0022-538X/92/031717-09$02.00/0
Copyright © 1992,AmericanSociety for Microbiology
Incompletely
Reverse-Transcribed
Human
Immunodeficiency
Virus
Type 1
Genomes in Quiescent Cells
Can Function
as
Intermediates in the Retroviral Life Cycle
JEROME A. ZACK,1* ALLYSON M. HAISLIP,"12PAULKROGSTAD,3ANDIRVIN S. Y. CHEN'2
Division of Hematology-Oncology, Department of Medicine' andDepartmentsof Microbiology and
Immunology2
and Pediatrics,3University of California atLosAngeles School of Medicineand Jonsson Comprehensive Cancer Center,LosAngeles, California 90024
Received 24 September 1991/Accepted 2 December 1991
Usingaquantitative polymerasechainreaction (PCR)method,wehave previously shown thatamolecularly
cloned isolate of human immunodeficiency virus type 1 (HIV-1) can efficiently enter quiescent primary lymphocytes; however, the reverse transcription process is notcompleted in these cells. In this study,we
furthercharacterizedthereversetranscription ofHIV-1in quiescent cells, andourresultsindicate that while initiation ofreverse transcription occurs simultaneously in both activated and quiescent lymphocytes, itnot
onlyends prematurely butalso proceeds more slowly in quiescent cells. We also performed experiments to address the role ofpartialreverse transcriptsasintermediatesinthe viral life cycle. Weusedazidothymidine
either beforeorafter infectionwith HIV-1to preventformation of and further DNA synthesisbypartialreverse
transcripts, respectively. Decreases in virus production from these cells following mitogenic stimulation indicated that partialreverse transcripts cancontribute significantlytovirus rescuefrom infected quiescent
cells stimulated subsequentto infection. Furthermore, weestablished that mitogenic stimulation of infected
quiescentcells induces reinitiation of DNAsynthesisfrompartialreversetranscripts. However, thevirusrescue isinefficient relativetothe initialmultiplicityof infection, and this is explained by inefficient completion of DNA synthesis fromthepartial reverse transcript. Thus,thearrest ofreverse transcriptionin quiescent cells may
playanimportantroleinHIV-1pathogenesis by contributingtothe inefficient infection of potentialtargetcells in theperipheral blood of HIV-i-infected individuals.
The stage of the cell cycle at the time of infection by retrovirusesgreatlyinfluences viralreplication. Both human and other animal retroviruses are capable of infecting and
persisting in quiescent cells without producing progeny
virions (7-9, 12, 13, 20, 23, 24, 26). Mitogenic stimulation subsequent to infection can induce progenyvirus
produc-tion. Following infection, incomplete species of viral DNA
canbe detected inquiescentcells(5, 8, 9, 22, 23). Ourrecent studies demonstrated that human immunodeficiency virus type1(HIV-1)canefficientlyenterquiescent primaryhuman lymphocytesbut that thereversetranscriptionprocessisnot
completedin these cells.Mitogenic stimulation ofquiescent cells harboring this partial reverse transcript induces virus
production, suggestingthatwecharacterized anovel latent
form of HIV-1. We further demonstrated that the HIV-1 DNA inquiescent lymphocytesis labile anddegradeswitha
half-life ofapproximately 1dayinvitro (23).Thislabilityof thepartialHIV-1reversetranscriptsuggests that theremay beamechanism for clearance of the virus in vivo. Thevast majorityofcirculating lymphocytesinvivoarein the quies-cent state;thus, this model mayexplain the low number of infected cells in theperipheralblood ofpatientswithAIDS (10, 11, 18).
In this study, we further characterized the nature ofthe partial HIV-1 reverse transcript in quiescent cells and per-formedexperimentstodeterminemorepreciselythe role of
this intermediate in the retrovirus lifecycle. Comparisonof
reverse transcription in both activated and quiescent
pri-mary T cells reveals that reverse transcription initiates at
* Correspondingauthor.
about the same time in both cell types, but there is an
apparent difference in the rate of thisprocess between the two cell types. Our results also indicate that the partial
reverse transcript in quiescent cells can be induced to complete the reverse transcription process following
mito-genic stimulation of the infected cells and is capable of leadingto progenyvirusproduction; however,when stimu-lation is applied 15 h postinfection, this rescue is
approxi-mately 20-fold less than infection of prestimulated cells duringa singlevirusreplication cycle.
MATERIALSANDMETHODS
Cells and viruses. Peripheral blood was obtained from
normal donors by venipuncture. Peripheral blood lympho-cytes (PBL) were isolated by centrifugation over
Ficoll-Hypaque and depleted of macrophages by adherence to plastic for 4 h. Quiescent lymphocytes were cultured in RPMI 1640 supplemented with 10% pooled human AB
serum, 100 U ofpenicillinperml,100,ugofstreptomycinper
ml, and 2 mM glutamine. To obtain activated cells, PBL from the same blood donorwere obtained and stimulated with phytohemagglutinin (PHA) (HA 15, 0.8 ,ug/ml; Well-come) for 3 days prior to infection. Following infection, these cellswerecultured in thesamemediumsupplemented with 30 U of recombinant interleukin 2 per ml following infection.
