0022-538X/85/030822-05$02.00/0
CopyrightC 1985, American Society forMicrobiology
In Vitro Transcription of a Cloned Vaccinia
Virus Gene by a Soluble
Extract Prepared from Vaccinia
Virus-Infected HeLa Cells
P. DAVID FOGLESONGt
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461
Received 20 August 1984/Accepted 16 November1984
Faithful transcription of avaccinia virus gene was accomplished in vitro by using a soluble extract prepared
from vaccinia virus-infectedHeLa cells. Specific transcription of the cloned vaccinia virus gene was detected by
using template DNArestrictedwithin the transcribed region. The vacciniavirus gene was not transcribed by
extracts prepared from uninfected HeLa cells even with supplementation by purified vaccinia virus RNA
polymerase, nor was a clone of adenovirus 2 DNA bearing the
major
latepromotertranscribed by the extractfrom vaccinia virus-infected HeLa cells. Thus, infection by vaccinia virus altered cellular transcriptional
specificity to favor expression of vaccinia virus genes. RNA synthesis by theinfected cell extract was resistant
toa-amanitinbut strongly inhibited by I,,y-imidoATP and novobiocin.
Vaccinia virus has been a very useful model systemfor analysis of eucaryotic transcription. Vaccinia virion cores
transcribe early viralgenesin vitro (13) and thus containall
ofthe enzymesrequired for early transcription.A number of
enzymes have been isolated from viral core particles and
have been characterized. Of these, the DNA-dependent
RNApolymerase (2, 32),guanylyltransferase and
7-methyl-transferase (18, 21), 2'-O-methyltransferase(1), and
polyad-enylate polymerase (6, 22)areclearlyrequired for transcrip-tion. Several other virion enzymes which may function in transcription have been described, including 5'-phosphate
polyribonucleotide kinase (31), protein kinase (14), two
distinct nucleic acid-dependent ribonucleoside triphosphate phosphohydrolases (25), asingle-strand-specific nuclease(s) (26, 28),endoribonuclease (24), twoabundantDNA-binding proteins (3, 11), and a type I DNA topoisomerase (3).
Purifiedpreparations of vaccinia virusRNApolymeraseare
similar to cellular RNA polymerase II isolates in their requirement forMn2+ and single-strandedDNAin vitro and theirinability to transcribe duplex DNA templates. Thus, otherproteins appear to berequired forproperinitiation of transcription from nativeDNAtemplates (19).Furthermore,
RNAsynthesis from intact vaccinia viruscore particlesbut notbypurified vaccinia virus RNA polymerase is sensitive
to
1,-y-imido
ATP(AMP-PNP) andalsotonovobiocin(7, 8,30). Thus, vaccinia virus is an ideal model system for
analysis of the factors required for initiation oftranscription. Recently, the development of soluble cellular extracts
capable
of faithful initiation oftranscription
in vitro has provided a powerful method for analysis oftranscriptional initiation events (16, 35). Defined duplex DNA templateshave beentranscribed invitrotoyieldtruncated transcripts characteristic of initiation from a given promoter. Cloning
and RNA mapping ofthe vacciniavirus genome (10) have
made possible analysis of transcription ofdefined vaccinia
virus genes invitro.Inthispaper Idescribethedevelopment
and characterization of a soluble extract from vaccinia virus-infectedHeLacells which faithfully initiates
transcrip-tion of a cloned vaccinia virusgene invitro.
tPresent address: Division ofVirology and MolecularBiology, St.JudeChildren's ResearchHospital, Memphis,TN 38101.
MATERIALS AND METHODS
Materials. Unlabeled nucleotides and novobiocin were
obtained fromSigma Chemical Co.AMP-PNP waspurchased from Boehringer Mannheim Biochemicals. [c-32P]GTPwas
purchased from Amersham Corp.
Preparation of nucleic acids. Plasmid pSmaI-F bearing
adenovirustype 2(Ad2)SmaIfragmentF(11.6to 18.2 map
units) was provided by H. Furneaux. Plasmid pAG4 (34) bearinga960-basepair (bp) insertion of vaccinia virusDNA wasprovided byB. Moss. Supercoiled plasmid DNAswere
isolated as described previously (20). DNA products of restriction endonucleasedigestswereanalyzed on4% poly-acrylamide gels under conditions describedpreviously (3).
