Analysis of Tat function in human immunodeficiency virus type 1-infected low-level-expression cell lines U1 and ACH-2.

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JOURNAL OFVIROLOGY, Mar. 1994.p. 1993-1997 Vol.68, No.3 0022-538X/94/$04.00+0

Analysis

of Tat Function in Human

Immunodeficiency Virus

Type

1-Infected Low-Level-Expression

Cell

Lines

Ul and ACH-2

PAULA

CANNON,'

SEON-HEE KIM,2CATHERINEULICH,3 AND SUNYOUNG KIM2*

Departmentof Biochemistry, Universityof Oxford, OxfordOX]3QU, United

Kingdom';

Institute for Molecular

Biology and Genetics andDepartmentof Microbiology, Seoul National University, Seoul 151-742, Korea-; andDivision of Molecular Virology, UniversityofTexasSoulthwestern Graduate School

of BiomedicalSciences, Dallas, Texas

752353

Received 28 December 1992/Accepted 24November 1993

The Ul and ACH-2 cell lines are subclones of human monocytic and T-lymphoid cells, respectively, persistently infected with human immunodeficiency virus type 1. These cell lines harbor the viral genome but produce only very low levels of viral progeny, which can be increased by stimulation with agents such as phorbol ester andcytokines. As such, they provide an in vitro model for human immunodeficiency virus type 1 latency. Inorder toexamine the basis for their latent state, we have analyzed the activity of endogenous Tat protein in these cells and investigated theeffect on viral replication of the addition of exogenous Tat protein. We find that

Ulcells seem tohave levels of Tatprotein that are suboptimal for long terminal repeat (LTR) transcription, becausetranscriptionfrom atransfectedLTR-chloramphenicol acetyltransferaseplasmidcanbeenhanced by cotransfection of a Tatexpression plasmid. Furthermore, viral replication can be stimulated in this cell line byincubation with purified Tat protein. In contrast, ACH-2 cells are not limited for LTR-chloramphenicol acetyltransferase transcription by endogenous levels of Tat, and virus production is not increased by the additionof exogenous Tatprotein.By semiquantitative PCR analysis of viral RNA, we have demonstrated that Tatprotein caused an increase in human immunodeficiency virus RNA expression inUlcellsbut had no effect inACH-2 cells. This suggests that adifferent mechanism underlies the latent state in Ul and ACH-2 cells.

Theability ofhumanimmunodeficiencyvirus type I (HIV-1) toestablishalatent phase ofinfectionin vivo is characteristic of the pathogenesis of AIDS (19, 20). Accordingly, much interest hasfocused onpossible mechanismsfor the establish-mentandmaintenance oflatentinfectionsby HIV-1(reviewed

in references 1 and 11). Latency can arise at apreintegration

stage ifconditionsin the host cell are unfavorable forreverse

transcription and integration of the provirus, as could occur upon infection ofaquiescenthostcell (28, 29). Alternatively, latency can be established when the provirus is already inte-gratedinthe genomeof thehost cell but is unabletoproduce high levels ofviraltranscripts orprogenyvirions.

TheUl and ACH-2celllinesareHIV-1-infected monocytic

and T-lymphocytic cells, respectively (3, 8). Ul and ACH-2

cells contain one and twocopies ofthe HIV genome,

respec-tively, but they express viral RNA and proteins at very low

levels. For thisreason, these cell lines have been used

exten-sively asin vitro models of HIVlatency.Because the parental

cell linesofUl andACH-2representthe twomajorhost cell types for HIV-1 infection in vivo, they are often studied in

parallel. Viralproduction from these cells can beincreasedby

avariety ofprocesses, including treatmentwith

cytokines

and mitogens andsuperinfection byotherviruses (3, 4, 7, 8, 12, 14, 23, 24). Such studies may provide an indication ofprocesses that could lead to activation of latent virus in the body and causeprogression todisease.

RNA transcription in U1 and ACH-2 cells is at a low level and shows an aberrant pattern, with the 2-kbmultiply spliced

*Corresponding author. Mailing address: Institute for Molecular

BiologyandGenetics,Building 105, Seoul NationalUniversity, Kwan-Ak-Gu, Seoul 151-742, Korea. Phone: 82-2-880-7529. Fax: 82-2-875-0907.

transcripts predominating (21, 24). Following stimulation, there is an initial increase in the levels of the 2-kb message, followed bya later increase in the singly spliced (4.3-kb) and unspliced (9.2-kb) species (12, 21, 24). This pattern of

tran-scription is reminiscent of the early stages of a synchronous

one-stepproductive infection (16)and has led to the hypoth-esis that latency could arise because of the inability of an

infection to progress beyond an initial transcriptional phase (16, 24). In HIV-1-infected butasymptomatic patients, it has been demonstrated thatatrulylatent stateofviralexpression

does not exist but that viral transcription and replication proceedat a low level throughout this period (22, 26).

