Differences in CD4 dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type 1.

0022-538X/94/$04.00+0

CopyrightC) 1994,AmericanSocietyforMicrobiology

Differences

in

CD4

Dependence

for

Infectivity

of

Laboratory-Adapted

and

Primary

Patient Isolates of

Human

Immunodeficiency

Virus

Type

1

DAVIDKABAT,I* SUSAN L.

KOZAK,'

KATHYWEHRLY,2 ANDBRUCE CHESEBRO2 Departmentof Biochemistry and MolecularBiology, Oregon Health SciencesUniversity, Portland, Oregon97201-3098,1 andLaboratoryofPersistent ViralDiseases, National InstitutesofAllergy

and InfectiousDiseases, Rocky MountainLaboratories, Hamilton, Montana598402

Received 15 October1993/Accepted 12January 1994

CD4 is known tobeanimportant receptor forhumanimmunodeficiencyvirustype1 (HIV-1)infectionof T

lymphocytesand macrophages.However,thelimiting stepsinCD4-dependentHIV-1 infectionsinvivoand in vitro are poorlyunderstood. To address thisissue,weproducedapanel of HeLa-CD4cellclones that express widelydifferent amounts of CD4 andquantitatively analyzedtheir infectionbylaboratory-adaptedandprimary patientHIV-1isolates.ForallHIV-1isolates,adsorptionfrom themedium ontoHeLa-CD4 cellswasinefficient and appeared to be limiting for infection in the conditions of our assays. Adsorption of HIV-1 onto CD4-positive peripheralblood mononuclear cells was alsoinefficient.Moreover,therewas astrikingdifference between laboratory-adapted and primary T-cell-tropic HIV-1 isolates in the infectivity titers detected on different HeLa-CD4 cells. Laboratory-adapted HIV-1 isolates infected all HeLa-CD4 cell clones with equal efficiencies regardless of the levels ofCD4,whereas primary HIV-1 isolates infected these clones in direct proportion tocellularCD4expression.Our interpretationis that forlaboratory-adapted isolates,abarrier step that preceedsCD4encounter waslimitingandthesubsequentCD4-dependentvirus capture process washighly efficient, even at very low cell surface concentrations. In contrast, for primary HIV-1 isolates, the CD4-dependent steps appeared to be much less efficient. We conclude that

primary

isolates of HIV-1 infect

inefficiently

followingcontact with surfaces ofCD4-positive cells,and we proposethat thisconfersaselective

disadvantage during passage in rapidly dividing leukemia cell lines. Conversely, in vivo selective pressure appears to favor HIV-1 strainsthatrequire largeamounts ofCD4 for infection.

Although CD4 is the principal cell surface receptor for humanimmunodeficiency virus type1(HIV-1),thepathwayby which it mediates infectionisnotfully understood (8,26,51). It is believed that HIV-1 binding to CD4 initiatesa series of steps that culminate in the fusion of viral and plasma

mem-branes,resulting in viral core transfer into the cytosol(28,30,

47). Bindingonto CD4 weakens associationbetween the viral gpl20 surface glycoprotein and itsgp4l transmembrane sub-unit(33-35,51), therebyexposingpreviouslyburiedregionsof gpl20 and gp4l (34, 42, 51) and enabling the gp4l amino-terminal fusion peptide to contact the plasma membrane.

CD4-dependent loosening ofgpl2O-gp4l subunitassociations apparently enables other specific cellular factors to interact with the virus-CD4 complexin amanner that isconducive to themembrane fusionprocess (2, 4, 9, 13, 15, 23).