Virus.Vaccinia virus strainWR waspurified from infected
HeLa cells through two sucrose gradients, as described previously (9).
Preparation of extractsfrom vaccinia virus-infected HeLa
cells. HeLacells at a
density
of 5 x105
cells per ml wereinfected with 280 PFUof vaccinia virus percell.At 1and5 h afterinfectionextracts were preparedas described
previ-ously
(16).Extracts werestoredat-70°C
in200-gilportions.
Preparation of vaccinia virus DNA-dependent RNA
poly-merase. Vaccinia virus RNApolymerase was purified from
virions through phosphocellulose and was assayed as
de-scribedpreviously (32).
Protein determination. Protein concentrationswere
deter-minedby
using
the methodof Bradford (5).Assay for in vitro transcription. The standard reaction
mixture (50
,ul)
contained 20 mM HEPES(N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.9),
5 mMMgCl2, 2 mMdithiothreitol,1 mMATP, 1 mM
CTP,
1 mMUTP, 0.2 mM
[a-32P]GTP
(20,000 to40,000cpm/pmol),
1.0gig
ofDNA, and 20 gil ofextract. Theassay mixtureswereincubated at30°Cfor 60min,andreactionswereterminated
byadding 1 gil of1 mMGTP and 150 gil of4 M urea-1.0% sodium dodecyl sulfate-20 mM EDTA. The RNAwas
ex-tracted with 200
gil
ofphenol-chloroform
(1:1), and theorganic phasewas extracted withasolution
containing
7Murea, 0.35 M NaCl, 10 mM
Tris-hydrochloride
(pH 8.0), 2mMEDTA,and0.5% sodiumdodecylsulfate. The aqueous
phases werepooled, extractedagain, and
precipitated
withethanol
by using
50gig
of EscherichiacolitRNAas acarrier.The pellets were suspended in 300 gil of 1 M ammonium
822
on November 10, 2019 by guest
http://jvi.asm.org/
E
0 I:
--Q) -0
>
450
FIG. 1. Restriction sites and truncated tran insertion region. A 960-bp SalI fragment of terminal repeatwasligated into the SalI site or The promoter of the 7,500-dalton gene is in( triangle.TranscriptionofpAG4 restrictedwith yielded transcripts of 450, 730, and 1,010 nucle
acetate andprecipitatedwith ethanol. The
suspended in300,ul of0.3 Msodiumacetal
with ethanol. Thefinal pellets were suspe
solutioncontaining50 mMboric
acid,
S rn10 mM sodium sulfate, and 0.2 mM ED'
mented
with
2,ulof0.2 MmethylmercuryIofasolution
containing
50%sucrose, 0.0: and0.02% bromphenol blue. Sampleswei on 4%polyacrylamide
gels in BBE cmethylmercury hydroxideat80Vfor3h.i
thedriedgelswere prepared byusingKo
RESULTS
Restricted DNAtemplatesfor in vitro trai
studiesof specific transcriptional events
by using DNA-dependent cell extracts ai
restricted within the coding sequence
transcripts of defined length are synthesi;
used in this study, plasmid pAG4, conta
fragment
isolated from the vaccinia virusrepeatand cloned into theunique SalI sil
Theinsertion contains thepromoterand7'
sequenceofageneencodingan earlypro
7,500, as well as about 230 bp of sequ
proximal to the site oftranscriptional inil
indicating
the relevant restriction sites in pAG4 insertion isshowninFig.
1. Restric SalI releases theinsertion, which,
if tiresult in a truncated 730-nucleotide transc
readily detected by denaturing polyacr trophoresis.