Inter-estingly, it has also been noted that there is a

qualitative

changeinthepatternof viral RNAexpression during

progres-siontothe

symptomatic

stageof thedisease,withasimilar shift

towardsthe production of

unspliced

messages (22).

While the establishment and maintenance of a low-level

infection probably depend on a combination of cellular and

viral factors, the pattern of transcription in these latent cell lines suggests in particular the involvementof the viral

regu-latory factors Tat and Rev

(11).

Transcription from the viral long terminal repeat (LTR) is strongly dependent on the presence of Tat

(reviewed

in reference

10),

so

limiting

amountsof this

key

viral

regulatory

protein

couldlead tolow

overalllevelsoftranscription. Rev hasalso been

proposed

as a candidate latency gene, because the

production

of the un-spliced genomic RNA isdependenton Rev and also because the pattern of viral RNA

expression

resulting

from mutations

in Rev is similar to that observed

during

the

early

transcrip-tional phase

(6, 27).

Although many studies have

investigated

the conditions

under which virus

production

can be increased in Ul and

ACH-2cells, little is known about

why

thesecellsare latent in 1993

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thefirst place or indeed whether they are latent for the same reasons. As aninitialanalysisof the molecular basis of latency, we have examined the role of Tat in the maintenance of low-level infections in these cells.

Tobecertain thatourACH-2and U 1 cellsdoindeed harbor latent HIV, we have examined the kinetics of HIV RNA

expression inthese cells at various time points after treatment

with50 ngof phorbolmyristateacetate (PMA [Sigma]) per ml.

Without stimulation by PMA, Ul and ACH-2 cells produce

few virus particles and a small amount of supernatant p24

(about100 to 200pg/ml),and RNA expression is at a very low level (Fig. IA and B, lane 1). (Note that the levels of

subgenomic RNAs are so lowthat the RNAbands are visible

onlyafter alongexposuretime,which is notshown here.) This

pattern of RNA expression is similar to the situation in the

earlytranscriptional phase ofaproductive infection (16). HIV RNAlevels began to risedramatically3 to 6h afterstimulation

with PMA, and the pattern of transcripts produced switched

from the multiply spliced to the singly spliced and unspliced species (Fig. 1), as has been reported by others (12, 21, 24). Thesechanges in the pattern ofRNA expression after PMA

stimulation faithfully recapitulate the transition from early to

late phases of transcription that occursduring asynchronous infection by cell-freevirus. Following PMAstimulation, there wasalsoacorresponding increasein viral p24production from the Ul and ACH-2 cells (data not shown). These results

confirm that the Ul and ACH-2 cells used in this study do indeedharbor latent HIV-1provirus, which ishighlyinducible byPMA.

An HIV LTR-chloramphenicol acetyltransferase (CAT) plasmid, p938 (17),canbeconsideredtobe a reporter plasmid for Tat function, because maximum transcription from a

TAR-containing LTRrequires thepresenceofTatprotein.By

comparing the levels of p938 CAT activity, both with and

withoutcotransfectionof a Tatexpressionplasmid, pCMVTat

(provided byD. Trono[2]),wecanobtainsomemeasureof the level of endogenous Tat activityin an infectedcell line. Ifthe

levels of Tat are high, then p938will not require supplemen-tation with pCMVTat to achieve maximum transcription.

However,ifthecellularlevels ofTat are so low as tolimit p938

expression, then cotransfection of pCMVTat will still boost

CAT activity. We can therefore use thissystem to quantitate endogenousTat activity.

