analyzed on 5% acrylamide/8 M urea gels or on 69/o acrylamide/8 M urea sequencing gels. Chem- ical degradation sequence reactions (Maxam and Gil- bert, 1980) were pet-formed on 5’ end-labeled Sl probes for markers; in other experiments molecular size standards were end-labeled Rsal fragments of 4x174 replicative form (RF) DNA.

MOUSE ADENOVIRUS El TRANSCRIPTION 525

1561 2890

1069 1114 1462

ElB

281 IAn/

2890

108.154 752 829 951 IE lA

6 IdO0 20bo 3obo 400hnt

FIG. 1. MAV-1 El mRNAs and or-k. The transcription map of MAV-1 ElA and El B mRNAs was determined by Northern blot analyses, nuclease protection and primer extension assays, and cDNA sequencing. The line at the bottom represents the DNA genome, measured in nucleotides, and corresponds to 0 to 12.7 m.u. of the MAV-1 genome. The bold lines represent mRNA exons interrupted by introns (carets). Open boxes represent the ElA coding regions; the hatched box, the smaller El B protein coding region; and shaded boxes, the larger El B protein coding regions. Nucleotide numbers below the mRNAs indicate start sites, splice junctions, and polyadenylation sites. Nucleotide numbers above the orfs indicate start and stop codons.

cDNA cloning

Cytoplasmic RNA was isolated from L cells 22 hr postinfection (p.i.), poly(A)-selected, and analyzed on a Northern blot to ensure that it did not contain detect- able amounts of late infected-cell RNA (see below). Double-stranded cDNAs were made according to the method of Gubler and Hoffman (1983) using reagents from Bethesda Research Laboratories. The cDNAs were then treated with EcoRI methylase and ligated to EcoRI linkers. Following digestion by EcoRI and purification away from excess linkers (Maniatis eta/., 1982) the cDNAs were cloned into the Lambda ZAP vector (Stratagene). Plaques which hybridized to MAV-1 DNA and pMBE were plaque-purified three times. Plasmids containing the cDNA inserts were excised from the Lambda ZAP vectors according to the manufacturer’s instructions. The resulting plasmid clones were characterized by restriction endonuclease analysis and DNA sequencing of both ends of an insert. For repre- sentative cDNA clones, nested sets of deletion clones were prepared using exonuclease III and mung bean nuclease (Henikoff, 1984) and sequenced by the dideoxy-chain termination method (Sanger et a/,, 1977).

RESULTS

Several types of experiments were performed to map El mRNAs expressed early during MAV-1 infection. These included genomic sequencing, Northern blot analyses, nuclease protection and primer extension assays, and cDNA sequencing. The transcription map in Fig. 1 is a summary of the data which is presented in the following sections. Nucleotide numbers are given for start sites, splice sites, and polyadenylation

sites of the mRNAs, and for proposed protein initiation and termination codons.

DNA sequence of 8.9 to 13.7 m.u. of MAV-1

The sequence of nt 1 to 2950 of MAV-1 El was determined previously (Ball et a/., 1988) and included the orfs of the El proteins but did not extend to the 3’ends of the mRNAs. In this paper we report the DNA sequence of nt 2801 to 4333 of MAV-1, which completes the sequence of El and includes adjoining sequences corresponding to predicted late MAV-1 proteins. The sequencing strategy is shown at the bottom of Fig. 2; both strands were sequenced in their entirety. Orfs longer than 100 aa were examined using protein homology searches, and ot-fs 5,6, and 7 appeared to correspond to human adenovirus proteins. Only the carboxy terminus of orf 5 is shown; this or-f could encode the 55K El B protein (Ball et a/., 1988). Orfs 6 and 7 were similar to Ad2 late proteins (data not shown). A limited homology of orf 6 to protein IX, a late structural protein of Ad2 (Alestrom et al., 1980) was detected. Or-f 7 was translated from nt 4333 to 3256, and the 360 aa of this or-f had 76% similar or identical amino acids when compared to the carboxy terminus of Ad5 late protein IVa2 (van Beveren et al., 1981). The genomic DNA sequence is presented in Fig. 3. The DNA sequence from nt 2801 to 2950, previously reported, is shown again here in order to include the entire or-f 6 and the El mRNA splice sites in this region. Translation and transcription signals for E 1 and for the possible late proteins in this region are underlined and indicated in the sequence.

526 BALL ET AL.

8.9 ma. 13.7 m.u.

I I I I I 2800 3000 3500 4000 4333 nt

5 II IIIIIII I 11111 I _

I I I I I11 III1 II I II I II I IIIIIIU

I I I I I IIIIII II III Ill/ II

z - -

FIG. 2. Map, open reading frames, and sequencing strategy of 8.9 to 13.7 m.u. of MAW1 DNA. The top line represents the MAV-1 genome; map units and nucleotide numbers are given. Nucleotide 1 is at the left end of the genome. Horizontal arrows (indicating the direc- tion of translation) in the middle portion of the figure represent the six translational reading frames. Vertical lines on the arrows indicate stop codons; the vertical line marked with an open circle indicates a methionine codon. Numbered, shaded boxes represent those orfs referred to in the text. Arrows at the bottom of the figure show the sequencing strategy.

