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Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle.

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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 of

HIV-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 after

infec-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 oligonucleotide

primers.

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 to

nucleotides (nt) 9048 to 9071 in the

HIV-1JRCsF

sequence, andAA946

(5'-CTCTGGATCAACTGGTACTAGC-3')

cor-responds to nt 9265-9244 (antisense). These two primers

amplify

a218-bpfragmentcorrespondingto sequences in the

HIV-lJRCSF

nef gene that proceed and overlap the U3

region

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 of

M667 AA55 M661

_0-_*_ LA8 LA9

AA943 AA946 LA45 LA64 _ _

_

_0-

- 0L3RIU5

gag 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 and

US

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 with

EcoRI,

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 (25

mM 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 a

1-min

denaturation step at

94°C

followed by a

2-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 with

HIV-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 of

gpl20.

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 x

106

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 M

MgCl2

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)

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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)

and

R/U5

(M667/AA55)

primer

pairs

wereutilized. These

primer

pairs detect different

regions

of the minus strand of viral DNA found in

partial

reverse transcripts. The

primer 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 reverse

transcription

must

proceed through

the first

ycles of PCR amplification

y

or

LTRIgag

(full-length)

template-switching

event to form DNA detectable

by

these

tracted

18 h postinfection. primers. The primer pair

LA45/LA64

amplifies a region of in all lanes. HI indicates the HIV-1 genome

corresponding

to tat/rev

coding

se-in parallel. quences

just

5' of

envelope coding

sequences. This

primer

pairwill detect furtherextension of the minus strand of viral DNA. We utilized these additional

primer pairs

to further

investigate

the kinetics ofreverse

transcription

in

quiescent

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 with

HIV-1JR-CSF

was harvested at various time th primer

pairs capable

points

postinfection

and

analyzed by

quantitative

PCR. All ormed

during

different

samples

were obtained

during

the first 20 h

postinfection,

rocessbyvirtueof their prior to progeny virus

production (23).

The

M667/AA55

me. The

resulting

virus primer

pair,

the indicator ofinitiation ofreverse

transcrip-ngle 3' LTR and addi-

tion,

revealed that reverse

transcription begins early

after DNAofheterogeneous infection,within the first 2.5 h in both activated and

quies-centcells

(Fig. 3).

The

primer

pair

that detects

complete

or

lengthHIV-1 DNAwas nearlycomplete reverse

transcription

(M667/M661) (Fig. 3,

nfection(19). Wetested

bottom)

indicates that

full-length

HIV-1 DNA is evident

:s

may account for the within 6 h

postinfection

in activated cells butisnotfound in -scent cells with three

quiescent

cells. We also

analyzed

these same

samples by

quiescent PBL from a

using

the

primers

specific

for the tat/rev

region

(LA45/LA64)

y cloned HIV-1 strain

(Fig. 1)

which detect the

approximate midpoint

of

elongation

irusisolatesobtainedby of the minusstrand of

HIV-lJR-CSF

(nt

5964 to

6079).

These

V-seropositive patients,

primers,

while

positive by

6 hin activated

cells,

resulted in PCR

(2,

16, 17,

23)

to no

signal

following

infectionof the

quiescent

cellsevenat24 lates to complete the h

(25).

ThesameDNA

samples

reveal thatreverse

transcrip-oligonucleotide primer

tion of the

nef region

of HIV-1

(detected by

the

AA943/

fic for the viral LTR AA946

primer

pair)

is

completed

within 6 h in theactivated first region synthesized cells but isnot

completed

until 9 h

postinfection

in

quiescent

imerpairwill detect the cells

(Fig. 3,

middle).

Similar

analyses

with cells from a DNA

synthesized

fol- differentnormal-blood donor exhibited thissame 3-h

delay.

ion.

Therefore,

anyand

Thus,

while therewas nodetectable difference in initiation of ild be detected with this reverse

transcription,

therewas a3-h

delay

inkinetics of the 661

amplifies

sequences

early

stages of

elongation

between activated and

quiescent

genome andflanks the cells.

Thus,

reverse

transcription

proceeds

more

slowly

and te an

amplified product

terminates

prematurely

in

quiescent

cells.

and gag sequences are Roleof

partial

reverse

transcripts

inthe virus life

cycle.

