MOLECULAR AND CELLULAR BIOLOGY, Sept. 1981, p. 807-8130270-7306/81/090807-07$02.00/0

Vol. 1, No. 9

Control of Adenovirus Early Gene Expression:Posttranscriptional Control Mediated by Both Viral and

Cellular Gene ProductsMICHAEL G. KATZE, HAKAN PERSSON, AND LENNART PHILIPSON*Department ofMicrobiology, The Biomedical Center, S- 751 23 Uppsala, Sweden

Received 13 April 1981/Accepted 9 June 1981

An adenovirus type 5 host range mutant (hr-i) located in region ElA andphenotypically defective in expressing viral messenger ribonucleic acid (RNA)from other early regions (Berk et al., Cell 17:935-944, 1979) was analyzed foraccumulation of viral RNA in the presence of protein synthesis inhibitors. NuclearRNA was transcribed from all early regions at the same rate, regardless ofwhether the drug was present or absent. As expected, low or undetectable levelsof RNA were found in the cytoplasm of hr-l-infected cells compared with thewild-type adenovirus type 5 in the absence of drug. When anisomycin was added30 min before hr-i infection, cytoplasmic RNA was abundant from early regionsE3 and E4 when assayed by filter hybridization. In accordance, early regions E3and E4 viral messenger RNA species were detected by the Si endonucleasemapping technique only in hr-l-infected cells that were treated with the drug.Similar results were obtained by in vitro translation studies. Together, theseresults suggest that this adenovirus type 5 mutant lacks a viral gene productnecessary for accumulation of viral messenger RNA, but not for transcription. Itis proposed that a cellular gene product serves as a negative regulator of viralmessenger RNA accumulation at the posttranscriptional level.

At early times after adenovirus infection, atleast five regions of the viral genome are tran-scribed into cytoplasmic ribonucleic acid (RNA)(2, 18). Studies with host range mutants havesuggested that a major element of genetic con-trol exists in early region EiA (1.5 to 4.5 mapunits) of the viral deoxyribonucleic acid (DNA).Jones and Shenk (11) isolated an adenovirustype 5 (Ad5) mutant, d1312, with a deletion from1.5 to 4.5 map units, which was defective bothfor growth in HeLa cells and for transformationof rat cells. Furthermore, d1312 was defective inthe accumulation of cytoplasmic early messen-ger RNA (mRNA) (12). However, since nuclearRNA was detected at a low level in d1312-in-fected HeLa cells, the defect in the mutant maybe at a posttranscriptional level such as process-ing, transport, or stability of the RNA.Another Ad5 mutant, hr-i, also unable to

replicate in HeLa cells and defective in transfor-mation, was isolated by Harrison et al. (10).Recently, the lesion in the hr-i mutant wasmapped between 1.3 and 3.7 units of the Ad5genome by marker rescue (8). Berk et al. (1)examined the accumulation of viral RNA in hr-1-infected HeLa cells by Si endonuclease map-ping. Both cytoplasmic and nuclear RNA fromearly regions ElB, E2, E3, and E4 were unde-

tectable in cells infected with the mutant. How-ever, early region ElA RNAs were present inthe nucleus and the cytoplasm. These investi-gators concluded that an early region ElA geneproduct, which is defective in the hr-1 mutant,is required for either the initiation of transcrip-tion or stabilization of early viral nuclear RNA.The regulation of viral mRNA expression has

been studied by using inhibitors of protein syn-thesis (13, 16). It was found that viral mRNAaccumulation was depressed when inhibitorswere added shortly before Ad2 infection of HeLacells. This observation, along with other evi-dence, led Persson et al. (16) to propose a modelin which a protein coded by early region E1regulates the accumulation of viral mRNA byinactivating a cellular component normally re-sponsible for turnover of viral mRNA. In a sep-arate report, we presented evidence that theprotein(s) encoded in early region El primarilycontrols the early region E4 of the viral genome(H. Persson, M. G. Katze, and L. Philipson, J.Virol., in press).The present study examined the control of

viralmRNA accumulation in hr-i-infected HeLacells by using inhibitors of protein synthesis.The results are consistent with the hypothesisoutlined by Persson et al. (16) and strongly

807

808 KATZE, PERSSON, AND PHILIPSON

suggest that the hr-i mutant lacks a mechanismto counteract the negative cellular control.