The virus strains
HIV-lJRCSF
(15) and HIV-lNL43 (1)were recovered following electroporation of molecular clones (3). Virus stocks were subsequently obtained from 24-h harvestsofsupernatantfrom infectedPBL and stored at -70'C. Patient isolates 1 and 2 were obtained from the 1717
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1718 ZACK ET AL.
peripheral blood of HIV-seropositive patients seen at the University of California at Los Angeles Medical Center. Patient cells were cocultivated with PHA-stimulated PBL from normaldonors. Twenty-four-hour culture supernatants were harvested on day 15 of coculture. Prior to use, virus stockswerefiltered and treated with RNase-free DNaseI (1
,ug/ml;
Worthington) for 30 min at room temperature in the presence of 0.01 M MgCl2 to remove contaminating HIV-1 DNAarising from the lysis of infected cells during prepara-tion.Infectionwasaccomplished by incubating cells with the DNase-treated virus in the presence of 10,ug
of Polybrene per mlfor 1 to 2 h at 37°C. Infections were standardized by enzyme-linked immunosorbent assay (ELISA) specific for HIV-1 p24"ag antigen (Coulter). Limiting-dilution experi-ments on PBL from various donors have indicated that approximately 1 to 30 pg of p24 is equivalent to one infectious unit ofHIV-1JRCsF.
Cells were rinsed twice prior to culturing to remove residual free virus. Heat-inactivated virus controls were prepared by incubation for 30 min at 60°C. Infections with heat-inactivated virus were performed inparallelwithinfections with live virus.Nucleic acidpreparation.All reagents used for nucleicacid preparationwere specifically prepared for polymerase chain reaction(PCR)useand tested to ensure that no HIV-1 DNA contamination was present. Nucleic acids from live and heat-inactivated virus infections were extracted in parallel. Cellswerewashed inphosphate-buffered saline (PBS), lysed in urea lysis buffer (4.7 M urea, 1.3% [wt/vol] sodium dodecylsulfate, 0.23 M NaCl, 0.67 mM EDTA [pH 8.0], 6.7 mMTris-HCl [pH 8.0]) and subjected to phenol-chloroform extraction and ethanol precipitation. Total nucleic acids resultingfrom this extraction procedure were used for PCR amplification.
To analyze viral DNA in quiescent lymphocytes infected with
HIV-lNLA-3,
clinical HIV isolates (see Fig. 2), and pseudotypedHIV-1 (see Fig. 7), an alternative DNA extrac-tion and PCR method was used. Eighteen hours afterinfec-tion,
PBLwerewashed once with PBS and resuspended at a concentration of 2.5 x 107 cells per ml in gelatinlysisbuffer(45
mMNaCl, 9 mMTris-HCI [pH 8.3], 2.2 mM MgCl2,0.1
mg of gelatin [Sigma] per ml, 0.4% Nonidet P-40, 0.4% Tween 20, 1 mg of proteinase K per ml). The samples were incubatedat60°C for aminimum of 3 h and then placed in a boiling water bath for 10 min. These lysates were used directlyin PCR amplification, asdescribed below.
PCR amplification. Quantitative PCR amplification with 32P-end-labeled primers was performed as previously de-scribed (2, 16, 17,23), except that denaturation was
accom-plished
by heating to 94°C. Twenty-five cycles of amplifica-tion were performed for HIV-1 and human 1-globin sequence analysis by using radiolabeled oligonucleotideprimers.
3-Globin-specific
primers and the HIV-1-specific primers M667, M661, LA8, LA9, LA45, LA64, and AA55 were previously described (23). The primer AA943(5'-TGACTTACAAGGCAGCTATAGATC-3')
corresponds tonucleotides (nt) 9048 to 9071 in the
HIV-1JRCsF
sequence, andAA946(5'-CTCTGGATCAACTGGTACTAGC-3')
cor-responds to nt 9265-9244 (antisense). These two primersamplify
a218-bpfragmentcorrespondingto sequences in theHIV-lJRCSF
nef gene that proceed and overlap the U3region
of the 3' long terminal repeat (LTR). The location of the HIV-1-specific primers used in these studies is depicted schematically in Fig. 1. Following amplification, radiola-beled products were resolved on a 6% polyacrylamide gel and visualized by autoradiography. HIV-1 DNA standards usedto quantitateviral DNA werederived from dilutions ofM667 AA55 M661
_0-_*_ LA8 LA9
AA943 AA946 LA45 LA64 _ _
_
_0-
- 0L3RIU5gag pol env
FIG. 1. Oligonucleotide primers used inPCRanalysis. The rel-ative locations and orientations ofoligonucleotide primers used to analyze reverse transcription are represented byarrows. The DNA genome of
HIV-1JR-CsF
is depicted schematically. Open boxes represent the LTRs of the viral DNA, U3 andUS
indicate the 3' and 5' sequences of HIV-1 RNA duplicated during formation of the LTRs, R is the region repeated at both ends of the viral RNA genome, and the solid circle represents the location of the viral primer binding site (nt 637 to 651). The shaded rectangle represents the polypurine tract (nt 9085 to 9099) which directs plus-strand priming. Figure is not drawn toscale.cloned
HIV-lJR-CsF
DNA (3) digested withEcoRI,
which doesnot cleaveviral sequences. This DNAwas diluted into carrier tRNA (4,ug/ml)
or PBL DNA (10,ug/ml)
where indicated inthefigure legends. Standard curves for 3-globin DNAwerederived from dilutions of PBL DNA. HIV-1- and 3-globin-specific primers were simultaneously incorporated into each reaction where indicated. For PCR analysis of samples in Fig. 2and 7, PCR mixtures consisted of 10,ul
of the cellularlysate added to 15,ul
of low-salt PCRbuffer (25mM Tris [pH 8.0], 2 mM
MgCl2,
30 mM NaCl, 0.1 mg of bovine serumalbumin perml, 0.25 mM dNTP).Twenty-five cycles of amplification were performed, each consisting of a1-min
denaturation step at94°C
followed by a2-min
anneal-ing-extension phase at
65°C.
