0022-538X/94/$04.00+0
Copyright © 1994, American SocietyforMicrobiology
Adenovirus
Type
40
and 41
Growth
In
Vitro:
Host Range
Diversity Reflected
by
Differences
in
Patterns
of
DNA
Replication
C. T. TIEMESSEN'* ANDA. H.KIDD2
Department of Virology, University of the Witwatersrand, andNationalInstituteforVirology, Johannesburg, SouthAfrica,'
andDivisionof Virology, Departmentof Medical Microbiology, Universityof Lund, Sweden2
Received 3August 1993/Accepted 1 November 1993
Subgroup Fadenovirusesadaptpoorly to cell culture, but the reasons for their fastidious nature are as yet illdefined. In an attempt to gain an overview of thedifferences inreplication between adenovirus type 2 (Ad2) andrepresentative strains ofAd4OandAd4l,celllines which show different degrees ofpermissiveness toAd4O
and Ad4lwere infected and examined with respect to three key functions in the Ad2 life cycle: hostprotein
shutoff, DNA synthesis, and late antigen synthesis. The complexity of growth patterns exhibited by the subgroup Fadenoviruses suggests that defectiveness is a multifactorial phenomenon not easily explainable by asingle aberrant function. Furthermore, results suggest that there may be replicative defects in subgroup F adenoviruses which are not shared by both serotypes or by all strains.
The subgroup F adenoviruses grow poorly in cell cultures
that are used to propagate other human adenoviruses (6). There arevariedreportsin the literature concerning the ability
of these virusesto replicate in different cell lines, such as 293 cells (2, 4, 18, 23, 26) and KB cells (17, 31). It has been
suggested thatpermissive adenovirustype40(Ad40)andAd4l
infection of the 293 cell line results from this line's expression of Ad5 El products, which might complement a defective
subgroup F adenovirus El function (23). The Ela regions of
Ad40 and Ad4l have been shown to have a relatively poor
capacity to transactivate other early genes (11, 29). There is also evidence from complementation studies with Ad5 Elb
productsthat Elbfunction mayalsobe compromised, atleast
inAd4O (17).
It maybesignificant that despite the similarity of Ad40 and Ad41 in terms of fastidiousness in culture (6), strategy of
infection in vivo (27), antigenicity (6), and having more than onefiber (12, 19), thegenomes of theseviruses appear quite
dissimilar by restriction enzyme mapping (28). Their overall
DNAhomology byliquid-phase hybridization is onlymoderate
(62 to 69%) (30). As some parts of the Ad4O and Ad4l
genomes appear more homologous than others (22), their
antigenic relatednessandalmost identicalfibersequences may
have come aboutthrough recombination between two parent
viruses.If this is the case, it wouldexplainsomeof thefindings
of this study, e.g.,that their apparently similargrowth
pheno-types may have come about, at least partially, for different
reasons.
Inthisstudywecompareddifferent aspectsofthegrowthof
Ad2,Ad40,andAd4l under identical conditions of infectionin
cells consideredpermissive (293 cells), semipermissive (Chang conjunctiva), and nonpermissive (human embryo fibroblasts)
for subgroup F adenovirus growth. The same stocks of
pas-saged virus (Ad2 prototype, Ad4O strain Hovi-X [28], and
Ad4l strain 26341-77 [28])were used in all experiments.
Hostprotein shutoffand intracellular viralstructural pro-teinsynthesis. Shutoffof hostproteinsynthesis is consideredto
*Corresponding author. Mailing address: National Institute for
Virology, PrivateBagX4,Sandringham 2131, South Africa. Phone: 11 882 9910. Fax: 11 882 0596.