MATERIALS AND METHODSCells and virus. HeLa cells were grown in suspen-

sion at concentrations of 3 x 105 to 5 x 105 cells per mlin Eagle spinner medium containing 7% calf serum.Wild-type Ad5 was grown and purified from infectedHeLa cells as previously described (18). Ad5 mutanthr-1 (10) was propagated in the permissive Ad5-trans-formed 293 cells (9). The hr-i mutant was titrated onboth 293 and HeLa cells. The mutant virus had titerson HeLa cells 103 to 104 times lower than the titers on293 cells.

Infection and labeling conditions. HeLa cellswere infected with wild-type Ad5 and mutants at amultiplicity of 1 plaque-forming unit per cell. Cyto-plasmic RNA was prepared from HeLa cells labeledwith [3H]uridine (50,uCi/ml) 2 to 5 h postinfection bythe method of Brawerman et al. (4). Nuclear RNAwas prepared from cells pulse-labeled with [3H]uridine(500 uCi/ml) for 5 min at 5 h postinfection by theprocedure of Smith et al. (20).Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis and in vitro translation. The pro-cedures for translation in the rabbit reticulocyte cell-free system have been described previously (17). Virus-specific RNA was selected on Ad5 DNA or purifiedrestriction enzyme fragments (18) bound to nitrocel-lulose filters as described by McGrogan et al. (14).Eluted RNA was then translated in the mRNA-de-pendent reticulocyte system. Samples were analyzedon 13% sodium dodecyl sulfate-polyacrylamide slabgels as described previously (15, 17). The gels wereanalyzed by fluorography (3).

Si endonuclease mapping of mRNA. Themethod originally described by Berk and Sharp (2)and modified by Persson et al. (16) was followed forS1 endonuclease mapping of mRNA. Briefly, 20,ug ofcytoplasmic RNA from each sample was hybridized ina high concentration of formamide (5) to 1 ,ug of Ad5DNA. The samples were treated with S1 endonuclease(225 U) and separated on a 2.0% alkaline agarose gel.The gels were blotted onto nitrocellulose sheets (21)and hybridized to 32P-labeled nick-translated probes(19).

Filter hybridization. Equal amounts of nuclear orcytoplasmic RNA were hybridized for 16 h at 65°C tonitrocellulose filters containing SalI and EcoRI frag-ments of Ad5 derived from 10 ,ug of viral DNA. Afterhybridization the filters were washed extensively andtreated with ribonuclease A (20 ,ug/ml). The amountof labeled RNA hybridized was quantitated by liquidscintillation counting.

RESULTS

Accumulation of cytoplasmic viral RNAin ceils infected with Ad5 host range mu-tant hr-i. Host range mutants with either de-fined deletions or point mutations in early regionElA have been constructed (10, 11). At lowmultiplicities of infection these mutants do notaccumulate cytoplasmic RNA from viral early

region E2, E3, or E4 (1, 12) in HeLa cells per-missive for the wild-type virus. We examinedthe accumulation of early viral RNA in HeLacells infected with a host range mutant in thepresence of inhibitors of protein synthesis. Pre-vious studies with inhibitors added before orafter infection ofhuman cells with wild-type Ad2have shown that inhibitors effect the accumu-lation of early viral mRNA (13, 16).

In the present experiments HeLa cells wereinfected with the host range mutant hr-i withand without the protein synthesis inhibitor ani-somycin added at 30 min before infection. Toavoid late expression from the control wild-typevirus all samples were collected at 5 h afterinfection at a time when the E2 region is not yetfully expressed. We have therefore not includedthe E2 region in our analysis. The cells werelabeled with [3H]uridine from 2 to 5 h postinfec-tion, and cytoplasmic RNA was isolated. Thelabeled RNA was subsequently hybridized tonitrocellulose filters containing different frag-ments of Ad5 DNA. As expected, the host rangemutant hr-1 accumulated substantially less earlyviral RNA from region 3 and 4 than did the wild-type virus (Table 1). The addition of anisomycin30 min before infection, however, allowed theaccumulation of early viral RNA from infectedcells. The amount of mutant early viral RNAaccumulated under these conditions reached alevel comparable to that in the wild-type infec-tions.Rate of transcription in mutant-infected

ceils. Infected cells were pulse-labeled with[3H]uridine for 5 min at 5 h after infection. Thenuclear RNA was extracted and hybridized tofilter-bound Ad5 DNA fragments representingdifferent early viral transcription units. The re-sults for wild-type Ad5 and the hr-1 mutant