Transfectionofplasmidswasby theDEAE-dextran method

(15).Tenmillioncells weretransfected by2

[Lg

ofp938and by 4

pLg

ofpCMVTatwhenrequired. Cellswereplatedat 2 x

105

cells per ml and harvested after 48 h, and CAT assays were

performed by standard procedures (2, 13). As controls for these experiments, we also transfected CEM and U937 cells,

whicharethe uninfectedparentalcelllines for ACH-2and

Ul,

respectively. When p938alone was transfected into CEM and

U937cells, therewasminimal CATactivity (Fig. 2, lanes1 and 3). However, when pCMVTat wasalso cotransfected, a

con-siderable increaseinCAT activitywasobserved because of the presenceof Tat. The same procedure was then applied to the

latently infected Ul and ACH-2 cell lines. CAT activity from the LTR construct alone was higher in these infected cell lines than in the uninfected parental cell lines because of the presence ofvirally produced Tat protein (lanes 5 and 7; note also the different assay conditions used for the different cell

lines). Even so, cotransfection of the Tat expression plasmid could boost CATactivityin Ul cells by afurther20-fold (lane

6), suggesting that, although Tatis present in U1 cells, it is at

suboptimal levels for maximum transcription from an endog-enous LTR. In contrast, LTR transcription from p938 in ACH-2 cells was not increased by cotransfection of the Tat

A

hours:

0 3 6.510 24

48 C

c*0

0

~w~:

fl

-

9.2 kb

*0,

--2

1 2 3 4 5 6

7 -28S

-

18S

B

hours:

S

0 3 6.5 10 24 48

i

0 3 6.5 10 24 48

L

- 9.2kb

---2

1 2 3 4 56 1 2 3 4 5 6

-28S

-18S

FIG. 1. Changes in the pattern of RNA expression inUI (A) and ACH-2 (B) cells on PMA induction.Ul and ACH-2 cellswere treated with 50 ng of PMA per ml, and RNA was harvested at several time points,asindicatedabove the lanes. Twenty micrograms of total RNA was electrophoresed and probed for HIV-1-specific transcripts as previouslydescribed (16). S and L are short and long exposures of the same Northern blot, respectively. Lane I is RNA fromunstimulated cells whose bands arevisible only after along exposure; lanes 2 to 6 contain RNAs from PMA-treated cells. (C) RNAs prepared from productivelyinfected H9 cells (lane 7 in panel A). Quantities of RNA weredetermined at an optical density at 260 nm, and 20

p.g

of total cellular RNA was loaded in each lane. Equivalent loading was confirmedby the intensity of theethidium bromide staining of the gel. 28S and18S indicate the positionsofrRNAs.

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NOTES 1995

*

_,

X*

S t__

*.

G

002~~-SV0

9'0

* t

1

2

3

4

5

6

7

8

0.2 3.2 0.3

90.6

1.4 30.1 12.2 8.5

L31

C1

L1 L1

U937

GEM

UI

AGH2

1

6

53

0

-cd

AC

CZ

.

1 C0

;_ 3

FIG. 2. Effect of cotransfection with Tat expression vector on

LTR-CAT activity. An HIV LTR-CAT construct, p938 (17), was

transfected intoUl and ACH-2cells and theiruninfected parentalcell lines U937andCEM, respectively,in thepresence(+)orabsence(-) ofaTatexpressionvector,pCMVTat.The resultsshowninthisfigure

representonly one ofmany independent transfections. CATactivity

wasmeasuredat37°C under the followingincubation conditions: U937 and CEM, 50 pLg ofprotein for 1 h; U1, 25 ,ug ofprotein for 1 h;

ACH-2, 10 ,ugofproteinfor 20min. Differentassayconditionswere

used with each cell line to keep the assays in the linear range for

[image:3.612.319.561.76.244.2]

quantitation. The most important parameter in each case is the magnitude ofinduction upon cotransfection with the Tatexpression

vector.C, unconverted ['4C]chloramphenicol substrate; Ac, acetylated products.

expression plasmid (compare lanes 7 and 8). This implies that the level or activity of Tat protein is not the primary factor limiting LTR expression and viral replication in ACH2 cells

andsuggests thatadifferent mechanismmay underlie the low level of virusproduction in these twocell lines.

Wenextexamined theabilityof Tatprotein toactivateviral production in these twocell lines by monitoring the effecton

viralp24production of incubation with purified Tat protein. It haspreviously been demonstrated that incubation of cells with Tat protein in the presence of protamine sulfate will allow

uptake of the protein and subsequent transactivation of an

HIV LTR(5). We hypothesized that if virus production from U1 cellswaslimited in partby insufficientlevels of Tat protein,

then the addition ofexogenousTat shouldbe able to

upregu-late virusproduction. Similarly,ourtransienttransfection data ledustopredict that ACH-2 cells wouldnot be stimulatedby the additionofmoreTat.

Ten millionUl andACH-2 cellswere incubated with 10 pLg

of purified Tat protein (provided by Alan Frankel at the Gladstone Institute)permlin I mlof medium containing 100

,ug ofprotamine sulfate per ml, as previously described (5).