be an important strategyfor ensuring an efficient, productive
adenovirus infection (8). It can therefore be used as an indicationofhowwelladaptedaparticular adenovirus strainis to ahost cell of interest. Changand 293 cells werechosen for study, as both are capable of supporting the replication of subgroupFadenoviruses, with293beinggenerallyregardedas
themorepermissive. Cellsgrowninflaskswereinfectedwitha
standard amount of Ad2, Ad40, or Ad4l determined by immunofluorescence in Chang cells (2.4 x 104focus-forming units [FFU] per ml, which was the highest infectivity of inoculum achievable for subgroup Fadenoviruses) and were
labeled with[35S]methioninefor 1,8,or20 hpostinfection and then harvested at specific time intervals. When Ad2-infected Chang cells were labeled for 1 h at 24-h intervals, virion structural proteins could be seen clearly superimposed on a
cellular protein profile. In contrast, no virion proteins were
detectedinAd4O-orAd4l-infected Chang cellsover6daysor when the cells were labeled for 20 h (data not shown) and
harvestedat42h. However, an extraprotein withan apparent
molecular mass (100 kDa) comparable to that of the hexon polypeptide (104 kDa) (25) was prominent in the profiles of Ad41-infected 293 cellslabeled inparallel for 20 h.
The same experiment was repeated, but with a labeling period of8hat48 hafterinfection andharvestingat56 h.The cellswerewashed twice withmethionine-free,L-glutamine-free
minimal essential medium and 1 ml oflabeling mixture
(me-thionine-free,L-glutamine-freeminimalessential medium
con-taining 2% dialysed fetal calfserum, 292.3 ,ugofL-glutamine perml,and afinal concentration of20
p.Ci
of[35S]methionine
[Amersham International, UnitedKingdom])perml (20 nmol final concentration) was added for 8 h. The medium wasremoved, and the cells were washed twice with
phosphate-buffered saline (PBS) and collected by scraping. They were
pelleted and resuspended in 100
p.l
ofbuffer containing 10% (wt/vol) sodiumdodecylsulfate(SDS), 10% (wt/vol) 2-mercap-toethanol, 40% (vol/vol) glycerol, and 0.02% (wt/vol)bromo-phenol blue in 1.25 MTris-HCl (pH 6.8). After incubation at
95°Cfor 5min,
20-[l
volumeswereloaded on 12%polyacryl-amide gels
(0.1%
[vol/vol] SDS). The electrophoresedgels
werefixed, driedat80°C,andautoradiographed for24to48h.
Ad2and
Ad4l
proteinsweredetectedin 293cells(Fig. 1),but 1239on November 9, 2019 by guest
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kDa 200
925 69
46
30
A B
M 1 2 3 4 5 6
21.5 E 0
=}-FIG. 1. Autoradiograph of [35S]methionine-labeled proteins of Ad2-, Ad4O-, andAd41-infected 293 cells (A) and Chang cells (B). Cellsgrowing in 25-cm2 flaskswerelabeled and proteinswere
electro-phoresed as described in the text. Lanes M, 14C marker; lanes 1, uninfected cell proteins; lanes 2, Ad2 (input concentration, 2.4 x 105 FFU/ml); lanes 3 and 4, Ad4O (input concentration, 2.4 x 104
FFU/ml); lanes 5 and 6, Ad4l (input concentration, 2.4 x 104
FFU/ml). Molecularmassesof marker proteinsareindicated.
there was no evidence of efficient host protein shutoff. Ad4O
proteins were not detected in either cell type, and Ad4l
proteins were not seen in Chang cells. This confirmed our
empirical observation that Ad4l growsbetterin293 cells and
suggested that Ad4O ismore fastidious.
Toclarifythetime- and dose-dependentparametersofhost protein synthesis, Chang cells were infected with different
concentrations of Ad2 and labeled for20 h at 24-h intervals
over a5-dayperiod. Complete shutoffof hostprotein synthesis
could be demonstrated only with Ad2 infection at levels
unattainable with Ad4O and Ad4l.Therefore, it couldnotbe
shownconclusively thatAd4Oand Ad4lfailedtocauseshutoff.
Itisperhapssignificant, however, that Ad4l grewmuchmore
efficiently in 293 cells (see below) than in the Changcells in which itwastitrated andyetfailedtoshow evidence of shutoff of host protein synthesis in the 293 cells.