TABLE 1. Effect of anisomycin on the accumulationof cytoplasmic early viral mRNA in mutant-infected

cellsRNA hybridized (cpm)a

PrepnE3 E4

Wild-type Ad5 6,047 6,598hr-i 802 1,648hr-i with anisomycinb 7,152 9,090

a Equivalent amounts of early RNA were hybridizedto filters as described in the text. The probe for earlyregion E4 was the EcoRI-C fragment of Ad5 (84.0 to100.0 map units), and the probe for early region E3was the EcoRI-B fragment (75.9 to 84.0 map units). Abackground of approximately 50 cpm to filters withoutDNA has been subtracted from each value.

b Ad5 mutant-infected cells were maintained in thepresence of 100lM anisomycin (Pfizer Laboratories)from 30 min before infection until 5 h postinfection.

MOL. CELL. BIOL.

CONTROL OF ADENOVIRUS EARLY GENE EXPRESSION 809

TABLE 2. Rate of transcription in Ad5- and hr-i-infected HeLa cells

RNA hybridized (cpm)aPrepn

El E3 E4

Ad5 793 575 142hr-i 1,219 598 381hr-1 with anisomycinb 770 100 280

a Ad5- and hr-1-infected cells were pulse-labeled for5 min at 107 cells per ml with 500 ,uCi of [3H]uridineper ml at 5 h postinfection. Equivalent amounts ofnuclear RNA were hybridized to filters as described inthe text. The probes of Ad5 DNA used were: earlyregion El, SalIB (O to 26.0 map units); early regionE3, EcoRI-C (75.9 to 84.0 map units); early region E4,EcoRI-B (84.0 to 100.0 map units). A background ofapproximately 40 cpm to filters without DNA has beensubstracted from each value.

b hr-1 mutant-infected cells were maintained in thepresence of 100 ,uM anisomycin from 30 min beforeinfection until 5 h postinfection.

sis of region E4 RNA also showed that the drugtreatment allowed the accumulation of viralRNA from hr-i-infected cells. As with region E3,no hr-1 RNA was detected in the absence of thedrug (Fig. 1B). Furthermore the major RNAsfrom region E4 accumulated in drug-treated, hr-i-infected cells had the same sizes as the RNAspecies obtained from wild type-infected cells.In vitro translation with RNA prepared

from hr-l-infected cells. To test whether theRNA that accumulated in hr-l-infected cells inthe presence of anisomycin was functional, the

CM C cno C-)C-) ot CE -

*0O rIC +

E aOC)._ 0

c

virus are shown in Table 2. Nuclear RNA fromtranscription units ElA, EiB, E3, and E4 wasdetected in hr-l-infected cells. Furthermore, theamount of labeled nuclear RNA was comparableto the amount obtained with the wild-type virus.The rate of transcription of hr-1 nuclear RNAfrom early regions ElA, EiB, E3, and E4 wasnot increased when anisomycin was presentthroughout the time of labeling (Table 2). Theresults with the hr-1 mutant suggest that theincreased accumulation of viral cytoplasmicRNA in the presence ofanisomycin is not causedby an increased rate of transcription in the pres-ence of the drug.Si blot analysis ofcytoplasmic RNA from

mutant-infected cells. Cytoplasmic RNA wasisolated at 5 h postinfection from cells treatedwith anisomycin 30 min before or 2 h afterinfection with the hr-1 mutant. RNA was alsoisolated from cells not treated with anisomycinand from wild type-infected cells. The cytoplas-mic RNA was hybridized to Ad5 DNA, treatedwith Si endonuclease, separated on an alkalineagarose gel, and blotted onto nitrocellulosesheets. The filters were subsequently hybridizedto 32P-labeled DNA fragments of Ad5 DNA rep-resenting different regions. The results for earlyregion E3 are shown in Fig. 1A. No viral RNAfrom region E3 was detected in hr-l-infectedcells in the absence of anisomycin. However,when anisomycin was present, substantialamounts of early region E3 RNA were detected.Four major RNA species were detected, corre-sponding in size to the ones previously identifiedfrom this region after wild-type infection (1).The same four RNA species were detected inwild type-infected cells. Si endonuclease analy-