Control cellsweretreated withprotamine sulfate alone. Cells wereplatedat2 x 105cellsperml, andsupernatantp24 levels

were measured atvarious times postaddition with a

commer-cially available kit (Abbott Laboratories, Chicago, Ill.). Viable cell numbers were monitored throughout to ensure that the

procedure didnotkillthe cells(datanotshown). The results of

onesuchexperimentareshown inFig.3.p24 productionfrom U1 cells incubated with Tatproteinwas increasedby

approx-imately 20-fold over that of the control population, whereas

*U1

*Ul Tat

z ACH2

E ACH2Tat

1 2 3 4

DAYS

FIG. 3. Effect of incubationwithpurified Tat proteinonviralp24

production. Ten million Ul and ACH-2 cellswereincubatedwith 10

,ugof Tatproteinin thepresenceofprotaminesulfate(UITat,ACH-2 Tat),asdescribedbyFeinbergetal.(5).Thecontrolpopulations (UI,

ACH-2) weretreated with protamine sulfate alone. Supernatant p24 levelsweremeasuredat I to4daysposttreatment.The resultsshown in thisfigurerepresentonlyone of fiveindependent experiments.

the ACH-2 cellswere not significantly induced. These obser-vationsaretherefore in goodagreementwith the data from the LTR-CAT transfections andsuggestthat the low level of virus production from U1 cells canbeboosted simply by increasing

the levelsof Tat. Incontrast,virusproduction in ACH-2 cells

appears tobe regulated bysome other mechanism.

We have also analyzed the viral RNA after addition of Tat protein. The levels of viral RNA induced by Tat were lower

than those induced by PMA and were not detectable by conventional Northern (RNA) blot hybridization analysis. Therefore, cDNAs were synthesized and amplified by PCR

with total RNAs prepared from Ul and ACH-2 cells treated with Tat. An 800-bp DNA fragment representing 1-actin mRNAwasalsoamplified from thesameRNAas acontrol for

the cDNA synthesis and subsequent amplification. We have previously reported that three major DNA bands are found with primer pair ART7-US (Fig. 4A) and that they are

approximately 400, 220, and 200 bp in length, representing cDNAsformRNAsspecifictoTat, Rev, and Nef,respectively (18). (In the gel shown inFig. 4B, the two DNAfragments of 200 and 220bp ran as one band. The specificity of each band was confirmed by blot hybridization analysis, as described

previously [18].) Untreated U1 and ACH-2 cellsproducedvery

little viral RNA, although a number of PCR analyses

repro-ducibly showed that ACH-2 cells expressed higher basal levels ofmultiply splicedmessagesthan U1cells(Fig. 4B, lanes1and

4; alsosee lane 1 ofFig. lA and B).The levels of viral RNAs

weresignificantly increased aftertreatmentwith PMA inboth cell lines (Fig. 4B, lanes 7 and 8). (Note that because the magnitude of induction by PMA is muchgreater thanthatby purified Tat protein, asmaller number ofcycleswasused for amplification ofcDNAprepared from PMA-treated cells. See the legend to Fig. 4 for details.) Treatment of Ul cells with purified Tat protein for 24 h strongly increased the level of viral RNAs, while there was no significant change in the controlculturecontainingnoTatprotein (Fig. 4B,lanes 2 and

3). On the contrary, addition of Tat protein did nothave any

significant effectonthelevel of viral RNA in ACH-2 cells(Fig.

4B, compare lanes 4and 5 with lane 6). These results again

TAT

%

Conversion

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1996 NOTES

-M Tat,Rev,Nef mRNA

~NetmRNA

II

B r-U1= rACH2i Ul AC

Tat Tat PMA

_-+ _+

-buis 0 24 24 0 24 24 24 24

:% wi;a02

.:~~~~

*--_

I

to

:H2

_400bp(Tat) -200/220bp (Nef,Rev)

1 2 3 4 5 6 7 8

~

g

. e Xi--800bp(Actin)

Tat RevNef

X O u

IX 0 S, S

|0 -. -.