Hexonproduction.Thevirtual absence of hexon polypeptide in gels derived from Ad4O-infected 293 and Chang cells suggested differences in levels of expression of this major
structuralprotein between theAd4O andAd4lstrainsused.To study this further,a group-specificenzyme-linked
immunosor-bent assay (ELISA) based on a polyclonal antihexon capture
antibody (15) was used to estimate levels ofunincorporated hexon overtime, the total free hexon antigen produced atany one time being considered thesumofintracellular and extra-cellular antigendeterminations. Our assumption that the
pro-ductionofa structural antigen reflects virus production isnot
ideal since the particle-to-free hexon ratios may not be the
same for Ad2, Ad4O, and Ad4l, but it is useful in the case of
viruseswhichcannotbequantified easily intermsofparticleor
infectivitycounts.
In 293cellsinfected withthestandardamountofAd2(2.4 x 104 FFU/ml), the intracellular Ad2 hexon level rose sharply
between days 2 and4 andreachedaplateau; thiswasfollowed
by a similar rise in the extracellular hexon level (Fig. 2A).
Intracellular Ad4O hexon accumulationwas the least
remark-able accumulation among the three viruses tested (Fig. 2B).
Thus, theAd4O strain studied appearedto growpoorly in293
cells, which is inagreementwith severalreports ofdifficulties in propagating Ad4O in these cells (1, 4, 26, 30). In sharp
contrast, and in keeping with the in vivo protein labeling resultsabove, intracellular Ad4l hexon reached alevel
com-parable to that of its Ad2 counterpart, and it did so even soonerthan intracellular Ad2 hexon did. Thiswasfollowedby
amore gradual increase in the extracellular Ad4l hexon level (Fig. 2C). The pronounced replicative block of Ad4l strain
Takin 293 cells described by Pieniazeketal. (18), withaloss of infectivity within two passages, was not seen with our common Ad4l strain, which had been passaged three times previously in 293 cells.
For Chang cells infected with the same dose of virus, the accumulation ofAd2 hexonintracellularly and in the medium occurred earlier than that for293cells(Fig. 3A).In contrastto
the results with293cells, the accumulationof free intracellular and hence also extracellular Ad4l hexonwasless marked than Ad4O hexon accumulation (Fig. 3B andC). Arapid
intracel-lular accumulation of Ad4O hexon occurred in Chang cells between days 1 and 2, which was reminiscent of, but less marked than, that seen for Ad4l hexon in 293 cells. That Chang cells have not been transfected with El sequences suggeststhat the replicationof the Ad4O strain studied isnot as
dependenton exogenous Elproductsasthe replication ofthe
Ad4l strain studied.
Infection of HEF cells (human lungfibroblasts) with either Ad4O or Ad4l yielded no evidence of hexon production
relative to uninfected controls. The accumulation of Ad2
hexon mirrored that seen with 293 cells, although at much
lowerlevels (data not shown).
DNA synthesis in semipermissive and nonpermissivecells. Replicate Ad2-, Ad4O-, and Ad4l-infected Chang and HEF
cellcultures wereharvesteddaily for 1 week. Acomparisonof the levels of total intracellular viral DNA and intracellular
packaged DNA (selected on the basis of DNase resistance conferred by capsid proteins) was made by dot blotwith an
Ad4O-derived plasmidprobe (Ad4O or Ad4l infections) or an Ad2-derived plasmid probe (Ad2 infections).
Asshown inFig.4A,replication and packaging of Ad2DNA weremuch moreefficient inChang cells than in HEFcells over time.Approximately 10 to20% of the Ad2 DNA synthesizedin Chang cells was packaged. Ad4O and Ad4l DNAsynthesis in
Changcellscould be detectedfrom day 2onwards(Fig. 4B and
C). The relative amount of Ad4O DNA packaged in Chang cells was less than 1%. It was first detected 3 days after infection, increased over the next 2 days, and then declined. Ad4l total DNA and packaged DNA levels were maximal 3 days after infection. Packaged DNA was detected earlier and over a longer period of time for Ad4l than for Ad4O. The
proportion of Ad4l DNA packaged wasestimated to be about 1% of the total. That Ad4l replicated its DNA better than Ad4O in Chang cells but failed to outperform Ad40's hexon production in the same host suggests that the two subgroup F
adenovirus strains have quite different growth characteristics. In contrast to the above results, which show that the Ad4l strainproduced more DNA, a different relationship was noted between Ad4O and Ad4l DNAsynthesis in the nonpermissive fibroblast cells (Fig. 4B and C). No DNA was synthesized or
packaged into virions in HEF cells infected with Ad4l.