-23002000 _ 1750

o-o1750 '*- 1750

a141

h rl Awt hr 1B

wt

FIG. 1. Sl endonuclease analysis of cytoplasmicRNA prepared from cells infected with host rangemutants. Cytoplasmic RNA was prepared from wild-type Ad5- or hr-l-infected HeLa cells at 5 h postin-fection. The cells were maintained in the absence ofanisomycin (no drug) or in thepresence ofanisomycin(100 pM) from 30 min before infection (-30 min) orfrom 2 hpostinfection (+2 hrs). The RNA was hybrid-ized in a high concentration of formamide to Ad5DNA, treated with SI endonuclease, and separatedon a 2.0% alkaline agarose gel. The gel was blottedto a nitrocellulose sheet and hybridized to restrictionenzyme fragments of Ad5 DNA labeled with 32P bynick translation. The filter was then washed andanalyzed by autoradiography. (A) Early region E3Ad5 DNA probe: EcoRI-C (75.9 to 84.0 map units).(B) Early region E4 Ad5 DNA probe: EcoRI-B (84.0-100 map units). The virus used is indicated below thegels. wt, Wild type.

VOL. 1, 1981

810 KATZE, PERSSON, AND PHILIPSON

RNA was selected by hybridization to Ad5DNA. The selected RNA was then translated ina cell-free, protein-synthesizing system. RNAprepared from hr-i-infected cells showed littleor no synthesis of early viral proteins. In con-trast, RNA prepared from hr-i-infected cellsmaintained in the presence of anisomycin di-rected the synthesis in vitro of a complex patternof early viral proteins (Fig. 2). These proteinswere also detected when RNA from wild type-infected cells was used to program the cell-freesystem. However, several proteins, such as EiB65K and E3 16K, were synthesized in greateramounts by the RNA obtained from mutant-infected cells.The hr-1 virus synthesizes two EiA proteins

(54K and 42K) encoded by a 12S mRNA. How-ever, the ElA 13S mRNA of the hr-1 mutant istranslated into a 26K protein instead of the 58Kand 48K proteins obtained with wild-type EiA13S mutant (7). To analyze early regions EiAand EiB in more detail, cytoplasmic RNA wasselected by hybridization to restriction enzymefragment SalI-B covering these two early re-gions. The 54K and 42K proteins were bothsynthesized from RNA selected from hr-i-in-fected cells, as was the 26K protein (Fig. 3).Addition of anisomycin at 30 min before hr-iinfection resulted in the accumulation ofmRNA's for these proteins as judged by in vitrotranslation. Furthermore, the drug treatmentgreatly enhanced the accumulation of early re-gion EiB mRNA's encoding polypeptides EiB58K, EiB 18K, and EiB 15K. Early viral RNAfrom region E4 capable of synthesizing viralproteins was also induced when hr-i-infectedcells were treated with anisomycin. None ofthese proteins was synthesized in response toRNA selected from hr-i-infected cells nottreated with anisomycin. Altogether, these datastrongly suggest that infection with the hr-1mutant in the presence of anisomycin allows theaccumulation of functional viral mRNA's fromat least three early regions.

DISCUSSIONHost range mutants in early region EiA of the

Ad5 genome have been isolated previously (10,11). These mutants do not accumulate earlyviral mRNA from regions E2, E3, and E4, sug-gesting that viral gene products from early re-gion EiA control the expression of RNA fromother early viral regions (1, 12). The deletionmutants (such as d1312) lack the entire earlyregion EiA and therefore do not express anygene products from this region. On the otherhand, the host range mutants belonging to com-plementation group I express some gene prod-

_,.ab...-.X U

P am

-I*?