A difficulty in understanding CD4-mediated infection de-rives from the enormous diversity of HIV-1 isolates and from differences in their abilities to infect different CD4-positive cells suchasmacrophages and Tlymphocytes (12, 16, 41,45, 51). Primary patient isolates ofHIV-1 infect normal

prolifer-ating CD4-positive T lymphocytes but not certain leukemic CD4-positive T-cell lines such as H9 orCEM, whereas labo-ratory-adapted (LA) viruses replicate in these immortalized leukemiccells(8,12,51). LAvirusisolatesarealso much more

easilyinactivated by soluble CD4 (sCD4) (3, 7, 14,21,22, 33,

34,37, 48, 49).This evidence suggests that different isolatesof

*Correspondingauthor. Mailing address: Department of Biochem-istryand Molecular Biology L224, Oregon Health Sciences University,

Portland,OR97201-3098. Phone: (503) 494-8442. Fax: (503) 494-8393.

Electronicmailaddress:[email protected].

HIV-1 interactdifferently withCD4 andpossiblywithcellular accessoryfactors.

Amajorgap inunderstanding CD4-mediated HIV-1 infec-tions and viraladaptationsandtropismsderives from a lackof informationconcerning the steps of the infectionpathway that are limiting in cell culture conditions. Knowledge of the limiting stepsof infectionmightlead tomore-focusedefforts at drugdevelopment, because inhibitingthese steps would have relatively large effects on viral replication. For the present experiments, we produced a panel of HeLa-CD4 cell clones that expresswidelydifferent amounts ofCD4,and we quanti-tatively analyzed their infection by different HIV-1 isolates. CD4-positiveHeLa cells were used for this work becausethey aresusceptibletobothprimaryandLAT-lymphotropic HIV-1 isolates (10-12). Our results suggest that for both types of viruses, adsorptionontothe cells isinefficientinpartbecause of a limitation that precedes encounter of virus with CD4. After thatstep, LAviruses efficiently infectcells even at very low cell surface CD4 concentrations, whereas infectivity of primary isolates is strongly dependent on the level of CD4 expression.

MATERUILSAND METHODS

Cells and viruses. Cell lines were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. HeLa cells(20)wereobtainedfromRobinWeiss(Institute of Cancer Research, London, England). Retroviral packaging cell lines t2 (31, 32) were used for retroviral vector amplification as previously described (6, 50). To construct the pSFF-CD4 vector,the 3.0-kbp EcoRI-EcoRI fragment of T4-pMV7 (30) 2570

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containing the entire CD4 cDNA was subcloned into the EcoRI site of pUC 1813 (25). The 1.8-kbp BamHI-BamHI

CD4-encoding fragment was then cloned into the BamHI site of pSFF (6) to obtain pSFF-CD4. This construction removed the transcription termination [poly(A) addition] signal from the CD4 cDNA, thus ensuring relatively efficient function of pSFF-CD4 as a retroviral vector. The resulting CD4-encoding virions (6, 50) were used to infect HeLa cells, and HeLa-CD4 clones were then isolated by limiting dilution cloning. Other HeLa-CD4 cell clones were previously described (9, 10). HIV-gpt virions were prepared by cotransfecting Cos-7 cells with pHIV-gpt and pHXB2-env in the presence of 10

,uM

chloroquine as described elsewhere (38, 39). Unless otherwise stated, assays of HIV-gpt infection were done on cells plated at 2 x 105 cells per 25-cm2 dish 24 h previously which were treated with 8 ,ug of Polybrene (Sigma, St. Louis, Mo.) per ml for 30 min before infection. A 2-ml volume of virus-containing culture medium was incubated with the cells at 37°Cfor 2 h, and the cultures were maintained for 48 to 72 h before trypsinizing and replating at 6 x 105 cells per 100-mm dish. The medium was replaced 24 h later with growth medium supplemented with 250 ,ug of xanthine, 3 ,ug of hypoxanthine, 4,ugof thymidine, 10 ,ug of glycine, and 150,ug of glutamine per ml, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid; pH 7.4), and 40 ,ug of mycophenolic acid perml. Drug-resistant colonies were stained with 0.1% tolui-dine blue in 30% ethanol 10 to 14 days later. Efficiencies of infection were increased approximately twofold by Polybrene pretreatment of the cells, but the Polybrene effect was the same for all HeLa-CD4 cell clones (results not shown). Rep-lication-competent HIV-1 LA isolates NL4-3 and Ala (9) and primary patient isolates (11) were assayed on the HeLa-CD4 cell clones by the focal infectivity assay method (10). Primary HIV-1 isolates were made as previously described (11). Virus was amplified by passage of undiluted culture supernatant in fresh phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells. Stocks were made from supernatant fluid taken at days 7 to 10 at the time of peak reverse transcriptase activity. Stocks used in these experiments were made at the following passages: 208K10 and 208K2 2nd passage; 13956 and 208K4, 3rd passage; 13231, 5thpassage; and 14558, 7th passage