Northern blot analysis of El mRNAs

Preliminary experiments examining the production of viral DNA in MAV-l-infected L cells vs Ad5-infected HeLa cells indicated that MAV-1 infection under our conditions was at least 8 hr slower than Ad5 infection (K. Spindler, unpublished data). Viral DNA labeled with 32P04 was observed at 16 hr p.i. in the Ad5-infected cells, whereas in MAV-l-infected cells, labeled viral DNAwas not observed until 24 hr p.i. The onset of DNA synthesis defines the beginning of the late phase of infection. We therefore used RNA isolated at 20 to 22 hr p.i. for early RNA, and RNA isolated at 48 hr p.i. for late RNA for the mapping experiments shown below. To confirm that RNA isolated at 22 hr p.i. did not contain late RNA, Northern blots with RNA isolated from cells mock-infected or infected with MAV-1 for 8 to 48 hr were probed with pMBA (30 to 64 m.u.) (Fig. 4A). This probe included a region of homology to the Ad2 hexon gene (Larsen et a/., 1979). At late times in Ad2 infection, many transcripts, including the hexon transcript, are produced from the major late promoter, resulting in a qualitative change in the transcription pattern. There was a striking difference in the pattern between MAV- 1 RNA isolated 48 hr after infection (lanes 7 and 10) and all earlier infected-cell RNA (lanes 2 through 6, 9). This suggested that RNA isolated at 48 hr p.i. repre- sented late RNA and that RNA isolated prior to 24 hr p.i. did not contain significant amounts of late RNA.

MAV-1 El mRNAs were detected by hybridizing a Northern blot of mRNAs isolated at various times p.i. to

pMSCL (MAV-1 nt 90 to 2556) (Fig. 4B). mRNAs which were 1400, 1900, and 2200 nt in length were first detected at 14 hr p.i., although the 1400-nt band is faint (Fig. 4B, lane 4). In other experiments, the 1400-nt band was more apparent at 14 hr p.i. (data not shown). In addition, at 48 hr a minor band of 900 nt was detected by this probe. This band, which might represent a minor late ElA mRNA, was not investigated further. More precise locations of the major early mRNAs (1400, 1900, and 2200 nt) were determined by hybridization of similar blots to shorter probes from the left end of MAV-1 (for example, Fig. 4C) and the results are summarized in Table 1. A 1400 nt mRNA hybridized to probes at the far left of the genome (nt 1 to 828) and to pMPl (nt 2556 to 4333). We identified this as an ElA mRNA because it included an or-f between nt 281 and 852 which could encode a peptide similar to the first exon of the primate adenovirus ElA proteins (Ball eta/., 1988). The hybridization to pMPl suggested, however, that the ElA mRNA splicing pattern was unusual for an adenovirus ElA message. The 1900- and 2200-nt mRNAs hybridized to several probes between nt 828 and 4333, and were identified as El B mRNAs since this region encompassed approximately the coding regions for MAV-1 proteins similar to human adenovirus El B proteins (Ball et a/., 1988). The 1900- and 2200-nt mRNAs differed in their intensity of hybridization to the Bglll-SalI fragment (nt 828 to 1 175) (Fig. 4C), suggest- ing that the two El B mRNAs might undergo alternate splicing in this region of the genome.

Nuclease protection and primer extension assays

El mRNA 5’ and 3’ ends and splice junctions were located by nuclease protection and primer extension assays. Using a primer extension assay, we detected multiple bands which indicated 5’ ends between nt 98 and 158 (Fig. 5, lanes 5 and 6; lanes 11 and 12 show a darker exposure). Similar fragments were observed with an Sl nuclease analysis, although the precise sites mapped differed by 1 or 2 nt (Fig. 5, lanes 8 and 9; lanes 14 and 15). These ends corresponded to 5’ start sites of the ElA mRNAs, since the primer extension and nuclease protection assays mapped the same positions. The probe used for the analysis shown in Fig. 5 was 5’end-labeled at nt 362. In Sl nuclease assays with a probe end-labeled at nt 654, the heteroge- neity and approximate location of the 5’ ends corresponded to that seen in Fig. 5 (data not shown). Similar 5’ ends were observed in both early and late MAV-1 RNA (compare lane 5 to lane 6, lane 8 to lane 9). At 48 hr p.i., more mRNA hybridized to the ElA region probes than at 22 hr p.i. (Figs. 4B and 4C, Fig. 5). Human adenovirus ElA mRNA levels also increase throughout the