How-ever, some DNA was synthesized in HEF cells infected with Ad4O, but there was no evidence of packaging. It could be argued that the amount of DNA packaged might have been below the level of detection if the proportion of DNA incor-porated paralleled that seen in Chang cells. However, longer exposure of Ad4O dot blots showed an increased signal for
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FIG. 2. Comparison ofintracellular andextracellular hexon levelsat varioustimesafterinfection of 293 cells. Cells in 24-well disheswere
infected induplicatewith 2.4 x 103FFUof Ad2 (A),Ad4O (B),orAd41 (C)perwell.Theuninfectedcontrols (D)receivedPBS.Intracellular
and extracellular hexon accumulation was monitoredby indirect ELISA asdescribed previously (15), andresultswere averaged. OD, optical density;pi..postinfection.
intracellular DNA but did not give any evidence of DNA
packaged into virions (data not shown). That no Ad4l DNA
synthesis at all was detected in these experiments suggests defectiveness in one or more early functions. This is in
agreementwithreportsofdifferences in the abilities of thetwo viruses totransform variouscell types(5, 30).
Persistence of Ad4O DNA in nonpermissive cells. As no
cytopathic effect occursin HEFcells, wedetermined whether subgroup F adenovirus DNA persists when the cells are
cultured further. Duplicate cultures in 75-cm2 flasks were
infected with 1.2 x 104 FFU of either Ad4O orAd4l perml,
and both these cells and uninfected controls were passaged
when they reached confluence. DNAwasextracted from one
of the cultures at each passage and the other was used to propagate the cells further. The cultures infected with Ad41 survived sixpassages, as didthe uninfected controls, whereas thoseinfected withAd4Osurvivedonly threepassages,the loss
of theculturecoinciding withacytopathic effect.Passagezero
(P0) DNAs from infected and uninfected flasksweredigested
withBamHI, EcoRI, HindIll, and SmaI. Blots hybridized with homologous viral DNA from CsCl-purified Ad4O or Ad4l
particles indicated the presence ofvirus-specific DNA
frag-mentsonly for Ad40-infected HEF cells (data not shown). DNAs from allsixpassages for Ad4l and threepassagesfor Ad4Oweredigested with EcoRI. In ethidium bromide-stained
gels, viral bands could beseenonly for Ad40 P3 DNA, with all four EcoRI bands present (Fig. 5A; note that the EcoRI D fragment of 2,518 bp cannot be seen), indicating that an
episomal rather thanan integratedform of the Ad4Ogenome
persisted. When extracted cellular DNA was blotted and hybridized with pSP64-Ad41EcoRI B, which carries Ad4l DNA from the E3 and fiber regions (13), viral DNA was
evident in all three passages for Ad40-infected cells, with a
weak reactioninDNA fromAd41-infected HEF cellspassaged
once(Fig. SB). TheamountofAd40DNAhadincreasedwith
passage,whereasthe amountof Ad4l haddecreased. Attemptstorescuethe Ad4Ogenomeinnonpermissivecells. It has already been shown that Ad2 can provide a helper
function for Ad4lgrowthin HEFcells(24).As thepersistence ofthe Ad40genomeinanepisomal formhadbeenshown(see above), the reactivation or rescue ofsubgroup F adenovirus
genomes on superinfection with Ad2wasinvestigated. In this experiment,HEFcellsseparatelyinfected withAd40and Ad4l
weresuccessfully passagedseventimesoveraperiodof 49days
of continuous culture.Ad40-infectedcells underwentastageof
apparentvirus-induced cell destructionasbefore,but thistime survivingcellswere abletogrowtoconfluence.A similarcrisis phase occurred at the fifthpassage, afterafurther 20 days of growth.