Addition of exogenous nucleosides. Quiescent cells (5 x
106)
werecultured in thepresence orabsence of50,uM
each nucleoside (2'-deoxycytidine [Calbiochem], 2'-deoxyade-nosine, 2'-deoxygua2'-deoxyade-nosine, and 2'-deoxythymidine [ICN]) or 2,uM
the nucleoside analog azidothymidine (AZT) (Sig-ma) for 3 h prior to infection withHIV-lJR-CsF.
Cells were infected for 2 h, rinsed, and cultured for 20 h in appropriate media. DNA was harvested, and 5% of the recovered amount was analyzed by PCR with various primer pairs.Generation of pseudotyped virus. To generate infectious replication-defective HIV-1 virions, an envelope-negative HIV-1 was pseudotypedwith the murine amphotropic enve-lope. An envelope-negative HIV-1 plasmid (pJRCSF-AFLA) was generated by digestion of pYK-JRCSF (4) with
AflIll
(which cleaves at nt positions 6513 and 7485 and once in the vector), followed by religation of the two larger fragments. This resulted in a viral clone deleted in the region encoding aminoacids 67 to 368 ofgpl20.
pJRCSF-AFLA (12.5,ug)
and 12.5,ug
of the amphotropic env expression vector pJD-1 (6) (obtainedfrom M. Emerman) were coelectroporated (3)into 5 x106
COS cells. Supernatants were harvested daily for 3 days following cotransfection, and virus stocks were stored at-70°C.
These stocks were assayed by ELISA for HIV p24(Coulter) prior to use. Prior to infection of PBL with this virus, virus stocks were subjected to treatment with DNase (4
,g/ml)
(Worthington) for 1 h at room temperature in the presence of 0.01 MMgCl2
to remove contaminating plasmid. DNase-treated virus stock was used to infect PBL in the presence of Polybrene, as outlined above for wild-type HIV.RESULTS
Infection of quiescent
lymphocytes
by HIV-1. We previ-ously showed that infection of primary quiescent PBL by a molecularly cloned isolate of HIV-1(HIV-lJR-CsF)
obtained J. VIROL.on November 10, 2019 by guest
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[image:2.612.319.554.82.117.2]NL4-3 Pt.1 Pt.2
-1L~L
wjT
H(IV-i
R
U5-140bp
(XIV-1
LTR
gag-200bp
FIG. 2. InfectionofquiescentPBL wil Three HIV-1 isolates, HIV-lNLA3 (1) isolates (Pt.1 and Pt.2),wereusedtoinfe
samenormal-blood donor.Twenty-fivec'
in the presence of the R/U5 (initiation) primer pairswereperformedonDNAex
DNA from 2.5 x 105 cellswas analyzed
cells treatedwith heat-inactivated virusi
from the cerebrospinal fluid of a p
resulted in efficient initiation of re'
inefficient completion of this proces.
by differential PCR amplification wil of distinguishing DNA structures fi stages of the reverse transcriptionpr relative positions in the HIV-1 geno:
genomic structure consisted of a si tionalsynthesisof minus-strand viral length.
Arecent study suggestedthatfull-] synthesizedinquiescentcells afterir the possibility that strain difference discrepant results by infecting quic other HIV-1 strains. Infection of (
normal donor with the molecularl'
HIV-1NIL43
(1),aswellaswithtwovi culture ofPBL fromtwodifferent HI'was analyzed by using quantitative
determine the ability of these iso
reverse transcription process. The pair M667/AA55 (Fig. 1) is speci
sequences in the R/U5 region, the I
duringreversetranscription. Thispri first 140 bases ofviral minus-strand lowinginitiation ofreversetranscript
allreverse transcriptsofHIV-1wou
primer pair.Theprimer pair M667/M in the LTR/gag region of the HIV-1 viral primer binding site. To general with theseprimers, contiguous LTR
necessary. Thus, completeornearly
scriptionisrequiredtogenerate the
using this primer pair. Analysis wil specific) primer pairresultedinPCR: of the heat-inactivated controls for
(Fig. 2, top). PCR analysis ofthe sa the LTR/gag (full-length) primer p
signals above those of the controls
HIV-1 STANDARDS while all three strains could enter quiescent cells and initiate reverse transcription, completionof this processwas ineffi-cient. We therefore conclude that premature termination of
CD ° C)° reverse transcription appears to be a general property of
HIV-1 infection ofquiescent lymphocytes.