Therefore,
the Sall digest ctinelyused as atemplate for in vitrotrans
deduced from the BamHI-DdeI double dif
not
shown)
that the orientation of the vainsertion within pAG4 was such that the
scription from the 7,500-dalton gene pr(
from the unique BamHI site ofthe vect(
Bgll site, asshowninFig. 1. Therefore,r(
with DdeI should have yielded a shortc
about 450
nucleotides,
andsimilarly,
resshouldhaveproduceda
transcript
of aboul(Fig. 1). RNA products of these predicto
served when these DNAs were transcril
vacciniavirus-infectedHeLacells as desc notshown). These RNAs were notobser
in vitro transcription assays programme
sponding
restricted pBR322 DNAs. The, the independently reported conclusion (2 thesis initiates at the same site on this ivirus DNA in vivo and in vitro in exi
virus-infected HeLa cells. The SmaI digest of a
plasmid
bearing Ad2 SmaI fragment F containing the major late
o v promoter wasused as thetemplate for invitrotranscription
cT) byuninfected HeLa extracts toyield a560-nucleotide
tran-script (35).
Comparisonofuninfected and vacciniavirus-infected HeLa cellextractsfortranscriptionof adenovirusandvaccinia virus
- 730 genes. Extracts were prepared from uninfected HeLa cells
- 1010 and alsofromHeLa cells infected with 280 PFU of vaccinia
virus per cell at 1 and 5 hafterinfection, as described above.
scripts
of the pAG4 The protein concentrations of these extracts weredeter-f
pBR322cDNA (v34)s
minedto be about 8mg/ml.
Thetranscription of adenovirusdicated by the open SmaI fragment F and
Sall-digested
pAG4 was determinedDdeI, Sall, and BglI for all three extracts as described above (Fig. 2). Assay
otides, respectively, mixtures lacking an added DNA template (Fig. 2, lanes A)
showed no RNAsynthesis, demonstrating the DNA
depen-,n the pellets were dence oftranscription by these extracts. Other assay
mix-teandprecipitated tures (lanes B) were programmed with Ad2 SmaI-F DNA
nded in 15 ,ulofa and showed the expected RNA product of 560 nucleotides
iMsodium borate, for theuninfectedextract. This band wasgreatlyreduced in
TA (BBE) supple- the assay mixture containing the 1-h-infected cell extract
hydroxide and 4,ul (VH1 extract) and completely absent in the assay mixture
2% xylene cyanol, containing the 5-h-infected cell extract (VH5 extract),
indi-reelectrophoresed cating that vaccinia virus infection eliminated RNA
polymer-ontaining 10 mM ase II transcription of SmaI fragment F. In other assays
Autoradiograms of (lanesC) the extracts were programmed with the Salldigest
dak XAR-5 film. of pAG4. The 730-nucleotide RNA product was observed
clearly only with the VH5 extract,indicating that the
vacci-niavirus gene can be transcribed most efficiently by extracts
nscription. In vitro from infected cells prepared several hours after infection.
can be performed These results are consistent with those reported by other
nd template DNA workers(27). Thehigher-molecular-weightbands(especially
so that truncated the 960-nucleotide band seen clearly in HeLa cell lane C)
zed. The template evidentin all lanesprogrammed withexogenous DNA were
iins a 960-bp SalI determinedto besensitiveto DNase(data not shown). Other
inverted terminal investigators havealsoconsideredsuch bandsto be artifacts
teof pBR322 (34). of endlabelingby crude lysates (17). The 560- and
730-nu-30bp ofthecoding cleotide RNAs were resistantto DNasetreatment.
tein withan Mrof Optimal reaction conditions for vaccinia virus transcription
ences that are 5' in vitro. Optimal synthesis ofthe 730-nucleotide RNA
re-tiation. A diagram quired the addition of20 ,u1 ofextract (8mg ofprotein per
the region ofthe ml) and 1.5 p.g of DNA per 50-,ul assay (data not shown).
tionof pAG4with
ranscribed, would
criptthatcould be __________ H1 VH5
ylamide gel elec- A B C A B C A B C M
f
pAG4
was rou- Uscription
studies. I_353
gest
of pAG4(data 960- 1078iccinia virus DNA 730- 1 872
direction of tran-
X3
60'
omoter was away
560-or and toward the
estriction ofpAG4
ened transcript of X -
31n
;triction with BglI
t1,010nucleotides
led sizes were
ob-bed in extracts of
cribedabove
(data
vedasproducts of
,d with the
corre-se results support
'7)
that RNAsyn-region
of vaccinia tracts of vacciniaFIG. 2. Invitrotranscription by uninfected andvaccinia virus-infectedHeLacellextracts.Products oftranscription by uninfected
(HeeLa), VH1, and VH5 cell extracts were programmed with no
DNA(lanes A),SmaI-digestedpSmal-F(lanes B),andSall-digested pAG4 (lanesC). Productsthatwere 560, 730,and 960nucleotides longareindicated.Lane McontainedHaeIII-digested4X174DNA thatwas5'-labeled with
[y-32P]ATP
andpolynucleotide kinase (usedassize markers).