06

21 n1

FIG. 4. PCR analysis of HIV mRNAs in UI and ACH-2 cells. (A) Locations of oligonucleotideprimers ART7 and US. The thin linerepresents

viral DNA with the flanking LTRs, and thick linesrepresent exons. Solidarrowheads indicate splice donors; openarrowheads indicate splice

acceptors. Nucleotidesare numbered according tothe numbering systemofRatneret al. (25). Specificities ofDNA bandswere confirmed as

previously described in detail (18). (B) PCR analysis of HIV mRNAs. TotalRNAswerepreparedfromU1 andACH-2cells priortoor24 h after

induction with Tat protein orPMA. Following cDNA synthesis with ART7, specificHIV mRNAsequences were amplifiedwith the upstream

primer US. In addition, ,B-actinmRNAwasalso amplifiedtocontrol for the cDNA synthesis and PCR reactions with thesameRNA samples. (The

oligonucleotide primers for ,3-actinwerepurchased fromClontech [Palo Alto, Calif.].) ThesamePCR conditions (30 cycles)wereusedtoamplify thecDNAs from untreated andTat-induced cells (lanes1to6), while only 25 cycleswereusedwith the cDNAs from the PMA-treated cells(lanes

7 and8). The results shown in thisfigureweretaken fromoneof fourindependent experiments, which produced virtually identicaldata.

suggested that Tat might be limiting in Ul cells but not in ACH-2 cells.

In thesimplest interpretation ofourresults, the Ul cell line

appears to direct low levels of LTR transcription because of

suboptimal levels of Tat. Transcription from a transfected

LTR-CAT reporter plasmid can be enhanced 20-fold by

co-transfection withaTat-expression plasmid.Furthermore, viral

p24 production from these cells can be boosted to a similar

degree by incubation with purified Tat protein. Incontrast,the ACH-2 cell line appears tobe abletodirect reasonable levels oftranscription fromatransfected LTR-CAT, andthiscannot be further increased by cotransfection of a Tat expression

plasmid. In addition, virus production is not stimulated in ACH-2 cellsby addition of Tat protein.

Addition ofexogenousTatprotein toUl cells may cause a

temporary increase intranscription, leadingtoincreased

pro-duction ofvirally encoded Tat. This would initiate a positive-feedback loop, and as a result, levels of other viral proteins would increase. The net result would be a shift in viral

transcription toaproductive infection. The underlying mech-anismmaintaining latency is therefore thestatusquolow-level

transcription, which could be broken by increasing levels of Tat. Insupportofthishypothesis,wenotedalarge increasein overall transcription levels in Ul cells upon induction,which

wasnotjust ashift in the types of RNA produced (Fig. IA). While some investigators have found asimilarincrease inthe overall levels of RNA after stimulation (24), others have described a more qualitative than quantitative change in the RNAproduced (21).

This description of Ul latency therefore argues against a

modelof virallatencybasedsimplyonsuboptimal levels of the

Revprotein, asbothwe andothershavepreviously suggested (16, 21, 24).Revprobably dependsonthepresence ofTat for itshigh-level expression, whereasTatexpressionisnot depen-dent on Rev. The apparent Rev- phenotype of latent cells couldalso be describedasalow-level-transcription phenotype.

Enhancing transcription byincreasinglevels of Tatallows the virus toescape from this situation andtriggersacascade into productive infectionwhich involvesother viral geneproducts.

Theunderlyingreasonfor the low level ofactivityof Tat inU1 cells, and therefore the reason for the establishment of the

latent state inthe first place, is still opento speculation.

The blocking ofproductive infection in the ACH-2 cell line isprobably also caused by subcritical levels of LTR transcrip-tion, because RNA levels are low. However, there is clearlya

fundamentally different underlying cause, because the

inte-grated proviral LTR does not behave in the same way as an

exogenous transfected LTR. The reasonable level of CAT

activity obtained with the transfected LTR argues against a

lack ofeither viral or cellularfactorsnecessary for high-level

viral transcription, yet the endogenous proviral LTR is only capable ofproducing small amountsofviral RNA in unstimu-lated cells (Fig. IB). We therefore think that ACH-2 cells probably contain reasonable levels of the cellular and viral factors necessary forefficient LTR transcription but that the

ACH-2proviral LTR somehowcannotusethem effectively. It

has been proposed that transcription from the proviral LTR

can be affected by local conditions at the site of integration, such aschromatin condensationormethylation (1, 9). In such

circumstances, the blocking of proviral transcription mayonly

beovercomeby general cellular activation, and the addition of

moreTatprotein alone hasnoeffect. Insummary,wepropose

thatthe latentstateinbothU1 and ACH-2cell linesis caused by low activity of the endogenous LTRs. The bases for this low-level activity, however, appear to be different in the two cell lines.

We thank Alan Frankel for his generous supply of purified Tat

protein.

This work was supported by the Korea Science and Engineering Foundation(S.K.)and Public Health ServicegrantAl 30897 (S.K.).

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