FIG. 2. In vitro translation with hybridization-se-lected RNA prepared from hr-l-infected cells. Cyto-plasmic RNA was prepared at 5 h postinfection fromHeLa cells infected with wild-type Ad5 or hr-i virus.Cells infected with hr-i were also treated with ani-somycin (100 pM) from 30 min before infectionthroughout the experiment (hr-i + drug). The RNAwas hybridized to filters containing Ad5 DNA, andthe hybridized RNA was translated in the mRNA-dependent reticulocyte cell-free system. The transla-tional products were analyzed on a 13% sodium do-decyl sulfate-polyacrylamide gel. The gel was ana-lyzed by fluorography. Abbreviations: -RNA, noRNA added to the cell-free system; ad2, [35S]methi-onine-labeled Ad2 marker virus.

ucts from early region EiA. The hr-i mutant,which belongs to complementation group I, ex-presses a functional EiA 12S mRNA encodinga 54K and a 42K protein. However, the EiA 13SmRNA is not translated into a 58K and a 48Kprotein as in the wild-type virus, but is insteadtranslated into a 26K protein (7). This suggeststhat the hr-i mutant contains a mutation intro-ducing a stop codon for protein synthesis in theEiA 13S mRNA. Additional lesions which havenot yet been detected in this mutant may exist.Berk et al. (1) suggested that the defect in thehr-i mutant is at the level of transcription sincethe mutant also fails to accumulate nuclearRNA

MOL. CELL. BIOL.

l--.--.

...a

"-1 .,

I

CONTROL OF ADENOVIRUS EARLY GENE EXPRESSION

v

CU

*0*

m-]Ha-

-El b/65K yf,58K1 Y-

: 54K >Ela\ 48KJ

Ela/26K E-

- Elb/18K

-- Elb/15K z -

-E./ ' ,

__: E4 / 164 Ka--a i_a

FIG. 3. In vitro translation with RNA selected by hybridization to different early regions of the Ad5genome. Cytoplasmic RNA wasprepared from HeLa cells infected with wild-type AdS or hr-i virus. The hr-l-infected cells were also treated with anisomycin (100 LM) from 30 min before infection to the time of harvestat 5 h postinfection (hr-i + drug). The RNA was hybridized to filters containing fragment SalI-B ofAdS DNA(O to 26.0 map units, leftpanel) or to filters containing fragment EcoRI-B ofAd5DNA (84.0 to 100 map units,right panel). The hybridized RNA was translated in the reticulocyte cell-free system, and the translationalproducts were analyzed on a 13% sodium dodecyl sulfate-polyacrylamide gel. The gel was analyzed byfluorography. The SalI-B DNA probe represents early regions ElA and EIB. The EcoRI-B probe representsearly region E4. Abbreviations: -RNA, no RNA added to the cell-free system; ad2, [36SJmethionine-labeledAd2 marker virus.

from other early regions. On the other hand,small amounts of nuclear RNA were found toaccumulate in d1312-infected cells (12). Thepresent results suggest that the defect is at aposttranscriptional level.When added after viral infection, inhibitors of

protein synthesis enhance the accumulation ofearly viral mRNA (6). Furthermore, this en-hancement may be caused by an increasedmRNA stability since the half-life of early viralmRNA is prolonged in the presence of inhibitorsof protein synthesis (22). Experiments in whichanisomycin was added before Ad2 infection ofHeLa cells showed a reduced accumulation offunctional mRNA from early regions E2, E3, andE4 (13, 16). These results were interpreted tomean that a viral protein from early region ElAcontrols the accumulation of functional viralmRNA's from other early regions. In accord-ance, the rate of transcription from the earlytranscription units was only weakly affectedwhen anisomycin was added before infection,whereas mRNA accumulation was strongly de-pressed (16). On the basis of these results wesuggested that the ElA proteins are involved ata posttranscriptional level, and the results ofWilson et al. (22) led us to propose that the ElAproteins control mRNA accumulation at the

level of RNA stability. A model was introducedin which a viral protein encoded in early regionElA inactivates a cellular protein that degradesviral mRNA. The ElA viral protein involved inthe stabilization of viral mRNA would then beabsent or not functional in the host range mu-tants, thereby allowing the cellular product todegrade viral mRNA. The addition of anisomy-cin before infection of HeLa cells with the mu-tants would prevent the synthesis of the cellulardestabilizing protein. In this way, the accumu-lation of mutant viral mRNA should be facili-tated. The present study was designed to testthis hypothesis.