I
on November 10, 2019 by guest
http://jvi.asm.org/
[image:2.612.63.299.75.163.2] [image:2.612.324.560.500.648.2]Higher concentrations of extract or of DNA resulted in synthesis of spurious products. Similar sharp optima for
extractand DNAconcentrations arecharacteristicof related
in vitrotranscription systems (16). Synthesis of the
730-nu-cleotide RNA required relatively high concentrations (1
mM) of ribonucleoside triphosphates, perhaps because of
the presence of vaccinia virus phosphohydrolases in the
infected cell extracts. Specific RNA synthesis occurred at
Mg2+concentrationsof5 to 10 mM, conditions similar to the
conditions required for transcription ofDNA within intact
vacciniaviruscores. However, addition of 5 to 10 mMMn2+
to assay mixturescontaining 5 mMMg2+ strongly inhibited
synthesisof the730-nucleotide transcript (data not shown),
whereas
Mn2+
was required for activity of the RNApolymer-asepurified fromvaccinia virions. Thus, the soluble infected
cell extract retained an important characteristic of viral
RNA synthesis in vivo which was lost upon purification of thevaccinia virion RNApolymerase.
a-Amanitinresistance of in vitro transcription by vaccinia
virus-infected HeLa cell extracts.a-Amanitin, apotent
inhib-itor ofRNA polymerase II, has been shown tocompletely
inhibit in vitro transcription ofAd2 SmaI fragment F by uninfectedHeLacell extracts (35). The a-amanitin
sensitiv-ity of transcription by the VH5 extract was examined.
Transcription ofAd2 SmaI-F and transcription of
Sall-di-gested pAG4 were determined as described above for the
HeLacell and VH5 extracts, respectively, in the presence
andabsenceof1,ugofao-amanitin per ml (Fig. 3). Synthesis
of the 560-nucleotide RNA was completely abolished by
ot-amanitin (Fig. 3, HeLa cell extract, lane C). However,
synthesis of the 730-nucleotidetranscriptbytheVH5 extract
wascompletely resistant to 1 ,ug ofa-amanitinper ml (Fig. 3,
VH5 extract, lane C), indicating that RNA polymerase HI
doesnotcatalyzethesynthesis of thistranscript. This RNA
product is synthesized by thea-amanitin-resistant vaccinia
virus RNA polymerase since its synthesis by infected cell
extracts is inhibited by antibody directed against purified
vaccinia virus RNApolymerase (27).
Supplementation of uninfected HeLa cell extracts with
vaccinia virus RNA polymerase. Both vaccinia virus RNA
HeLa
VH5
A
B
C
A
B
C
-0
Om~~~~~~~~~~~~~~~~~~~~~
-W;4:....
730-
560-FIG. 3. a-Amanitinsensitivity of transcriptionby uninfected and vaccinia virus-infectedHeLaextracts.Uninfected(HeLa)and VH5 cellextractswereassayedfortranscriptionofpSmal-Fand
Sall-di-gested pAG4, respectively, in thepresenceandabsence of 1 ,ug of ox-amanitinperml.Lanes A,noDNA; lanesB,DNA; lanesC,DNA plus1 ,uofa-amanitinperml.