AGGAATTGAG Tu/mTG TACGAGACCA ATATGGGTGA GGGAAAATGT TTACTGTGTA ATTAAAAATT CTTTGTGTTT GCI&/ATGm 2890 met orf 6 splice acceptor

splice donor

qGCCGTATGC GACGGTGGGC CGCTACTTCT CGGCTGTTGC ACGAAGATCC GCCTGCGACA CCGCCCAGCC AAGACCAACA GGCTGAAGTA 2980 term orf 5 (ElB 55kd and ElA)

CTTCGAAGAG ATATTCACAT TCTGAATGCG AAAATTTCTG AGTTGGAAAA TCAAATGGAA CGTCTGTGCC GCTCTTTGGA ATCGACCTTT 3070

AACAGAATAG AACTGCTTCA TTCTATGCTT GCACAAGGAG AGGAAGAGGA GGAAGAGGAG GACGGAGCTG AAGACATTGA GGAAAACGGG 3160

GAAGAAAGTG ATmTCCCA AATAAAGATG CATGCTCACG CGCTTTATGT TTAATA?@AA TGCACACACG CTTATGCTTA ATmGGTGT 3250 term orf 6 poly A signal poly A signal

poly A signal

TTAmCTTA TTTGCGAGTT TGTCACATTT TTTACTATAT GCGGTGTTCC ACCTTTGTCG CTGACGAAGG ACTTTGTCTA TTTTTTCTAG 3340 poly A site

term orf 7 (complement)

TAGCTGGTAA AGAATAGTTT GCACATTTAG GTACATAGGC ATTAAACCGT TTTGGGGGTG AAGGTACATC CATTGCATAC ATTCATGTTC 3430

GGGGGAGGTG TTATAGATGA TCCAGTCGTA ATTTATGTGG TTAGCGTTAT GATGAAAAAT ATCTTTTAGC AAAAGGGTAA TAGCAGTAGG 3520

CATGGCTTTA GTGTAGGTGT TAATGAAGCG AGCAACTTGG GAAGGTTGCA TTTTGGGGCT AATGATGTGG CATTTGCTTT GGATTTTTAA 3610

AGTAGCAATG TTGCCCGCCT GGTCTTTTCT GGGGTTCATG TTGTGCAGCA CTACGAAAAC AGAGTAACCG GTACACTGGG GAAAACGATC 3700

GTGTAGCTTT GAAGGAAAGG CATGGAT+AAA TTTTGCAATG CTTTTATGTT TTCCCAATTC CTCCATACAC TCATCCACAA TAATACAAAT 3790

GGGACCTCTT TGGGCCGCTT GGGCAAAAA T GTTTTGGGGG TGTGTAACAT CGTAGTTGTG GTCTGCTGTA AGGTCATCGT AGGTGAGTGT 3880

AATGAGTCGA GGTTTTAAAG TTCCGCTTTT AGGAACAATG GTCTGTTGCG GACCGCATGT ATAGTTTCCT TCAACTAGTT GTGCATTCCA 3970

CGCTGACATT TCTTGCGGGG GTATCATGTC GATTTGAGGA ACCACAAAA~I AAACGGTTTC GGGTGAAGGA TCTATTAAAT GACAGGAAAG 4060

AAGGTTGCGG AGCAATTGCG ACTTTCCGCT TCCTGTGGGC CCGTACACAA TTGCAATCAG GGGTTGCAAA GAATAGTTTA ACGATTCGCA 4150

GTTGCCTTCT GGACTTAAAA GCGGAGCTGC ACTATTTAGC GATTTACATA GGATCTCATA ATCATGTTTT AGTTCTTGAA GGAGACCTTT 4240

GCCTGCTAAG GCCGCTAAGT CTTCAACAGT CTCAAAGTTC ATGAGGGGCT TTAGTCCTTC GGAAAGGGGC AAGTTGGATA GAGTGTGCTG 4330

CAG 4333

FIG. 3. DNA sequence of 8.9 to 13.7 m.u. of MAV-1. Several transcription or translation features are underlined, including initiator (met) and termination (term) codons, splice donor and acceptor sites, polyadenylation signals, and pclyadenylaticn sites.

course of infection (Osborne and Berk, 1983; Shaw and Ziff, 1980).

The two major Ad2 El A mRNAs have identical 5’and 3’ ends and an identical second exon, but differ in the position of the donor splice site (Perricaudet er a/., 1979). We expected to find two donor splice sites within the region of the MAV-1 genome corresponding to the first exon of primate adenovirus El A mRNAs. A fragment was 3’end-labeled at nt 359 and Sl nuclease protection assays were performed (Fig. 6). Only one fragment about 390 nt in length was protected, indicating only one donor splice site, near nt 750. The alternate splice site is found between conserved regions 2 and 3 in primate adenoviruses (Kimelman et a/., 1985) which would correspond to about nt 675 in the MAV-1 genomic sequence and would have been indicated by a protected fragment about 315 nt in length. No such fragment was detected, even after long exposures of

the gel. To confirm this result, the experiment was re- peated several times with different early and late RNA preparations, including RNA isolated 14 hr p.i. (data not shown), the earliest time that El mRNAs are seen on Northern blots (Fig. 4B). In every case only one protected fragment was seen. DNA sequence analysis indicated that there were two possible donor splice sites at nt 744 and 753; the exact one used was determined by sequencing cDNA clones (see below). A minor protected fragment 140 nt in length which would map a 3’ end at about nt 500 was seen only in late RNA (Fig. 6, lane 4) and was not investigated further.