Kinetics ofreversetranscription.Wehave takenadvantage of the orderedsequence ofevents in the reverse
transcrip-.4 *tion
process (forareview, see reference 21) to analyze the stages of reverse transcription by using oligonucleotide primers specific for DNA structures present at various stages of reverse transcription. To analyze the kinetics of reverse transcription in activated and quiescent cells, two primer pairs in addition to the LTR/gag(M667/M661)
andR/U5
(M667/AA55)
primer
pairs
wereutilized. Theseprimer
pairs detect different
regions
of the minus strand of viral DNA found inpartial
reverse transcripts. Theprimer pair
AA943/AA946detects sequencesatthe 3' end of the HIV-1 th differentHIV-1 isolates. genome, corresponding to the region of the nef gene imme-and two primary patient diately 5' to and overlapping the U3 region of the 3' LTR.
ct
quiescent
PBL from the Viral reversetranscription
mustproceed through
the firstycles of PCR amplification
y
or
LTRIgag
(full-length)
template-switching
event to form DNA detectableby
thesetracted
18 h postinfection. primers. The primer pairLA45/LA64
amplifies a region of in all lanes. HI indicates the HIV-1 genomecorresponding
to tat/revcoding
se-in parallel. quencesjust
5' ofenvelope coding
sequences. Thisprimer
pairwill detect furtherextension of the minus strand of viral DNA. We utilized these additionalprimer pairs
to furtherinvestigate
the kinetics ofreversetranscription
inquiescent
patient with AIDS (15) versus activated human T
lymphocytes.
verse transcription but Cellular DNA from both
quiescent
and activated cells s(23).
This was shown infected withHIV-1JR-CSF
was harvested at various time th primerpairs capable
pointspostinfection
andanalyzed by
quantitative
PCR. All ormedduring
differentsamples
were obtainedduring
the first 20 hpostinfection,
rocessbyvirtueof their prior to progeny virus
production (23).
TheM667/AA55
me. The
resulting
virus primerpair,
the indicator ofinitiation ofreverse transcrip-ngle 3' LTR and addi-tion,
revealed that reversetranscription begins early
after DNAofheterogeneous infection,within the first 2.5 h in both activated andquies-centcells
(Fig. 3).
Theprimer
pair
that detectscomplete
orlengthHIV-1 DNAwas nearlycomplete reverse
transcription
(M667/M661) (Fig. 3,
nfection(19). Wetested
bottom)
indicates thatfull-length
HIV-1 DNA is evident:s
may account for the within 6 hpostinfection
in activated cells butisnotfound in -scent cells with threequiescent
cells. We alsoanalyzed
these samesamples by
quiescent PBL from ausing
theprimers
specific
for the tat/revregion
(LA45/LA64)
y cloned HIV-1 strain
(Fig. 1)
which detect theapproximate midpoint
ofelongation
irusisolatesobtainedby of the minusstrand of
HIV-lJR-CSF
(nt
5964 to6079).
TheseV-seropositive patients,
primers,
whilepositive by
6 hin activatedcells,
resulted in PCR(2,
16, 17,23)
to nosignal
following
infectionof thequiescent
cellsevenat24 lates to complete the h(25).
ThesameDNAsamples
reveal thatreversetranscrip-oligonucleotide primer
tion of thenef region
of HIV-1(detected by
theAA943/
fic for the viral LTR AA946
primer
pair)
iscompleted
within 6 h in theactivated first region synthesized cells but isnotcompleted
until 9 hpostinfection
inquiescent
imerpairwill detect the cells
(Fig. 3,
middle).
Similaranalyses
with cells from a DNAsynthesized
fol- differentnormal-blood donor exhibited thissame 3-hdelay.
ion.
Therefore,
anyandThus,
while therewas nodetectable difference in initiation of ild be detected with this reversetranscription,
therewas a3-hdelay
inkinetics of the 661amplifies
sequencesearly
stages ofelongation
between activated andquiescent
genome andflanks the cells.Thus,
reversetranscription
proceeds
moreslowly
and te anamplified product
terminatesprematurely
inquiescent
cells.and gag sequences are Roleof
partial
reversetranscripts
inthe virus lifecycle.
Thecomplete
reverse tran- formation ofincomplete
reversetranscripts
following
infec-200-bpPCRproduct by
tion ofquiescent
cells appearstobea commonphenomenon
th the R/U5
(initiation-
exhibitedby
multiple
HIV-1isolates(see Fig.
2).
Quiescent
signals
well above those Tlymphocytes harboring
such structures arecapable
ofall three virus isolates
producing
viable progeny virus ifstimulatedtoproliferate by
imeDNAsamples
with amitogen
added in vitro(23,
24).
However,
we had notair resulted in minimal
formally
proven that thispartially
reverse-transcribed spe-(Fig. 2,bottom). Thus,
cies isresponsible
for therescueof progeny virusfollowing
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[image:3.612.74.307.80.238.2]1720 ZACK ET AL.
QUIESCENT STIMULATED CELLDNA(4'g)
x r
9 16 20 1 2.5 6 9 16 20 1 3
0~~~I
o
C_
° c 0 !nQ cu'1'
~*
0 :HIV-1
R/U5-140bp
Bglobin
110bp
HIV-1
net
21Bbp
.z.W
w Il:p *iF
I
*
'*
*.* :
110 bp 4
4I.
HIV-1 .