~730-
560-A
B
CD
EF
G
HJ
w
K
FIG. 4. Supplementation ofextracts with vaccinia virus RNA
polymerase. Uninfected and vaccinia virus-infected HeLa cell ex-tractswereassayedfor transcription of Sall-digested pAG4 with or withoutsupplementation with purified vaccinia virus RNA polymer-ase.Uninfectedextractassaymixtures containedno DNA(lane A), SmaI-F DNA (lane B), and Sall-digested pAG4 (lanes C,1D,andE). VH5extract assay mixtures contained no DNA (lane F) or Sall-di-gestedpAG4 (lanes G, H, and I).LanesDand Hcontained assay mixturessupplemented with 0.02 U ofvaccinia virus RNA polymer-ase, and lanesEandIcontainedassay mixturessupplemented with 0.1UofRNApolymerase.LanesJ andKcontained assay mixtures ofSall-digested pAG4 transcription by 0.02 and 0.1 U of vaccinia virus RNApolymerase, respectively.
polymerase and RNA polymerase II require additional
fac-tors toutilize duplexDNA as atemplate.Therefore, it was
of interest to determine whether those factors present in
extracts ofuninfected cells could substitute for the factors
present ininfectedcells or whetherthere werevirus-specific
proteins other than the RNA polymerase which were
re-quired forinvitro transcription. Assays were performed as
described above, using both HeLa cell and VH5 extracts
with Sall-digested pAG4 as the template with or without supplementationwith 0.02 or 0.1 Uof
purified
vacciniavirus RNA polymerase (Fig. 4). Supplementation with vacciniavirus RNApolymerase didnotresultin detectable
synthesis
of the 730-nucleotide transcript by the HeLa cell extract
(Fig. 4, lanes D and E), nor did it significantly alter the
transcription of infected cellextracts(lanes H andI). RNA
polymerase alone did not synthesize any detectable 730-nucleotide RNA (lanes J and K). Therefore, in vitro
tran-scription of pAG4requires
virus-specific
factors other thanvacciniavirus RNA polymerase.
Sensitivity oftranscriptionby HeLa cellandVH5extracts to
AMP-PNP and novobiocin. Transcription by vaccinia virus
core
particles
requires hydrolysis
ofATPtoADPandPi
andthereforeisinhibitedbyATPanalogs, suchasAMP-PNP, in
whichhydrolysis of the
P,y
phosphatebond isrestricted (8,30). This analog inhibits the vaccinia virus ribonucleoside
triphosphate
phosphohydrolases
but notthe vacciniavirus RNA polymerase (30). Novobiocin, an inhibitor of thevaccinia virus DNAtopoisomerase, also inhibits
transcrip-tionbyintact vaccinia viruscores (7).
Therefore,
thesensi-tivity of in vitro transcription to these compounds was
determined. Assays ofthe VH5 extract with
Sail-digested
pAG4 were performed as described above in the presence
and absenceof 1 mMAMP-PNP or novobiocin. The assay
with the ATPanalogwasperformed intheabsenceofATP.
The results are shown in Fig. 5. Transcription ofthe
730-nucleotide RNA bythe VH5 extract was
strongly
inhibitedon November 10, 2019 by guest
http://jvi.asm.org/
[image:3.612.319.551.72.236.2] [image:3.612.106.260.477.661.2]A B
C
D
730-FIG. 5. Transcription ofSall-digested pAG4 by vaccinia virus-infected HeLaextracts in thepresence and absence of AMP-PNP
and novobiocin. Lane A containedanassay mixture programmed
withnoDNA, and lane B contained the 730-nucleotide transcript
synthesizedfromSall-digested pAG4. LaneC shows the results of
an assayperformedinthe absence ofATPandin thepresenceof 1 mMAMP-PNP. LaneDshowsthe results ofan assayperformedin
thepresenceof1mMnovobiocin.
by 1 mMAMP-PNP (Fig. 5, lane C). Thisresult is consistent
witharequirement for vaccinia virus phosphohydrolaseIor
IIactivity for in vitrotranscriptionin thissystetn. Complete
inhibition of transcription by 1 mM novobiocin was
ob-served(lane D), indicatingapossible requirement forDNA
topoisomerase Iactivity forinvitrotranscription. Synthesis
of the560-nucleotide RNA by uninfected HeLa cellextracts
programmed with Ad2 Smal fragment F DNA was also
completely inhibited by 1 mM novobiocin (datanot shown).