The

complete

reverse tran- formation of

incomplete

reverse

transcripts

following

infec-200-bpPCR

product by

tion of

quiescent

cells appearstobea common

phenomenon

th the R/U5

(initiation-

exhibited

by

multiple

HIV-1isolates

(see Fig.

2).

Quiescent

signals

well above those T

lymphocytes harboring

such structures are

capable

of

all three virus isolates

producing

viable progeny virus ifstimulatedto

proliferate by

imeDNA

samples

with a

mitogen

added in vitro

(23,

24).

However,

we had not

air resulted in minimal

formally

proven that this

partially

reverse-transcribed spe-(Fig. 2,

bottom). Thus,

cies is

responsible

for therescueof progeny virus

following

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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 cells

weredivided 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 #)

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0

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.1

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i

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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.Culturesupernatantswere

harvesteddailyandanalyzed 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.AZT

pre---- 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.

Virus

production

should result

only

from

* _ viral genomes that did not initiate reverse

transcription prior

to mitogenicstimulation,asthose thatdid initiate would be terminated by the AZT. (Bottom)VirusproductionfromAZT-pretreated

qui-* - 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 1

jLg

ofsolubleCD4 per mlwasaddedtolimit virusspread(day 1).Culture supernatants

wereharvested

daily

and

analyzed by

ELISA for viral

p24 antigen.

*

ii.

(B) AZT pretreatment inhibits reverse transcription in quiescent PBL. QuiescentPBL(5 x

106)

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.

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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 infectedcellswere

incubated 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 donor

yielded

similarresults: a two-and-a-half-fold decrease in virus pro-duction from AZT-treated cells.

On

the bases of these resultsand the data

indicating

that completeviral DNAisnot

responsible

formostof the virus rescue (Fig. 4), we conclude that the

partially

reverse-transcribed viral species is an intermediate that can be induced toform progeny virusfollowing mitogenic stimula-tion ofinfectedquiescent

lymphocytes.

Efficiency of virus rescue following mitogenic stimulation. Our

previous

studies have shown that

quiescent

cells stim-ulatedatlater timepoints

postinfection produce

less progeny virus (23, 24) than those stimulated early afterinfection. In thoseexperiments,reinfection andnumerousrounds ofvirus productionoccurredfollowingstimulation. Todeterminethe

efficiency

ofrescue ofprogenyvirus from cells stimulated followinginfection inasingle-cycle infection,stimulatedand quiescent PBL were infected with a

replication-defective

HIV-lJRCsF

pseudotypedwithamurineamphotropic

enve-lopeglycoprotein. The entry of virus into thetwocell types wasequivalentas

assayed

byPCR

(25). Quiescent

cellswere stimulated with PHA 15 h

postinfection.

Maximal virus productionwasapproximately7

ng/ml

in

prestimulated

PBL and 0.4ng/mlin cellsstimulated

following

infection

(Fig.

6).

These results indicate that rescue of virus

production by

stimulation 15 h

following

infection is

approximately

20-fold less efficient than virus

production

in

preactivated

cells. Thus, the intermediate described

above,

although

biologi-callyactive,is much lessefficientat

completing

the virus life cyclethan avirus

infecting

an activated cell.

Partiallyreverse-transcribed viral genomescanbeinduced toreinitiate DNA

synthesis.

The

partially

reverse-transcribed viral genome isbiologicallyactive yet

apparently

inefficient ingivingrisetoaroundof virus

production.

To

investigate

thisphenomenon,we

performed

amolecular

analysis.

Qui-escent cells were infected with the

replication-defective

HIV-lJRCsF

and the infected cellswere

split

intotwo

pools,

one of whichwas left

quiescent.

The otherwas stimulated with PHA 15 h

postinfection,

afterformationof the interme-diate. DNAwasharvested

prior

toand 2 and4

days

after the PHA was

added,

and DNAwas

subjected

to

quantitative

PCR analysis by

utilizing

primer

pairs

that

distinguish

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 is

comparable

to that in cells leftquiescent

(Fig. 7), again

indicating

that de novo initiation of DNA

synthesis

fromviral RNA isnot

responsible

forinductionof virus

production.

Primer

pairs

specific

for the tat/rev

region

show

only

a

slight

increaseinviralDNA

following

stimula-tion,

as most

transcripts

have

already proceeded

through

this

region; however,

the

primer

pair specific

for the gag

region

of the viral genome

(LA8/LA9)

shows

approximately

afivefold increase in copy number in cells stimulated

follow-ing

infection

compared

with cells left

quiescent.