LTR/gagw- o
200 bp
3 globin e 110 bp
FIG. 3. Kinetics ofreversetranscriptioninprimarycells.Quiescentand stimulatedcells from thesamenormal-blood donorwereinfected
withHIV-1JR-CSF(2 ,ugofp241ag antigenper107 cells).DNAwasharvestedatvarious timepointswithin the first 20h,asindicated. DNA
from5 x 104cellswassubjectedto25cyclesofamplificationwiththeR/U5-, nef-, orLTR/gag-specificoligonucleotide primer. Standard
curvesof HIV-1 DNA from 10to25,000 copieswere runinparallel.HIV-1 standards contained tRNA(4>g/ml)ascarrier. ,-Globin-specific primerswereincluded in each reactiontostandardizeinputcell DNA.Dilutionsofuninfected PBL DNAwereanalyzedinparallelascontrols. DNAsamplesareidentical in all threepanels. HIindicates DNAfromcellstreated with heat-inactivated virus.
activation of the infected cell. There arethree viralgenomic
structuresthat could be found in infectedquiescentcells: the incompletely reverse-transcribed viral species, viral
ge-nomic RNAthat has notinitiated the reverse transcription
process,andasmallamountofwhatappearstobefull-length
viral DNA which islikelyformed in the few activated cells that exist in fresh PBLpopulations.We undertookaseriesof experiments to determine which of these structures was
contributing to the production of progeny virus following stimulation ofaninfected quiescentcellpopulation.
Full-length viral DNA isnot responsiblefor progenyvirus
rescue.The threepossibleviral DNAstructures inquiescent cells mentioned above are illustrated schematically in Fig. 4A. After formation ofpartial reverse transcripts in
quies-cent cells, reverse transcription that is newly initiated fol-lowing mitogenic stimulation should be inhibited by the
presence of the nucleoside analog AZT. Therefore, AZT
treatment following infection would block DNA synthesis andreplicationofviruseither fromtheincompletely
reverse-transcribed species or from de novo synthesis from viral
genomic RNA but not from completely synthesized viral DNA. Thus, anyprogenyvirus producedwould bederived from expression of previously completely reverse-tran-scribed viral DNA. Quiescent cells were infected with the
HIV-1NL4N3
isolate, and thecellswereincubated for 15hto allow formation of partially or completely reverse-tran-scribed viral DNA. After this incubation period, the cellsweredivided intotwopools,and AZTwasaddedtooneof
thepools. Both cultureswere subsequentlystimulated with
PHA and cultured in thepresence of soluble CD4(obtained from Eric Daar, Cedars Sinai Hospital) to inhibit virus spread. Supernatantsderivedfrom these cultureswere
ana-lyzed daily for virus production. Infected cells treated inthis
mannerexhibitedcompleteinhibition of virus production in
thepresenceofAZT,while cells cultured in the absenceof
AZTproduced large quantities cf progenyvirus (Fig. 4B). Thesensitivity of thep24ELISA is such that virus produc-tioncanbedetectedeveninthe absence of virusspreadand
reinfection(seebelow).Asimilarexperimentwith cells from adifferent normal donor resulted innanogram quantitiesof viruspermilliliterbyday4in untreatedcells,withnovirus
productionfrom cells treated with AZTfollowing infection. Thus, assuming that AZT is not affecting integration, the viral intermediate in this system does not appear to be completelyreverse-transcribedviral DNAbutmaybeeither non-reverse-transcribed viral genomic RNA or incomplete reversetranscripts.
Thepartialreversetranscriptininfected quiescent cellscan
function as an intermediate in the virus life cycle. We next determined whether viral genomic RNA that had not yet initiated the reverse transcription process or whether the
incompletely reverse-transcribed genomic species was the
intermediate responsible for virus rescue. A second AZT inhibition experiment was performed (schematically
illus-trated in Fig. 5A). During infection of quiescent cells by HIV-1, formation ofboth partially andcompletely
reverse-transcribedspecieswouldbe blockedbyAZT pretreatment, but viralgenomicRNAthat didnotinitiatereverse
transcrip-F- 1 2.5 6
HIV-1STANDARDS COPY #)
J. VIROL.
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[image:4.612.128.477.82.361.2]HIV-1 REVERSE TRANSCRIPTION IN QUIESCENT CELLS 1721
[image:5.612.137.497.87.626.2]/ VIRAL RNA \
|PRTIAL
RT+PHA
FULL-LENGTH DNA
INFECTED QUIESCENTTCELL
NOAZT
w VIRUSPRODUCTION
PARTIAL
RT)--C:
FULL-LENGTH DNA I
INFECTED QUIESCENTTCELL
NOAZT
+PHA
+ AZT
POST
INFECTION
RNA
INTERMEDIATE _
PARTIAL REVERSE TRANSCRIPT
INTERMEDIATE FULL-LENGTH
DNA
INTERMEDIATE-p
NO
VIRUSPRODUCTION NO
VIRUS PRODUCTION
VIRUSPRODUCTION
B
200-1504
0. ffi
100-N
0.
50±
n-+VIRUS
0
.
4
PHA
-AZT
+AZT
5
+/-AZT
DAYS POST
INFECIION
FIG. 4. Full-lengthreversetranscriptsare notresponsiblefor virusrescuefollowingmitogenicstimulation of infectedquiescentcells.(A) Schematicrepresentation ofexperimental strategy ofAZT treatment postinfection. (Top) Infected quiescentcellscontain threepotential forms ofthe HIV-1 genome: viral RNA that doesnotinitiate reverse transcription,thepartialreversetranscript, andaminuteamountof full-length viralDNA.Ifaquiescentcellpopulationharboringthesestructuresis stimulated withPHA,virusproductionensues.(Bottom)If the infectedquiescentcellistreated withAZTpostinfectionandsubsequentlystimulated withPHA(inthe presence of soluble CD4toblock virusspread),newlyinitiatedreversetranscriptionwillbeblocked.Consequently,progenyvirus should be releasedonlyfrom cellsharboring
full-lengthviral DNApriortostimulation.(B)Virusproductionfrom cells treated with AZTpostinfection. QuiescentPBL(107)wereinfected with 1.5 p.gof
HIV-lNL4-3
onday0. Sixteen hourspostinfection,the cellsweretreated for 2 h in thepresenceorabsenceof AZT(5 FxM). PHAstimulationwasthenapplied(day 1), and soluble CD4(10 pg/ml)wasaddeddailytopreventvirusspread.Culturesupernatantswereharvesteddailyandanalyzed byELISAspecificfor HIV-1p24$ag protein. VOL. 66,1992
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1722 ZACK ET AL.