The Ad40- orAd4l-infected HEF cellswere then superin-A
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FIG. 3. Comparisonofintracellular andextracellularhexonlevels(monitored by ELISA)atvarioustimes afterinfectionof Chang cells.Cells in24-welldisheswereinfected induplicate with2.4 x 103FFUofAd2(A),Ad4O(B),orAd4l(C)perwell. The uninfected controls(D)received PBS. OD,optical density; p.i., postinfection.
fected with Ad2 after the seventhpassage(2.4 x 105FFU/ml; 0.1FFU percell). Asacontrol forpersistence of theoriginal
adenovirus genome, DNA was extracted fromrepresentative
cells at this passage and digested withEcoRI. As before, all four EcoRI fragments of Ad40were detected, but no Ad4l
DNA.Byanindirect immunofluorescencetestbasedon
mono-clonal antibodies to subgroup F adenovirus hexon (lla), no
reactivationof Ad4O or Ad4l growth could be detected 44 h
after superinfection with Ad2. The same experiment was
repeatedwithanAd2multiplicityofinfection of1.0,whichled
toamoreenhancedcytopathic effectbutnosignificant
detec-tionofsubgroupFadenovirus late antigens.
Coinfection of nonpermissive cells with Ad4O and Ad4M. Since Ad4O DNA replicates in HEF cultures and persists in
passaged cells, and since it has been shown that Ad4l can
replicate to the stage of late antigen synthesis in cells
coin-fected with Ad2 (24), the possibility of complementation
between Ad4Oand Ad4l inthese cellswasinvestigated. HEF
cells were coinfected with Ad4O and Ad4l at concentrations
andinputratios(Ad4OtoAd4l)of0.25:1, 0.5:1,1:1,1:0.5,and
1:0.25. At 44 handat7dayspostinfection, replicate coverslips,
each with 106 cells, were examined by immunofluorescence
with Ad4O-specific and Ad41-specific monoclonal antibodies
directed against hexon. No complementation to the stage of
lateantigensynthesiswasdetectedbythismethodatanyofthe
virus input concentrations. This can have more than one
interpretation. The simplest is that the two viruses share the
samedefect. This isclearlynotthe wholereason,astheAd4l
strainappearedmoredefective in DNAsynthesisinthese cells.
Itisalsopossiblethattheirdefective functionsmaptodifferent
genes but that their respective "healthy" functionswere not
adequately expressed in these cells to allow the detection of
complementation.
It appears from the above results that Ad4l replication profitsmorefromexogenously suppliedElproductsthan does
Ad4Oreplication.Theseearly productsmaybe what is needed in orderto allow anyDNA synthesis in HEF cells, andtheir
productionmaybethefunctionthatcanbesupplied byAd2 in
these cells in ordertoallow theprogressionof infectiontolate
antigen synthesis (24). Thismayalso be the functionlimiting
efficient Ad4l hexonproductioninChangcells.Ad4O, onthe
other hand, undergoes DNA replication on a reduced scale
(comparedwith Ad2)inbothsemipermissive and
nonpermis-sivecells, suggestingthatanyearlydefect inreplicationinthis virusmaynotbeasimportanttothefinaloutcomeof infection
as the existence of one or more defects which prevent the
expression of newly synthesized DNA. Such defects may be relatedtothedecreasedlevelsof lateproteins expressed from the latetranscriptionunit in abortive infections ofmonkeycells
byhuman adenoviruses (7, 16, 20).
Defectiveness in subgroup F adenoviruses may thus be a
multifactorialphenomenon involvinggenefunctionsatseveral A
C
,'I/'
D
8
1
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Cells
A Chang
HEF
ii Chang
HEF
daysP.l.
1 2 3 45 6 7 U
~*
Amk-.