DISCUSSION
Significant insights into the molecular mechanisms of
eucaryotictranscription have been obtained from studies of
vaccinia virus transcription, notably the discovery of
polyad-enylation (12) and thecharacterization of themechanism of
RNA capping (29). Since vaccinia virus encapsidates the
enzymesrequired for earlygeneexpression, the virion core
particles have been used to analyze transcription in vitro.
However, understanding the precise mechanisms of
tran-scription required the development of a soluble system
capable of faithful transcription in vitro. Cloningand mRNA
mappingof thevaccinia virusgenomehaveprovided
numer-ous potential templates for transcriptional analyses (10).
Plasmid pAG4 was selected for these studies since it
con-tains anearly vaccinia virus gene which has been mapped
precisely (34). Thepromoterof thisgenehasbeen shownto
function in vivo (15), and the DNA sequence of the
5'-proximal region has been determined (33). Transcription of
SalI-digested pAG4 results in a 730-nucleotide transcript
whichcanbereadily detectedbydenaturinggel
electropho-resis.
Extracts for transcriptional assays were prepared from
vaccinia virus-infected HeLa cells by a procedure which
yields transcriptionally activeextractsfrom uninfectedHeLa
cells(16). The infected cell extract synthesizedtheexpected
730-nucleotide product which initiatesat the same site as the
in vivo transcript, as shown by other workers (27).
Interest-ingly, uninfected HeLa cell extracts fail to transcribe the
vaccinia virus gene, and conversely vaccinia virus-infected
HeLa cell extracts do not transcribe the adenovirus clone
bearing the RNA polymerase II major late promoter. Thus,
a virus-specific function(s) is required for vaccinia virus
transcription, and infection by vaccinia virus inactivates RNApolymerase II transcription. Greatly reduced levels of
cellular mRNA have been reported for vaccinia
virus-in-fected cells (4). SynthesisofRNA by infected cell extracts is
resistant to a-amanitin (27), as is RNA synthesized by
purified vaccinia virus RNA polymerase (2, 32). The
vacci-nia virus RNA polymerase is required for synthesis of the
730-nucleotide RNA since antibody directed against it
elim-inates pAG4 transcription (27).
Supplementationof uninfected extracts by purified
vacci-nia virus RNApolymerase did not result in any detectable
synthesisof the730-nucleotide transcript. Thus, a
virus-spe-cific factor(s) other than RNA polymerase is required for
specific transcription in vitro. Inhibition oftranscription by
the VHS extract by AMP-PNP indicates a requirement for
ATP hydrolysis to ADP and
Pi,
as has been observed fortranscription in vaccinia virus core particles (8, 30).
There-fore,phosphohydrolase I or II may berequiredfor
transcrip-tion by theinfected cell extract. AMP-PNPalsoinhibits the
vaccinia virustopoisomerase(7) but atconcentrationshigher
than 1 mM, which was used in thesestudies. Novobiocin, a
potent inhibitor of the topoisomerase, completely inhibits
transcriptionby bothinfected and uninfected HeLaextracts, suggesting a requirement for topoisomerase activity for in
vitrotranscription. It should be noted that novobiocin may
not bespecific for the topoisomerase, but it does notinhibit
vaccinia virus RNApolymeraseorphosphohydrolases I and
II (D. Foglesong and R. Guggenheimer, unpublished data).
The apparent requirement for topoisomerase activity is
surprising sincethetemplatesused werelinearDNAswhich
are not subject to any topological constraint. However,
replication of linear duplex adenovirus DNA in vitro also
requires a type I topoisomerase (23). Thus, topoisomerase
activity may facilitate the movement of transcription and
replication forks through duplexDNA.
Since vaccinia virus encapsidates all of the enzymes
required for earlytranscription,virion extracts were assayed
foractivity in transcribingpAG4 in vitro. Initial attempts in
which I used extracts prepared by conventional methods
(32) wereunsuccessful even when the extracts were
supple-mented with purified vaccinia virus RNApolymerase.