If our hypothesis is correct, the defect in thehost range mutants should act at a posttran-scriptional level. We first determined the rate oftranscription by labeling infected cells for 5 minwith [3H]uridine and measuring the amount oflabel incorporated into viral nuclear RNA byfilter hybridization to viral DNA fragments rep-resenting different early viral regions. The re-sults (Table 2) show that nuclear RNA is tran-scribed in HeLa cells infected with the hr-imutant from early regions El, E3, and E4 at thesame rate as in cells infected with the wild-typevirus. The rate oftranscription was not increasedin hr-l-infected cells treated with anisomycin

VOL. 1, 1981 811

ON OM _F

< , .TCN z -L D'O M

-c- -o.l

II 40 RIMPOTMIS

812 KATZE, PERSSON, AND PHILIPSON

shortly before infection. Since viral nuclear RNAwas detected in cells infected with host rangemutants at a level comparable to that in wildtype-infected cells, we conclude that the defectin the mutant is probably at a posttranscrip-tional level. The discrepancy between our resultsand those of Berk et al. (1) may be a reflectionof the fact that we analyzed the rate of transcrip-tion, whereas Berk et al. examined the accumu-lation of nuclear RNAs by Si analysis.The accumulation of mutant viral RNA was

determined by filter hybridization, Si endonu-clease analysis, and in vitro translation. Theaddition of anisomycin shortly before infectionof HeLa cells with hr-1 allowed the accumula-tion of viral RNA from both early regions E3and E4 at a level comparable to that of the wild-type virus (Table 1). Very little RNA from thesetwo early regions accumulated in cells infectedwith the hr-1 mutant in the absence of the drug.The viral RNA accumulated in hr-i-infected

cells maintained in the presence of anisomycinhad the same structure as RNA obtained fromwild type-infected cells, as revealed by Si map-ping analysis (Fig. 1). Early viral RNA accu-mulated from regions E3 and E4 in hr-1-infectedcells treated with anisomycin 30 min before or 2h after infection (Fig. 1). The RNA from cellstreated from 30 min before infection appeared tobe more abundant than RNA prepared fromcells treated with anisomycin from 2 h afterinfection. This result may reflect a level of thecellular destabilizing protein in cells treated withanisomycin from 30 min before infection that islower than the level in cells treated from 2 hpostinfection.The RNA accumulated in hr-l-infected cells

maintained in the presence of anisomycin wasfunctional when assayed by in vitro translation(Fig. 2). Hybridization selection of RNA pre-pared from hr-i-infected cells maintained in theabsence or presence of anisomycin to DNA frag-ment SalI-B, representing early regions EiAand EIB, followed by in vitro translation provedthe mutant character of the hr-i virus. The EiA58K and 48K proteins were not synthesized byeither of these two hr-1 RNA preparations, butinstead we detected a 26K protein. This result isin agreement with the results ofEsche et al. (16),and it demonstrated that the virus used in thisstudy had the expected lesion in the EiA 13SmRNA.However, viral proteins from early regions

EiB, E2, E3, and E4 were readily detected fromhr-1 RNA preparations maintained in the pres-ence of anisomycin. The amount of hr-1 viralmRNA accumulated under these conditionsreached a level comparable to that of the wild-type virus.

The results presented in this report suggestthat the defect in the hr-1 host range mutant isat a posttranscriptional level. However, we can-not exclude the possibility that other EiA prod-ucts may enhance transcription since the EiAproteins, of course, are not expressed in theabsence of protein synthesis. We propose thatthe protein synthesis inhibitors prevent the syn-thesis of a cellular protein which, when present,reduces the accumulation of viral mRNA. Theresults of Wilson et al. (22) suggest that mRNAstability could be a possible level for this regu-lation. However, further experiments involvingcell-free systems are necessary to characterizethe proposed cellular protein.

ACKNOWLEDGMENTSWe thank Britt-Marie Johansson and Eva Hjertson for

excellent technical assistance and Christina Sjoholm andMarianne Gustafson for expert secretarial help.

This work was supported by grants from the SwedishSociety against Cancer. M.G.K. was supported by a long-ternEuropean Molecular Biology Organization fellowship.

LITERATURE CITED1. Berk, A. J., F. Lee, T. Harrison, J. Wiliams, and P.