A second short ElA exon of approximately 125 nt with a 5’end near nt 828 was identified by Sl nuclease protection experiments (data not shown). A third ElA exon was inferred because of the Northern blot analyses in which the 1400 nt ElA mRNA hybridized to pMPl (nt 2556 to 4333) (Table 1). Furthermore, the 1400 nt

528 BALL ET AL.

A 5 5 r” 8 11 141822 48hg 22 48h

- 8.2

8.2

5.3, 5.6

4.6 3.4

2.8, 2.9

1.0

= 5.3, 5.6

- 4.6

- 3.4 = 2.8, 2.9

- 2.0

-1.0

123456 7 89 10

c 5 r” 8 11 141822 48h

2.8

1.0

B ‘: r” 8 11 141822 48h

2.8 -

j/ -2.2 2.0 - ,!, !‘, ,, / - 1.9

- 1.4

l.O- - 0.9

123456 7

123456 7

FIG. 4. Northern blot analysis of MAV-1 mRNA. Poly(A)+ RNA extracted from cells infected with MAV-1 for 0 to 48 hr was analyzed on a Northern blot as described under Materials and Methods. Hours p.i. are shown across the top of the figures. (A) Time course of MAV-1 late gene expression. mRNA preparations from two different MAW1 infections were analyzed (lanes 1 to 7 and lanes 8 to IO). The blots were probed with random primer-labeled pMBA (30 to 64 m.u.). Lane 7 was exposed one-fourth as long as the other lanes. Numbers on the sides are the sizes of the Ad5 HindIll markers kbp. (B) A Northern blot similar to that shown in (A), lanes 1 to 7, was probed with pMSCL (nt 90 to 2556). Numbers on the left are the sizes of the Ad5 HindIll markers; the numbers on the right indicate the sizes of the major bands. (C)The same blot as(B), stripped and then probed with a Bglll-Sal1 fragment (nt 828 to 1175).

size of the mRNA was larger than could be accounted for by the 750 nt identified in the exons between nt 125 to 750 and nt 825 to 950, and a poly(A) tail. The second and third exons were confirmed and located precisely by sequencing in all three of the E 1 A cDNA clones (see below).

The Northern data indicated that the two El B mRNAs differed from each other only by about 300 nt in size, and by their intensity of hybridization to a probe covering MAV-1 nt 828 to 1175 (Fig. 4C, Table 1). Sl nuclease analyses with probes 5’ end-labeled at nt 1544 and at nt 1274 indicated 5’ends at about nt 1460

and 1089, respectively (Figs. 7A and 7B). To determine if the sites were mRNA start sites or acceptor splice sites, the results from Sl nuclease protection and primer extension assays were compared (Fig. 7C). Both the Sl nuclease probe and the primer extension probe were 5’ end-labeled at nt 1642. The products of primer extension and nuclease protection which map to about nt 1089 were of equal length (550 nt fragment in Fig. 7C, compare lanes 3 and 4 with 6 and 7) indicating that this was the 5’ start site for an El B mRNA. However, for the 5’ end mapped by Sl nuclease analysis to about nt 1460, the primer extension product


TABLE 1

SUMMARY OF NORTHERN BLOT ANALYSES

Probe”

mRNA Time* 90-2556 I-360 l-828 828-l 175 1175-2556’ 2556-4333

900 Early -d - - - - -

Late +/- +1- +I- +/- - -

1400

1900

2200

850e

Early + + + + - +

Late + + + + - +

Early + - - -I/- + +

Late - - - +I- + +

Early + - - + + +

Late + - - + + +

Early - - - - - -

Late +I- - - - +I- ++

a MAW1 DNA either as an isolated fragment or in a plasmid was labeled by the random primer method and used to probe Northern blots as described under Materials and Methods. See Figs. 38 and 3C for examples.

b RNA was extracted from mock-infected cells or 22 hr (early) or 48 hr (late) after MAV-1 infection. c Several probes from this region were used, with identical results. d - indicates no hybridization; +I- indicates weak hybridization; + indicates strong hybridization; and ++ indicates very strong hybridization,

similar to the intensity seen in Fig. 3A for late mRNAs. e The 900- and 850-nt bands are probably not from the same mRNA, because of the great difference in intensity of hybridization at late times

and their differential hybridization to the indicated probes.

mapped approximately 30 nt longer than the nuclease protection product (210 and 180 nt fragments in Fig. 7C, lanes 3 and 4 compared to lanes 6 and 7). If the two mRNAs used the same 5’star-t site at nt 1089, possible donor sites at nt 1106, 1109, 1115, or 1129 could splice to an acceptor splice site at nt 1462. The sequence of an El B cDNA clone clarified the position of the splice sites (see below).