0*.---B i Chang
HEF
ii Chang 0 * t
HEF
C Chang
0
HEF
ii Chang *0
HEF
FIG. 4. Dot blot hybridizations showing the timecourses ofDNA
synthesis (rows i) andpackaging ofviral DNA(rows ii)in Ad2 (A)-, Ad4O (B)-, and Ad4l (C)-infected Chang and HEF cells. At the indicated timesinfected cellswereharvested and the totalorpackaged
viral DNAwasextractedanddottedonnylonmembranes (14,21).For the detection ofpackagedDNA thecellswerefrozen and thawed four times and treated withDNase,andthen the DNAwasextracted from
the virions. The Ad2 blotswerehybridizedtopSPT18-Ad2SmaI A,and the Ad4O andAd4l blotswerehybridizedtotheAd40-derived probe, pBR322-Ad4OPstI H. Hybridizationswere carried out as previously described (14).
stagesof thereplication cycle. Ad4O and Ad4l having multiple defects, only some of which may be shared, would make it
difficult to implicate any single aberrant function, since the
final phenotype may depend on several. The presence of
multiple defects might also explain why different cell lines
appear to alleviate defectiveness to different extents. For example, HeLa cells appearunable torelease these virusesto surrounding cells andcausefoci of infection(3). However, this
does not match the experience of others with Ad4O in A549 cells (10) orAd4l in PLC/PRF/5 cells (9).
We conclude that the Ad4O and Ad4l strains we studied have behaviors quitedifferent from each other inculture, the interpretation of which is confounded by hostrange restriction
to different extents in different cell types. A model system which is nearer to the in vivo situation, with differentiated intestinalcells, is much needed in ordertodetermine whether these viruses are evercapable ofa fully productive infection
and, if so, to map normal events especially with regard to transcription and posttranscriptional processing. The
signifi-cance of genomic persistence in human cells might then be
morethoroughly assessed.
Ad4l Ad4O
bp
1 2 3 4 5 6 1 2 3M
A 23130
9416
6557
4361
2322 2027
B
FIG. 5 Detection of adenovirus-specific restriction enzyme
frag-mentsin DNAfrom Ad40- andAd4I infected HEFcellsatdifferent passages postinfection. (A) Ethidium bromide-stained 0.8% agarose
gelwith DNA extracted from infected cellsat P1 (lanes 1), P2(lanes 2), P3 (lanes 3), P4 (lane 4), P5 (lane 5), and P6 (lane 6). (B) Autoradiograph of Southern blot from panel A hybridized with
pSP64-Ad4lEcoRIB.Sizes ofHindlll-digestedlambda DNA(M)are
indicated.
This workwassupported byagrantfrom the PoliomyelitisResearch Foundation of South Africa.
We thank J. C. de Jong for supplying Ad40 strain Hovi-X and monoclonal antibodies MA3-20 andMA5-15. We thankN. K. Black-burn for helpandadvice in producingother monoclonal antibodies.
ADDENDUM IN PROOF
Wehave
recently
shown thatsubgroup
Fadenoviruses have asingle
VA RNAgene withhigh homology
tothat ofamonkey
adenovirus(A.
H. Kidd and C. T. Tiemessen, J. Gen. Virol. 74:1621-1626,1993).
Thisfinding
may have relevance to theorigin
ofsubgroup
F adenoviruses and to the abortive nature of infection ofmost human cells.REFERENCES
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3. Brown, M.,H. L.Wilson-Friesen,and F. Doane. 1992. A block in releaseofprogenyvirusandahighparticle-to-infectious unit ratio contribute topoor growthofentericadenovirustypes40and41 in
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[image:5.612.83.278.72.388.2] [image:5.612.331.544.73.397.2]cell culture.J.Virol. 66:3198-3205.
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![FIG.1.Ad2-,phoresedCellsFFU/ml);FFU/ml);FFU/ml).uninfected Autoradiographof [35S]methionine-labeledproteinsof Ad4O-, and Ad41-infected 293 cells (A) and Chang cells (B)](https://thumb-us.123doks.com/thumbv2/123dok_us/1296145.82639/2.612.60.298.69.257/phoresedcellsffu-uninfected-autoradiographof-methionine-labeledproteinsof-infected-chang-cells.webp)