Primer

pairs

that

amplify full-length

viral DNA

(M667/M661)

did not

detect a difference above

background

between stimulated

and

quiescent cells, indicating

that

although

further

exten-sion is

induced,

the

completion

ofreverse

transcription

is very inefficient. Cells from a second normal-blood

donor,

infected with

pseudotyped

virus

generated

in a separate

transfection,

behaved

similarly.

This resultaccountsfor the

low

efficiency

of virus rescue seen when stimulation is

applied

15 h

postinfection (Fig.

6).

Thus,

the

partially

reverse-transcribed viralgenome can beinduced tofurther

DNA

synthesis by

a

mitogenic signal applied

well after

infection,

but

completion

of DNA

synthesis

is very ineffi-cient.

DISCUSSION

Mechanism

responsible

for arrestofreverse

transcription.

Thecardinal feature of retrovirus infection isconversion of

the

single-stranded

RNAgenomeintodouble-strandedDNA

by

theaction of viralreverse

transcriptase.

Studiesinitiated

by

Temin

(20)

and

subsequently

pursued by

Temin and others

(5, 7-9, 12, 13, 20, 22, 24,

26)

indicated that thestate ofactivation ofacellatthe time ofinfection

by

retroviruses influences reverse

transcription

and virus

expression.

Our

previous

study

identified an

inability

of HIV-1to

complete

thereverse

transcription

processin

quiescent

T

lymphocytes

and more

completely

defined the structure of the viral

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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 DNA

extension 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.

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(9)

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.

REFERENCES

1. Adachi, A., H. E.Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986.Production of acquired immu-nodeficiency syndrome-associated retrovirus in human and non-human cells transfectedwith aninfectious molecular clone. J. Virol. 59:284-291.

2. Arrigo, S. J., S. Weitsman, J. D. Rosenblatt, andI.S. Y. Chen. 1989.Analysisofrev genefunctiononhumanimmunodeficiency virus type 1replicationinlymphoidcellsby using aquantitative polymerase chain reaction method. J. Virol.63:4875-4881. 3. Cann, A. J., Y. Koyanagi, and I. S. Y. Chen. 1988. High

efficiency transfection of primaryhumanlymphocytes and stud-iesof geneexpression. Oncogene3:123-128.

4. Cann,A.J., Y.Koyanagi, and I. S. Y. Chen. 1988.Experimental approaches to studies of primary clinical isolates of human immunodeficiency virus, p. 127-133. In R.Franza, B. Cullen, and F. Wong-Staal (ed.), Control of human retrovirus gene expression. Cold Spring Harbor Laboratory, Cold Spring Har-bor,N.Y.

5. Chen, I. S. Y., and H. M. Temin. 1982. Establishment of infectionby spleennecrosis virus:inhibition instationary cells andthe role ofsecondary infection.J. Virol.41:183-191. 6. Dougherty, J. P., R.Wisniewski, S.Yang, B. W. Rhode, and

H. M. Temin.1989. New retrovirushelper cellswith almostno nucleotide sequence homology toretrovirusvectors. J. Virol. 63:3209-3212.

7. Folks,T., J.Kelly,S. Benn, A.Kinter, J.Justement,J.Gold, R. Redfield, K. W. Sell, and A. S. Fauci. 1986. Susceptibility of normal human lymphocytesto infectionwith HTLV-III/LAV. J.Immunol. 136:4049-4053.

8. Fritsch, E. F., and H. M. Temin. 1977. Inhibitionofviral DNA synthesisinstationary chickenembryo fibroblasts infectedwith avianretroviruses. J.Virol. 24:461-469.

9. Harel, J.,E.Rassart, and P.Jolicoeur.1981.Cellcycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected withmurineleukemia virus.Virology 110:202-207. 10. Harper, M.E., L. M. Marselle, R. C.Gallo,and F.Wong-Staal.

1986. Detection oflymphocytes expressing human T-lympho-tropic virustype IIIinlymphnodes andperipheralblood from infected individualsbyinsitu hybridization.Proc. Natl.Acad. Sci.USA 83:772-776.