3’ and 5’ ends (donor and acceptor splice sites, see cDNA sequencing, below) near nt 2815 and 2885, respectively, were identified in the experiments shown in Figs. 8A and 8B. In Fig. 8A, the probe was 3’ end-labeled at nt 1846. Only one major protected fragment of about 1000 nt was identified in the analysis of both early and late mRNA, which indicated a 3’end at about nt 2800. The ends indicated by minor protected fragments about 250 nt in length (Fig. 8A) mapped near T- rich regions and may have been artifacts of the nuclease protection assay (see Materials and Meth- ods) or minor mRNAs. In the experiment shown in Fig. 8B, the probe was 5’ end-labeled at nt 3190. Only one fragment of 305 nt was protected, which identified a splice site near nt 2885. Determination of the 3’ end of the mRNAs is shown in Fig. 8C. The probe was 3’end- labeled at nt 2936; two fragments about 305 nt in length which differed by about 10 nt indicated 3’ ends at approximately nt 3250.

cDNA sequencing

The results of the MAV-1 El analysis presented in the preceeding sections suggested that the transcription map of MAV-1 El was unlike that of Ad2 and other primate adenoviruses. To further support the transcription mapping data, we isolated cDNA clones from a li- brary made from early infected-cell RNA. Three ElA and 10 El B cDNAs were examined; all of the ElA cDNAs and four of the ElB cDNAs were completely sequenced (Table 2). The three ElA clones appeared to represent the same mRNA. One El B clone corresponded to QW 1900-nt mRNA (Z117), eight repre- sented the 2100-nt mRNA, and one could have been either El B mRNA (295).

The cDNA sequencing confirmed the transcription and Northern data and identified the precise splice sites and the 3’ end for the three major El mRNAs (see Fig. 1 for the transcription map). The DNA sequences near the splice sites match the consensus splice sequences (Mount, 1982). All of the mRNAs spliced to the same exon which extended from nt 2885 to 3260. The cDNA sequence analyses identified a polyadenylation site at nt 3260 in one ElA and five ElB mRNAs; no clones extended beyond nt 3260 (Table 2). However, in the Sl nuclease protection assay (Fig. 8C), two fragments were protected, indicating two possible 3’ ends. Just 5’ of the polyadenylation site mapped at nt 3260 by the cDNA se-

530 BALL ET AL.

5’3 *AT

CO GClOO TA GC

.GC TA GC

GC rTA

** CG AT110 GC

* GC TA GC

*AT *TA

GC TA

rTA I GC 120

AT GC TA

tTA TA

*TA TA AT TA AT130 CG

*AT *TA

GC TA

*TA TA GC TA cc140 TA GC TA GC TA TA TA TA GC TA150 GC

*CG ‘TA

** TA GC

*TA GC

*TA AT AT160 Y5

62

I 1 2 3 4 5 6 7 8 9 IO 111213141516

Primer Primer Extension Sl Extension Sl ----

5 --- RNA

* Si Probe 90 Smal -I Primer

279 362 Hid BamHl

FIG. 5. Sl and primer extension analysis of 5’ends of El A mRNAs. RNA was isolated from L cells mock-infected or infected with MAV- 1 for 22 (early) or48 hr (late) and hybridized to the DNA probes shown at the bottom of the figure. The probes were 5’ end-labeled at the BarnHI site at nt 362; the Sl probe extended to the Smal site at nt 90 and the primer extended to the Hinfl site at nt 279. Hybridizations were done at 45” and 42” for Sl and primer extension analyses, respectively. Maxam-Gilbert sequence reactions are shown in lanes 1 to 4. The genomic sequence is given on the left and asterisks indicate prominent 5’ ends. Lanes 5 to 7 are primer extension products and lanes 8 to 10 are Sl nuclease protected fragments. Lanes 1 1 to 16 are the same as lanes 5 to 10 but exposed four times longer. The arrows on the right indicate major start sites near nt 109 and 154.

quence analysis, the genomic sequence was A or T for 10 of 11 nt, so it is possible that the smaller band in Fig. 8C was an artifact of the S 1 nuclease protection assay.

In the MAV-1 El transcription unit, there are three perfect polyadenylation signals, AATAAA (Proudfoot and Brownlee, 1976) at nt 3188, 3219, and 3247 (Fig. 3). Since all MAV-1 El mRNAs ended at nt 3260, apparently only the last signal at nt 3247 is used. Sim- ilarly, Ad7 has three polyadenylation signals in the El B mRNAs and one apparent polyadenylation site (Dij- kema et a/., 1982, 1981). There are no other perfect polyadenylation signals anywhere upstream of nt 3 188 in the MAV-1 genome (Ball eta/., Fig. 3). A GU- or U-rich region downstream of the polyadenylation site required for efficient 3’ end formation (McLauchlan et al., 1985) can be identified in MAV-1 El between nt 3265 and 3283.