RONA
INTF RAE DIAIN
DNA NIMNDIAl P
±PHA -- _
VIRUSPRODUC0TION
NO
VIRUSPRODUCT1011
QUIESCENTTCELL
t AZTPRETREATMENT
2000
-1500
Cg 1000 -(N
0-500
-0
-0 + VIRUS
-AZ
+AZT
4 PHA
+/-AZT
-.Q U~~~QIEFSCFNT
BI
LU N
z> <z
-i + + +
HIV-I nef
218bp ,44
ngloberN _ _ _ IS4_
110bp
.-~~~~~~~~~~~~~~~~~~~~~~~~..
H IV-i lat
rev-115bp V:v
DAYS POST INFECTION
FIG. 5. Pretreatment of quiescent PBL with AZT. (A) (Top)
%'CO1PY
;If
Schematicrepresentationof AZTpretreatment strategy.AZTpre---- treatmentwillprematurelyterminatereversetranscriptionin
quies-eDC-' centcells,resultinginabortedreversetranscripts. Viral RNA that O G didnotinitiatereversetranscriptionwillremain unaltered. Atatime subsequent to infection, cells arerinsed and treated with nucleo-sides to compete with any remaining intracellular AZT. P1-A
treatmentis then
applied.
Virusproduction
should resultonly
from* _ viral genomes that did not initiate reverse
transcription prior
to mitogenicstimulation,asthose thatdid initiate would be terminated by the AZT. (Bottom)VirusproductionfromAZT-pretreatedqui-* - escentcells.QuiescentPBL(2 x 107)werecultured in the presence
orabsenceof5pLMAZTfor 3 hon day0. Cellswerethen infected with
HIV-1NLA-3
(2 jLgofp24)for 2h, rinsed,and cultured for 16 h in RPMI 1640 supplementedwith 10% pooled human AB serum in - the presence or absence of AZT. Following culture, cells were rinsedthree times and cultured for 2 h in the presence of 50 ,uM nucleosides. The cellswere then split intotwo pools. Cells (107) from eachpoolwerestimulated withPHA,and 1jLg
ofsolubleCD4 per mlwasaddedtolimit virusspread(day 1).Culture supernatantswereharvested
daily
andanalyzed by
ELISA for viralp24 antigen.
*
ii.
(B) AZT pretreatment inhibits reverse transcription in quiescent PBL. QuiescentPBL(5 x106)
werecultured in thepresence(+N)
orabsenceof 50,uMall four nucleosidesor2jiMAZT(+AZT)or
both treatments (+N/AZT) for 3 hpriortoinfection with 1 jigof
HIV-1JR-CsF.Heat-inactivated(HI)controlswereinfected in paral-lel.Twentyhourspostinfection, DNAwasharvested and quantita-tive PCR was performed with 1i5 cell equivalents by using the primer pairs indicated. HIV-1 copy number standards were ana-lyzedinparallel.
J. VIROL.
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[image:6.612.102.473.102.602.2]tion would remain uninhibited. Assuming that AZT treat-ment does not directly affect the stability of the viral genomic RNA, following the removal of the AZT subsequent mitogenicstimulation would beexpected to induce produc-tionof progeny virus only from viralgenomic RNAnewly initiating the reverse transcription process, since the other genomic specieswould be aborted. Quiescent lymphocytes were preincubatedin the presenceor absence of AZT for 2 hpriortoinfection with
HIV-1NL4N3.
The infectedcellswereincubated overnight under the sameconditions in the pres-ence or absence of AZT, rinsed extensively, and then exposed to high concentrations of nucleosides to compete with any residual AZT that might remain inside the cells. Figure5B illustrates that AZT pretreatment inhibits reverse
transcription in quiescent cells and that addition ofexcess exogenous nucleosides efficiently prevents this inhibition. Theinfected cellswerestimulated withPHAandassayedfor virus production by ELISA, as described above. Infected quiescent cells treated in this manner exhibited a three- to fivefold decrease in virus production following AZT pre-treatment(Fig.5A, graph), indicatingthat the
majority
ofthe progenyvirusproduced inthe absence of AZT pretreatment was notderived fromnew reversetranscriptionfrom 'naive' viral RNA. A duplicate experiment utilizing cells from a different HIV-1-seronegative normal-blood donoryielded
similarresults: a two-and-a-half-fold decrease in virus pro-duction from AZT-treated cells.
On
the bases of these resultsand the dataindicating
that completeviral DNAisnotresponsible
formostof the virus rescue (Fig. 4), we conclude that thepartially
reverse-transcribed viral species is an intermediate that can be induced toform progeny virusfollowing mitogenic stimula-tion ofinfectedquiescentlymphocytes.