11. Ho, D. D., T. Moudgil, and M. Alam. 1989. Quantitation of

human immunodeficiencyvirus type 1 in the blood of infected persons. NewEngl. J.Med. 321:1621-1625.

12. Humphries, E. H., and H. M. Temin. 1972. Cell cycle-dependent activation of Rous sarcomavirus-infected stationary chicken cells: avian leukosis virus group-specific antigens and ribonu-cleic acid. J. Virol. 10:82-87.

13. Humphries, E. H., and H. M. Temin. 1974. Requirement for cell division for initiation of transcription ofRous sarcoma virus RNA. J.Virol. 14:531-546.

14. Kim, S., R. Byrn, J. Groopman, and D. Baltimore. 1989. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection:evidence fordifferentialgene expression. J.Virol. 63:3708-3713.

15. Koyanagi, Y., S. Miles, R. T. Mitsuyasu, J. E. Merrill, H. V. Vinters,andI. S. Y. Chen. 1987. Dualinfection ofthecentral nervoussystemby AIDS viruses with distinctcellulartropisms. Science 236:819-822.

16. Lee, H., P.Swanson, V.S.Shorty,J. A. Zack, J.D.Rosenblatt, and I. S. Y. Chen. 1989. High rate of HTLV-II infection in seropositive IVdrugabusersfromNewOrleans. Science 244: 471-475.

17. Pang, S., Y. Koyanagi, S. Miles, C. Wiley, H. Vinters, and I. S. Y. Chen. 1990. The structure of HIV DNA in brain and bloodof AIDSpatients.Nature(London)343:85-90.

18. Schnittman, S. M., M. C. Psallidopoulos, H. C. Lane, L. Thompson, M. Baseler, F. Massari,C.H.Fox, N. P. Salzman, and A. S. Fauci. 1989. The reservoir for HIV-1 in human peripheral blood isaTcellthatmaintainsexpression of CD4. Science 245:305-308.

19. Stevenson, M., T. L. Stanwick, M. P. Dempsey, and C. A. Lamonica.1990. HIV-1replicationiscontrolledatthelevelofT cell activationand proviral integration. EMBOJ. 9:1551-1560. 20. Temin, H. M. 1967.Studiesoncarcinogenesis by aviansarcoma viruses. V. Requirement for new DNA synthesis and forcell division. J.Cell. Physiol. 69:53-64.

21. Varmus, H., and R. Swanstrom. 1984. Replication of retrovi-ruses, p. 369-512. In R. Weiss, N. Teich, H.Varmus, andJ. Coffin (ed.), RNAtumorviruses. Cold Spring Harbor Labora-tory,ColdSpring Harbor,N.Y.

22. Varmus, H. E., T. Padgett, S. Heasley, G. Simon, and J. M. Bishop. 1977. Cellular functionsare required for thesynthesis and integration of avian sarcoma virus-specific DNA. Cell 11:307-319.

23. Zack, J. A., S. J. Arrigo, S. R. Weitsman, A. S.Go,A.Haislip, andI. S. Y. Chen. 1990. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral

structure. Cell 61:213-222.

24. Zack, J. A., A.J. Cann,J. P.Lugo,and I. S. Y. Chen. 1988. AIDS virusproduction from infected peripheralblood T-cells following HTLV-I-inducedmitogenic stimulation. Science240: 1026-1029.

25. Zack,J. A., and I. S. Y. Chen.Unpublished data.

26. Zagury, D.,J.Bernard,R.Leonard,R.Cheynier,M.Feldman, P. S. Sarin, and R. C. Gallo. 1986. Long term cultures of HTLV-III infected cells: a model of cytopathology of T cell depletioninAIDS. Science231:850-853.

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Figure

FIG.1.primerpriming.thegenome,genome5'LTRs,representanalyzeative sequences Oligonucleotide primers used in PCR analysis
FIG. 2.Three Infection of quiescent PBL wil HIV-1isolates, HIV-lNLA3(1)th
FIG. 3.withfromDNAprimerscurves Kinetics of reverse transcription in primary cells. Quiescent and stimulated cells from the same normal-blood donor were infected HIV-1JR-CSF (2 ,ug of p241ag antigen per 107 cells)
FIG. 4.formsSchematic Full-length reverse transcripts are not responsible for virus rescue following mitogenic stimulation of infected quiescent cells
+4

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