Predicted ElA protein coding sequence

The proposed coding sequences for MAV-1 El proteins were identified by similarity to human adenovirus proteins (Ball et al., 1988). However, only the amino terminal portion of a MAV-1 ElA protein was similar enough to be identified. From the transcription map presented here, the entire ElA protein is predicted to be 200 aa long (Fig. 9). The second exon, encoded be-

.392

1 2 3 4 5

s---=3’ RNA

3’* Sl Probe

BamH I San 359 1175

FIG. 6. Sl nuclease analysis of the first splice site of the ElA mRNA. The probe was 3’end-labeled at the BamHl site at nt 359 and extended to the Sal1 site at nt 1 175. Hybridizations and Sl nuclease digestion were done at 50” and 37’, respectively. Early RNA from two different infections is shown (lanes 3 and 5). The marker lane is an Rsal digest of &Xl 74 RF DNA and nucleotide sizes are shown on the left. The size of the major protected fragment is shown on the right.

SE ADENOVIRUS El TRANSC :RI PTION 531

1450

* 1460*’

1234567

5 --+ RNA -.

t3glll 828

891011 12 1234567

--- 5

RNA

5 =: RNA *5 Sl Probe ^ m* *fcs Sl probe *5 Sl probe tfgn I

Aval Bg/ll Nhel 828 -*5 Primer 1544 828 1274 1579 1642

HilXll sstl

3’5 GC TA CO CG CG c ci* 1080 AT CG GC TA AT AT TA AT* AT** AT 1090 CG CG AT AT AT* GC AT CG

472

-210

-180

GC 1100 CG CG GC 5’3

-Primer

FIG. 7. Sl nuclease and primer extension analysis of 5’ ends and splices of the El B mRNAs. The marker lanes of chemical degradation sequencing reactions or @Xl 74 RF Rsal fragments are as in Figs. 5 and 6, respectively. (A) Sl nuclease analysis of the acceptor splice site at 1462 of the El B 1900nt mRNA. The probe was 5’ end-labeled at the Aval site at 1544 and extended to the Bglll site at nt 828; hybridizations and Sl nuclease analyses were done at 42” and 37”, respectively. The 5’end at nt 1089 was detected on this same gel but is not shown. (B) Sl nuclease analysis of the 5’end of the El B 2200 nt mRNA. The probe was 5’end-labeled at the Nhel site at 1274 and extended to the Bglll site; hybridizations were done at 47”. (C) Sl nuclease and primer extension analysis of the 5’ ends of the El B mRNAs. The probe was 5’end-labeled at the Sstl site at nt 1642 and extended to the Bglll site at nt 828 for the Sl probe and to the Hincll site at nt 1579 for the primer. Hybridizations were done at 50” and 46” for Sl nuclease protection and primer extension assays, respectively.

tween nt 829 and 951, would include the amino acid sequences similar to the second exon of Ad2 ElA proteins. Remarkably, the portion of the MAV-1 ElA protein which was encoded in the third exon is predicted to be only two amino acids long. The El B 55K protein would also terminate with these two amino acids because its mRNA has the same final exon.

DISCUSSION

The organization of primate adenovirus Els is remarkably conserved, although the DNA sequences are not very similar. The data presented in this report indicated that MAV-1 El transcripts extended from 0.3 to 10.3 m.u., approximately the extent of the Ad2 El re-

gion (1.3 to 11.2 m.u.). Other 01% in the sequenced region were examined, and two orfs, 6 and 7 (Fig. 2) showed homology to late proteins IX and IVa2, respectively. The 850-nt mRNA, which hybridized strongly to nt 2556 to 4333 at 48 hr p.i. (Table l), may encode protein IX. The locations and orientations of these predicted late proteins IX and IVa2 corresponded to those of Ad2.

However, the detailed transcription map of MAV-1 El was strikingly different when compared with those of primate adenoviruses. In particular, primate adenovirus ElA and El B transcription units are discrete, and the locations of splice sites relative to the protein coding sequences are conserved. MAV-1 ElA and ElB transcription units appeared to be unique among the