Efficiency of virus rescue following mitogenic stimulation. Our
previous
studies have shown thatquiescent
cells stim-ulatedatlater timepointspostinfection produce
less progeny virus (23, 24) than those stimulated early afterinfection. In thoseexperiments,reinfection andnumerousrounds ofvirus productionoccurredfollowingstimulation. Todeterminetheefficiency
ofrescue ofprogenyvirus from cells stimulated followinginfection inasingle-cycle infection,stimulatedand quiescent PBL were infected with areplication-defective
HIV-lJRCsF
pseudotypedwithamurineamphotropicenve-lopeglycoprotein. The entry of virus into thetwocell types wasequivalentas
assayed
byPCR(25). Quiescent
cellswere stimulated with PHA 15 hpostinfection.
Maximal virus productionwasapproximately7ng/ml
inprestimulated
PBL and 0.4ng/mlin cellsstimulatedfollowing
infection(Fig.
6).
These results indicate that rescue of virus
production by
stimulation 15 h
following
infection isapproximately
20-fold less efficient than virusproduction
inpreactivated
cells. Thus, the intermediate describedabove,
although
biologi-callyactive,is much lessefficientat
completing
the virus life cyclethan avirusinfecting
an activated cell.Partiallyreverse-transcribed viral genomescanbeinduced toreinitiate DNA
synthesis.
Thepartially
reverse-transcribed viral genome isbiologicallyactive yetapparently
inefficient ingivingrisetoaroundof virusproduction.
Toinvestigate
thisphenomenon,we
performed
amolecularanalysis.
Qui-escent cells were infected with the
replication-defective
HIV-lJRCsF
and the infected cellsweresplit
intotwopools,
one of whichwas left
quiescent.
The otherwas stimulated with PHA 15 hpostinfection,
afterformationof the interme-diate. DNAwasharvestedprior
toand 2 and4days
after the PHA wasadded,
and DNAwassubjected
toquantitative
PCR analysis byutilizing
primer
pairs
thatdistinguish
dif-8
5
4
I--(N1
c
0-O d .ea y Iy
0 1 2 3 4 5 6 7 8
DAYS POST INFECTION
FIG. 6. Quiescentorstimulated PBL(107)wereinfected with 2.5 ml ofJRCSF-AFLA stock(100 ng/ml)onday0 andanalyzedforp24 by ELISAon thedays indicated. Prestimulated cultures are indi-catedbysolidsquares.Theinfectedquiescentcellswerestimulated withPHA15 hpostinfection (on day1) andare indicatedbyopen triangles.
ferent
regions
of the viral genome. The level ofR/U5DNAin stimulatedcells iscomparable
to that in cells leftquiescent(Fig. 7), again
indicating
that de novo initiation of DNAsynthesis
fromviral RNA isnotresponsible
forinductionof virusproduction.
Primerpairs
specific
for the tat/revregion
show
only
aslight
increaseinviralDNAfollowing
stimula-tion,
as mosttranscripts
havealready proceeded
through
this
region; however,
theprimer
pair specific
for the gagregion
of the viral genome(LA8/LA9)
showsapproximately
afivefold increase in copy number in cells stimulated
follow-ing
infectioncompared
with cells leftquiescent.
Primerpairs
that
amplify full-length
viral DNA(M667/M661)
did notdetect a difference above
background
between stimulatedand
quiescent cells, indicating
thatalthough
furtherexten-sion is
induced,
thecompletion
ofreversetranscription
is very inefficient. Cells from a second normal-blooddonor,
infected with
pseudotyped
virusgenerated
in a separatetransfection,
behavedsimilarly.
This resultaccountsfor thelow
efficiency
of virus rescue seen when stimulation isapplied
15 hpostinfection (Fig.
6).
Thus,
thepartially
reverse-transcribed viralgenome can beinduced tofurther
DNA
synthesis by
amitogenic signal applied
well afterinfection,
butcompletion
of DNAsynthesis
is very ineffi-cient.DISCUSSION
Mechanism
responsible
for arrestofreversetranscription.
Thecardinal feature of retrovirus infection isconversion of
the
single-stranded
RNAgenomeintodouble-strandedDNAby
theaction of viralreversetranscriptase.
Studiesinitiatedby
Temin(20)
andsubsequently
pursued by
Temin and others(5, 7-9, 12, 13, 20, 22, 24,
26)
indicated that thestate ofactivation ofacellatthe time ofinfectionby
retroviruses influences reversetranscription
and virusexpression.
Ourprevious
study
identified aninability
of HIV-1tocomplete
thereverse
transcription
processinquiescent
Tlymphocytes
and more
completely
defined the structure of the viralon November 10, 2019 by guest
http://jvi.asm.org/
[image:7.612.340.542.82.282.2]1724 ZACK ET AL.
HIV-1
RIU5 140bp
HIV-1
tat/rev 115bp
HIV-1 gag 95 bp
DNA STDS
DAY 1 DAY 3 DAY 5 (COPY#i
o
oo
I
O(o0
~~~~~~~~~~~C
.~ w
HIV-1
[image:8.612.94.263.86.325.2]
LTR/gag-200bp
FIG. 7. Extension ofHIV-1DNA synthesis following mitogenic stimulation. Quiescent lymphocyteswere infected with Env-minus
HIV-1 pseudotyped with the amphotropic murine leukemia virus envelope (asdescribed in Materials and Methods)onday 0. On day
1postinfection, cellsweresplit intotwopools andPHA stimulation
was applied to one pool (S). The other pool of cells remained
quiescent (Q). DNAwas harvested from each pool(105 cell equiv-alents) on the days postinfection indicated and analyzed by PCR using various primer pairs. HI indicates cells treated with heat-inactivated virus in parallel and harvested onday 1postinfection.