How-ever, virion extracts concentrated by ammonium sulfate
precipitation or negative pressure dialysis synthesized the
730-nucleotide
product (unpublished data). Thus, highpro-teinconcentrations may berequiredfor this in vitrosystem,
asfor othertranscriptionalsystems (16). Virion extracts may
be moreuseful thaninfectedcellextractsforsome
transcrip-tion studies since the proteins required fortranscriptionare
ofmuch greater
specific
activityinvirion extractsthan thoseof infected cellextracts. Otherinvestigators have also
tran-scribed cloned vaccinia virus genes in vitro by using virion
extracts (B.Moss,personal
communication;
F. Golini and J.Kates, Int. Symp.Pox/Iridoviruses, Abstr. ThA6,presented
26 July 1984 atMadison, Wis.). Thedevelopmentofsoluble
in vitro systems for vaccinia virustranscription willgreatly
facilitate the analysis of the protein factors required for
accurate initiation of transcription and also the DNA
se-quences required forrecognition by
those
factors.on November 10, 2019 by guest
http://jvi.asm.org/
[image:4.612.108.248.71.288.2]ACKNOWLEDGMENTS
Ithank B. Mossfor communicatingunpublished results andH. Furneaux and J. Hurwitz for helpful discussions.
This work was supported by Public Health Service grant 5T32 CA09176 from the National Institutes of Health.
LITERATURE CITED
1. Barbosa, E., and B. Moss. 1977. mRNA (nucleoside-2'-)-methyltransferase from vaccinia virus. J. Biol. Chem. 253: 7692-7697.
2. Baroudy, B. M., and B. Moss. 1980.Purification and character-ization of aDNA-dependent RNA polymerase from vaccinia virions. J. Biol. Chem. 255:4372-4380.
3. Bauer, W. R., E. C.Ressner, J. R. Kates, and J. N. Patzke. 1977. A DNAnicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties. Proc.Natl.Acad.Sci. U.S.A. 74:1841-1845.
4. Boone, R. F., and B. Moss. 1978. Sequence complexity and relative abundance ofvaccinia mRNAs synthesized in vivoand invitro. J. Virol. 26:554-569.
5. Bradford, M. M. 1976. Arapid arid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 6. Brakel, C., and J. Kates. 1974. Poly(A)polymerase from
vacci-nia virus-infected cells. J. Virol. 14:715-723.
7. Foglesong,P. D., and W. R. Bauer. 1984. Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. J. Virol. 49:1-8.
8. Gershowitz, A., R. F. Boone,and B.Moss. 1980. Multiple roles for ATP in thesynthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (,B,y-imido) tri-phosphate. J. Virol. 27:399-408.
9. Hruby,D. E., L. A.Guarino, andJ. R. Kates. 1979. Vaccinia virus replication. I. Requirement forthehost cell nucleus. J. Virol. 29:705-715.
10. Isle,H. B.,L.Venkatesan, and B. Moss. 1981. Cell-free trans-lation ofearly and late mRNAs selected by hybridization to cloned DNAfragments derived from the left 14million to72 million daltons of the vaccinia virus genome. Virology 112:306-317.
11. Kao,S., E. Ressner, J. Kates, and W.Bauer. 1981. Purification and characterization ofasuperhelixbinding protein from vac-cinia virus. Virology111:500-508.
12. Kates,J. 1970.Transcription of the vaccinia virus genomeand the occurrence ofpolyriboadenylic acid sequences in messenger RNA. ColdSpring Harbor Symp. Quant. Biol. 35:743-752. 13. Kates, J. R., and B. R. McAuslan. 1967. Messenger RNA
synthesis bya "coated" viral genome. Proc. Natl. Acad. Sci. U.S.A.57:314-320.
14. Kleiman, J. H., andB.Moss. 1975.Characterization ofaprotein kinase and two phosphate acceptor proteins from vaccinia virions. J. Biol. Chem.250:2420-2429.
15. Mackett, M., G. L. Smith,and B. Moss. 1982.Vaccinia virus:a selectableeukaryoticcloning andexpressionvector.Proc.Natl. Acad. Sci. U.S.A. 79:7415-7417.
16. Manley, J. L.,A.Fire, A.Cano,P. A.Sharp, andM. L.Gefter. 1980. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859.