532 BALL ET AL.

1560 - 964 - 645 -

472525 392 -

247 -

197-

157- 138-

1 2 3 4

X-3 RNA

3’* Sl Probe

BarnHI Smal

247 -

- 305

SSfl 1642

1 2 3 4

RNA z+ 5’ St Probe

Sphl 3190

1846

1 2 3 4

3’* Sl Probe Bsffl Spel 2936 3955

FIG. 8. Sl nuclease analysis of the splice sites for the last intron and of the 3’ end for all El mRNAs. Marker and major protected fragments are as indicated in Fig. 6. (A) St nuclease analysis of the donor splice site at nt 2815 of the El B mRNAs. The probe was 3’ end-labeled at the BamHI site at nt 1846 and extended to the Smal site at 15.5 mu., about nt 4900; hybridizatjons and S1 nuclease digestions were done at 47” and 37”, respectively. (6) Sl nuclease analysis of the acceptor splice site at nt 2885 of the El A and El B mRNAs. The probe was 5’ end-labeled at the Sphl site at nt 3190 and extended to the .%I site at nt 1642; hybridizations were done at 47”. (C) St nuclease analysis of the 3’ end of ElA and El B mRNAs. The probe was 3’ end-labeled at the BstYI site at nt 2936 and extended to the Spel site at nt 3995; hybridizations were done at 40”.

adenoviruses in that they were 3’-coterminal: they had different major start sites but a common final exon and polyadenylation site. An intron near the 3’ end of the EIA and EIB mRNAs interrupted the predicted

MAV-1 El A and 55K El B coding sequences; both proteins would terminate (in the same reading frame) in the last exon with two amino acids and a stop codon. Similarly, there is an intron near the 3’ end of the Ad2


TABLE 2

SUMMARY OF El cDNAs

cDNA clone Exon 1 a Exon 2’ Exon 38

ElA Z1506 144-752 829-951 2885-3247 z112* 178-752 829-951 2885-3260 (AAAA)” Zllb 183-752 829-95 1 2885-3255

ElB 2117’ 2136’ Z127* Z143d 2134 2115 Z128* 2151 2130 295

1099-l 114 1462-2815 2885-3260 (AAAMAAA) 1100-2815 2885-3260 (AAAA) 1101-2815 2885-3257 1101-2815 2885-3257 1117-2815 2885-3260 (AAAAAA) 1293-2815 2885-3252 1411-2815 2885-3260 (AAAAAAAA) llOl-2373* 1393-2373 1493-2373

a The nucleotide numbers given correspond to the genomic sequence found in the cDNA sequence. * Indicates cDNA clones that were completely sequenced. c The number of A’s reflect the length of the poly(A) tail that was sequenced. d Other clones which were not completely sequenced were analyzed by restriction endonuclease digestion patterns and enough sequencing

to determine the 5’and 3’ ends and the splice sites. ’ Nucleotide 2373 is at an EcoRl site; some clones ended here, indicating incomplete EcoRl methyiase protection during the cDNA cloning

procedure.

ElB mRNAs; however, it does not interrupt the ElB coding sequences. Other distinctive features are (1) there is only one major El A mRNA, and therefore only one predicted ElA protein; (2) there are heterogeneous early and late start sites for the ElA mRNA; and (3) the smaller Ad2 El B mRNA encodes only the 21 K protein whereas the smaller MAV-1 El B mRNA encodes only the 55K protein.

In contrast to the primate adenoviruses, there was only one major MAV-1 ElA mRNA, and therefore only one predicted MAV-1 ElA protein. Some conserved amino acid sequence features are present, including similarities within the conserved regions 1, 2, and 3

(Ball et al., 1988; Kimelman et al., 1985). For example, the Ad5 ElA protein binds zinc in a region which includes a potential metal-binding domain consisting of Cys-X2-Cys-X,--Cys-X2-Cys (Berg, 1986; Culp et a/., 1988) and a similar sequence is present in the MAV-1 ElA predicted coding sequence beginning with Cys- 136. However, there are other sequences which are conserved in primate adenovirus El As which are not found in the predicted MAV-1 ElA protein coding sequence. These human adenovirus ElA protein features include (1) the Arg-2 shown to be important for the rapid El A turnover (Slavicek et a/., 1988) and coop- erativity with the T24 H-ras oncogene in transformation

MSRLLRLSLSSRVWLAAQEATRNVSEDPWCRTPWDGSPT

<----------------l-----------------> CTAVRVVRAEVLADGTMDLDIVFPEAAVQAVFSRTPWQDS

<---m---2 TTATSAEEPSASTDSISSDPLPISCVESFEDMDLRCYEQL

----------><----------3---------->

SPSPESIETIEVFPPCSTCGGHEVNGFCSLCYLRGLTDLL A

PQADDAGEAEVPDESAKDLCFMDLLTWAMEDKTECSRHDE h

40

80

120

160

200

FIG. 9. Predicted coding sequence for the ElA protein. The amino acids at splice sites are in bold and marked with a caret. Conserved regions 1,2, or 3 are indicated by the arrows above the sequence (see text).

534 BALL ET AL.

(Whyte et al., 1988b), although the predicted MAV-1 ElA protein does have an Arg at position 3; (2) the sequence Tyr-Ser-Pro-Val-Ser (aa 184 to 188) conserved at the splice junction in the 289-aa ElA protein, where the Ser-185 has been shown to be critical for the transactivation function (Glenn and Ricciardi, 1987); and (3) a basic pentapeptide sequence at the ElA carboxy terminus shown to be important for nuclear localization (Lyons et a/., 1987).