STDS, standards.
genome in these cells (23). The current study was
under-taken to further characterize this phenomenon. Our data demonstrate thatreverse transcription initiates with similar
kinetics in both activated and quiescent T cells. Reverse transcription in mitogen-stimulated T cells is completed within6 hfollowing infectionwith HIV-1 (Fig. 3), in
agree-mentwithobservations reported recentlybyKimetal., who used Southern blot hybridization (14). Our data further indicate that in quiescent cells, synthesis of U3 DNA and
someadditional nef-encodingsequences5' tothe polypurine tract (a region synthesized immediately following the first template-switching event during reverse transcription)
oc-curs with delayed kinetics. In addition, there is decreased extensionin quiescent cells andprematuretermination ofthe
reverse transcription process. It is still not known what factor(s) causes the premature termination ofreverse
tran-scription in quiescentcells.Possible mechanismstoconsider include low concentration or sequestering of deoxyribonu-cleotides in quiescentTcells, certain structures in the viral RNA, binding of virion core proteins that halt reverse
transcription, thepresence ofreverse transcriptase
potenti-atorsin activatedcells, or inhibitors inquiescent cells. Our
previous study showedthatagraded decrease in the extent
ofreverse transcription seems to occur in these cells (23),
suggesting that there isnotadiscrete stopping pointsuchas
would be seen if RNA secondary structure or binding of
virionprotein to specific regions of the RNAwasblocking
theprogression ofreversetranscriptase. We have analyzed
reverse transcription in quiescent cells by using additional primer pairs, and these also indicated a graded decrease occurring in this process (25). The current studies do not address the possible interaction of a cellular factor(s) with the viral capsid or directly with reverse transcriptase to influence reverse transcription.
Biologic role of partial reverse transcripts. A question of critical importance is whether the incompletely reverse-transcribed structure is a viable intermediate in the virus life cycle. Our previous studies indicated that quiescent cell populations harboring this structure also harbor a labile virus intermediate which can be rescued to produce virus follow-ing stimulation. Here, we demonstrate directly that the incompletely reverse-transcribed species is likely to be an intermediate for further reverse transcription and subse-quent virusproduction. Postinfection treatment of quiescent cellswith AZTfollowed by mitogenic stimulation of the cell abrogated rescue of progeny virus. This indicates that HIV which completed the reverse transcription process prior to stimulation is not responsible for the majority of virus production, as expression from completely reverse-tran-scribed DNA should be unaffected by treatment with nucle-osideanalogs. In addition, AZT treatment of quiescent cells prior to infection resulted in minimal virus production, suggesting that the majority of progeny virus was not pro-duced by RNA that newly initiates reverse transcription after cell activation. Thus, the partial reverse transcript contributessignificantly to production of progeny virus, and, therefore, we conclude that it is an intermediate in the virus life cycle.
Lability
of partial reverse transcripts. The further DNAextension of partial reversetranscripts, as well as the rescue ofprogeny virus frominfected quiescent cells, is inefficient. This is consistent with previous results which indicate that rescue of progeny virus is less efficient with stimulation at later times following infection (24). This inefficient rescue may be due to lability of the partially reverse-transcribed genome. Our previous work indicated that the viral DNA has ahalf-life ofapproximately 1 day in quiescent cells (23). Our current resultssuggest that DNA loss is not the only cause of inefficient rescue from cells stimulated within 1 day of infection. We find that although the minus strand of viral DNA isstill present, furtherextension of the minus strand is inefficient. This could be explained by degradation of the viral RNA template, or alternatively, by lability of reverse transcriptase or other viral proteins that remain in associa-tionwith the viral genome and are required for further exten-sion tooccur. On the basis of the virusrescue and PCR analysis ofreversetranscription in a single-cycle infection, we estimate that only 5% of the initial infectious dose of virus can be rescued 15 hfollowing infection of quiescent cells.
Extrapolation of these in vitro results to the in vivo situation is informative. We previously hypothesized that the lability of the partially synthesized viral DNA could explain the lowproportion of infected cells in theperipheral blood ofinfected individuals. The even more extreme func-tional lability of the partially reverse-transcribed viral ge-nome inquiescent cells indicates that these structures would be onlymarginally involved in virus latency but would play acritical role inclearance of the virus from infected quies-cent cells. Onlypreviously activated cells or cells that are activated shortly after infection by the virus would sustain a productive infection. In the remainder of the infected quies-cent cells, viralreplication would be aborted. This scenario could explain the lowproportion of infected cells observed in infected individuals, and the resultant lowering of the J. VIROL.
on November 10, 2019 by guest
http://jvi.asm.org/
virus load wouldprovidea meansfor self-limiting infection. Suchalow-levelpersistent infection would thereby contrib-ute toprolonging clinical latency in infected individuals.
ACKNOWLEDGMENTS
We thank S. Arrigo, P. Green, G. Feuer, and W. O'Brien for criticalcommentsand W.Aft for manuscript preparation.
This work wassupported by National Research Service Award IF32 AI 08145-01 (J.A.Z.), NIH grants NS 25508 and Al 29107 (I.S.Y.C.), aLeukemiaSociety of AmericaScholarship(I.S.Y.C.), the UCLA Center for AIDS Research award (CFAR), and the California UniversitywideAIDSResearchProgram.
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