17. Manley,J. L.,and M. L. Gefter. 1981. Transcription of mam-malian genesinvitro, p.369-382. InG. U.Chirikjianand T. S.
Papas (ed.), Geneamplification and analysis, vol. 2. Structural analysis of nucleic acid. Elsevier, New York.
18. Martin, S. A., E. Paoletti, and B. Moss. 1975. Purification of mRNAguanylyltransferase and mRNA (guanine-7-)methyltrans-ferase from vaccinia virions. J. Biol. Chem. 250:9322-9329. 19. Matsui,T., J. Segall,A.Weil,andR.G.Roeder.1980.Multiple
factorsrequired foraccurateinitiation oftranscription by puri-fiedRNApolymerase II. J. Biol. Chem.255:11992-11996. 20. Mickel,S.,and W. Bauer. 1976.Isolation by tetracycine
selec-tion of smallplasmids derived from R-factor R12 in Escherichia coliK-12. J. Bacteriol. 127:644-655.
21. Monroy, G., E. Spencer, and J. Hurwitz. 1978. Purification of mRNAguanylyltransferase from vaccinia virions. J. Biol. Chem. 253:4481-4489.
22. Moss, B.,E. N.Rosenblum, andA.Gershowitz. 1975. Charac-terization ofpolyadenylate polymerase from vaccinia virions. J. Biol. Chem.250:4722-4729.
23. Nagata, K.,R.A.Guggenheimer, and J. Hurwitz. 1983. Adeno-virus DNA replication in vitro: synthesis offull-length DNA with purified proteins. Proc. Natl. Acad. Sci. U.S.A. 80: 4266-4270.
24. Paoletti, E., andB. R.Lipinskas. 1978. Soluble endoribonucle-aseactivityfrom vaccinia virus: specific cleavage of virion-as-sociatedhigh-molecular-weight RNA.J. Virol.26:822-824. 25. Paoletti, E., and B. Moss. 1974. Two nucleic acid-dependent
nucleoside triphosphate phosphohydrolases from vaccinia vi-rus: purification and characterization. J. Biol. Chem. 249:3287-3291.
26. Pogo, B. G. T., andM.T.O'Shea. 1977. Further characteriza-tion of deoxyribonucleases from vaccinia virus. Virology 77:56-66.
27. Puckett, C., and B. Moss. 1983. Selective transcription of vaccinia virus genes intemplatedependent solubleextractsof infected cells.Cell 35:441-448.
28. Rosemond-Hornbeak, H.,E.Paoletti,andB. Moss.1974. Single-strandeddeoxyribonucleic acid-specific nuclease from vaccinia virus. J. Biol. Chem. 249:3287-3291.
29. Shuman, S., and J. Hurwitz. 1981. Mechanism of mRNA capping by vaccinia virusguanylyltransferase: characterization ofan enzyme-guanylate intermediate. Proc. Natl. Acad. Sci. U.S.A. 78:187-191.
30. Shuman, S.,E.Spencer,H.Furneaux,andJ.Hurwitz.1980. The role of ATP in in vitrovaccinia virusRNA synthesis. J. Biol. Chem. 255:5396-5403.
31. Spencer, E., D. Loring, J. Hurwitz, and G. Monroy. 1978. Enzymatic conversion of 5'-phosphate-terminated RNA to 5'-di- and triphosphate-terminated RNA. Proc. Natl. Acad Sci. U.S.A.75:4793-4797.
32. Spencer, E., S.Shuman,andJ. Hurwitz.1980.Purification and properties of vaccinia virusDNA-dependentRNApolymerase. J. Biol. Chem. 255:5388-5395.
33. Venkatesan,S.,B. M.Baroudy,andB.Moss. 1981. Distinctive nucleotide sequences adjacenttomultiple initiation and termi-nation sites ofanearly vaccinia virus gene. Cell 125:805-813. 34. Venkatesan,S.,andB.Moss.1981.Invitrotranscriptionof the
inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites. J. Virol. 37:738-747. 35. Weil, P. A., D. S. Luse, J. Segall, and R. G. Roeder. 1979. Selective and accurate initiation of transcription at the Ad2 major late promoter inasolublesystemdependentonpurified RNApolymerase IIand DNA.Cell18:469-484.