Because the single MAV-1 El A mRNA has an orf with sequences similar to conserved regions 1, 2, and 3 of the human adenovirus ElA proteins, the MAV-1 ElA protein would be equivalent to the Ad2 289-aa ElA protein. Since no alternative spliced mRNA was seen, no MAV-1 protein equivalent to the Ad2 243-aa protein is predicted. A number of functions for the Ad2 243-aa protein have been demonstrated (Monte11 et al., 1984; Quinlan et a/., 1988; Spindler et a/., 1985), including transcriptional repression (Lillie et a/., 1986; Velcich and Ziff, 1985) and transformation (Hurwitz and Chinnadurai, 1985; Monte11 et a/., 1984). It has been suggested that the repressor activity of the 243-aa protein may contribute to transformation by Ad2 (Lillie et a/., 1986; Schneider et a/., 1987; Velcich and Ziff, 1985). The single MAV-1 ElA protein may fulfill the functions of both the 243-and the 289-aa ElA proteins of Ad2; alternatively, MAV-1 may be deficient in the particular activities of the 243-aa protein. For example, no MAV-1 transformation activity has been detected (Dussaix et a/., 1982; K. Spindler, unpublished data).

In Ad2, the major cap site for the ElA mRNAs is at nt 499 (+l), with a TATA box at -31 and the initiator AUG at +61 (Baker and Ziff, 1981). There are heterogeneous upstream start sites, and additional upstream sites are used after the onset of DNA replication, but these represent only a minor fraction of the El A mRNA start sites (Osborne and Berk, 1983). In MAV-1, there was no comparable single major cap site, since heterogeneous start sites for the MAV-1 ElA mRNAs were observed at both early and late times. Adenovirus El transcription signals have been extensively analyzed (for review, see Jones et a/., 1988) and we examined the upstream region of the E 1 A gene in MAV- 1 for puta- tive transcriptional control signals. A TATA homology (Breathnach and Chambon, 1981) is located at nt 127, a possible Spl binding site (Dynan and Tjian, 1983) is located at nt 66, a CCAAT box homology (Efstratiadis eta/., 1980) is found at nt 55, and a possible activating transcription factor (ATF) binding site (Lee and Green, 1987; Lee et a/., 1987) is found at nt 104. The TATA homology identified in the MAV-1 El A apparently does not direct precise initiation of transcription, however. The initiation of mRNAs between nt 98 and 158 is con- sistent with the observation that plasmids lacking the

first 90 nt of MAV-1 are unable to transactivate the Ad2 E3 promoter in a transient expression assay (Ball et a/., 1988).

MAV-1 and Ad2 differ in their strategies for encoding the proteins from the El B mRNAs. InAd2, the 22 S El B mRNA can encode both the 21 K and the 55K proteins; internal initiation is required for translation of the 55K protein (Bos et al., 198 1). The Ad2 13 S El B mRNA can only encode the 2 1 K protein (Bos et al,, 198 1). In MAV- 1, as in Ad2, the larger El B mRNA could encode both El B proteins, and in both viruses, the immediate con- texts of the 21 K and 55K AUGs are similar (Kozak, 1986). However, in MAV-1 the 55K protein need not be translated by internal initiation from the larger bicis- tronic mRNA because the first AUG encountered in the smaller El B mRNA is the predicted initiator AUG for the 55K protein.

In order to study mouse adenovirus genes in viva, it was first necessary to identify transcription units analo- gous to the human adenovirus early regions involved in viral-cellular interactions. MAV-1 El mRNAs were mapped and orfs similar to Ad2 El proteins were identified. There are distinctive features of MAV-1 which can be exploited to study the functions of conserved ElA and El B sequences. For example, since MAV-1 apparently lacks a message that could encode a 243-aa-like protein, it may provide a system in which to examine functions of the 243-aa protein in in vivo mouse infections. In addition, human adenovirus El A proteins have been shown to associate with cellular polypeptides (Harlow et a/., 1986; Yee and Branton, 1985); one, the 105K protein, was recently identified as the retinoblastoma gene product (Whyte et a/., 1988a). Using MAV- 1, it should be possible to examine the importance of this and other associations for natural infections.

ACKNOWLEDGMENTS

We thank Drs. Kenn Buckley, Carol Condit, Ralph Dean, Lois Mil- ler, David O’Reilly, and Richard Schwartz for helpful comments on experimental methods and this manuscript. We thank Mary Stringer and Mark Williams for providing part of the MAV-1 cDNA sequence analysis. We are grateful to Keena Lowe for editorial comments. This was supported by grants from the American Cancer Society (MV 302), and the University of Georgia Research Foundation, and by Public Health Service Grant (AI-2376202)from the National Institutes of Health. K.R.S. is the recipient of an American Cancer Society Ju- nior Faculty Award.

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