A. Sharp. 1979. Pre-early adenovirus 5 gene productregulates synthesis of early viral messenger RNAs. Cell17:935-944.

2. Berk, A. J., and P. A. Sharp. 1977. Structure of theadenovirus 2 early mRNAs. Cell 14:695-711.

3. Bonner, W. M., and R. A. Laskey. 1974. A film detectionmethod for tritium-labeled proteins and nucleic acids inpolyacrylamide gels. Eur. J. Biochem. 46:83-88.

4. Brawerman, G., J. Mendecki, and S. Y. Lee. 1972. Aprocedure for the isolation of mammalian messengerribonucleic acid. Biochemistry 11:637-641.

5. Casey, J., and N. Davidson. 1977. Rates of formationand thermal stabilities of RNA-DNA and DNA-DNAduplexes at high concentrations of formamide. NucleicAcids Res. 4:1539-1552.

6. Eggerding, F., and H. J. Raskas. 1978. Effect of proteinsynthesis inhibitors on viral mRNA's synthesized earlyin adenovirus type 2 infection. J. Virol. 25:453-458.

7. Esche, H., M. B. Mathews, and J. B. Lewis. 1980.Proteins and messenger RNAs of the transforming re-gion of wild-type and mutant adenoviruses. J. Mol. Biol.142:399-417.

8. Galos, R. S., J. Williams, T. Shenk, and N. Jones.1980. Physical location of host-range mutations of ade-novirus type 5: deletion and marker-rescue mapping.Virology 104:510-513.

9. Graham, F. L., J. Smiley, W. C. Russel, and R. Nairu.1977. Characteristics of a human cell line transformedby DNA from human adenovirus type 5. J. Gen. Virol.36:59-72.

10. Harrison, T., F. Graham, and J. Williams. 1977. Hostrange mutants of adenovirus type 5 defective for growthin HeLa cells. Virology 77:319-329.

11. Jones, N., and T. Shenk. 1979. Isolation of adenovirustype 5 host range deletion mutants defective for trans-formation of rat embryo cells. Cell 17:683-689.

12. Jones, N., and T. Shenk. 1979. An adenovirus type 5early gene function regulates expression of other earlyviral genes. Proc. Natl. Acad. Sci. U.S.A. 76:3665-3669.

13. Lewis, J. B., and M. B. Mathews. 1980. Control ofadenovirus early gene expression: a class of immediateearly products. Cell 21:303-313.

14. McGrogan, M., D. J. Spector, C. J. Goldenberg, D.

MOL. CELL. BIOL.

CONTROL OF ADENOVIRUS EARLY GENE EXPRESSION 813

Halbert, and H. J. Raskas. 1979. Purification of spe-cific adenovirus 2 RNAs by preparative hybridizationand selective thermal elution. Nucleic Acids Res. 6:593-607.

15. Persson, H., M. Jansson, and L. Philipson. 1980. Syn-thesis of and genomic site for an adenovirus type 2 earlyglycoprotein. J. Mol. Biol. 136:375-394.

16. Persson, H., H. J. Monstein, G. Akusjirvi, and LPhilipson. 1981. Adenovirus early gene products maycontrol viral mRNA accumulation and translation invivo. Cell 23:485-496.

17. Persson, H., U. Pettersson, and M. B. Mathews. 1978.Synthesis of a structural adenovirus polypeptide in theabsence of viral DNA replication. Virology 90:67-79.

18. Pettersson, U., C. Tibbetts, and L Philipson. 1976.Hybridization maps of early and late mRNA sequenceson the adenovirus type 2 genome. J. Mol. Biol. 101:

479-502.19. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P.

Berg. 1977. Labeling deoxyribonucleic acid to highspecific activity in vitro by nick translation with DNApolymerase I. J. Mol. Biol. 113:237-251.

20. Smith, M. M., A. E. Reeve, and R. C. C. Huang. 1978.Analysis of RNA initiated in isolated mouse myelomanuclei using purine nucleoside 5' (y-S) triphosphates as

affinity probes. Cell 15:615-626.21. Southern, E. M. 1975. Defection of specific sequences

among DNA fragments separated by gel electrophore-sis. J. Mol. Biol. 98:503-517.

22. Wilson, M. C., J. R. Nevins, J. M. Blanchard, H. S.Ginsberg, and J. E. Darnell, Jr. 1979. The metabo-lism of mRNA from the transforming region of adeno-virus type 2. Cold Spring Harbor Symp. Quant. Biol.44:447-455.

VOL. 1, 1981