(two of these passages were done at end point dilutions). CD4 expression assays. CD4 expression on cells was de-tected by incubating the cells with a CD4-specific monoclonal antibody, ADP318 (kindly provided by Paul Clapham and RobinWeiss, Institute of Cancer Research) (4 ,ug/ml), at 37°C for 1 h. The cells were washed with medium, incubated with a 1:500dilution of rabbit anti-mouseimmunoglobulin G-fluores-ceinisothiocyanate(Tago, Inc., Burlingame, Calif.) at 37°C for 1 h, and then they were washed with medium, fixed in cold 100% methanol, and mounted for observation by immunoflu-orescence microscopy. Quantitative CD4 analysis employed

the same CD4 monoclonal antibody diluted 1:500 and incu-bated with confluent 9-cm2 monolayers for 1 h at 37°C. The cells were rinsed with culture medium several times and were

then incubated inmedium with

[1251I]protein

A (0.2 ,uCi/ml, 2

to 10

jiCi/jig;

DuPont NEN Research Products, Boston,

Mass.) for 1 h. The cells were then washed for 5 min three times with fresh medium. The cells were dissolved in 1 ml of 0.1 NNaOH andwere counted inagamma counter. Aliquots wereassayed forprotein concentrations by the Coomassie dye method (Bio-Rad Laboratories, Richmond, Calif.).

Cell clones were also analyzed for CD4 by quantitative

fluorescence-activated cell sorting (FACS) as previously de-scribed (9). SIM.4 monoclonal anti-CD4 antibody was ob-tained from the National Institute of Allergy and Infectious

Diseases AIDS Research and Reference Program, Rockville, Md., and was used instead of OKT4A as the first antibody for indirect immunofluorescence. The second antibody was fluo-rescein isothiocyanate-conjugated goat anti-mouse immuno-globulin (Organon Teknika Corp, West Chester, Pa.).

RESULTS

HeLa-CD4 cell clones with different quantities of CD4. Retroviral vector pSFF-CD4 was amplified and expressed in packaging cells (6,50), resulting in high titers of CD4-encoding amphotropic host range virions. Infections of HeLa cells resulted in CD4 synthesis by nearly all cells in the cultures. As seen by two-stage immunofluorescence microscopy of viable cells (see Materials and Methods), certain clones had abun-dant cell surface CD4, whereas others had trace amounts (Fig. 1). For the clones that expressed the least amount, the immunofluorescent clusters of CD4 were tiny and few, and they occupied only a minute proportion of the cell surfaces (Fig. lb, upper clone). The quantity of cell surface CD4 expression in each clone was analyzed by FACS analysis (Fig. 2). Mean fluorescence intensity results demonstrated a wide range of CD4 expression (Table 1). Similar relative estimates of CD4 quantities were obtained by the

[125I]protein

A binding method (50) (results not shown).

Infection of HeLa-CD4 cells by a molecularly cloned LA

HIV-1

(HIV-gpt).

CD4 expression was more homogeneous in each clonal cell population produced by the pSFF-CD4 vector (Fig. 2) than in HeLa-CD4 clones previously prepared with the T4 pMV7 retroviral vector (9, 30). Therefore, the new clones were used for infectivity studies. Initially, we studied infection by the HIV-gpt method (38, 39). Replication-defective HIV-gpt virions contain an RNA genome encoding the gpt gene for resistance to mycophenolic acid in virions that contain gpl2O-gp4l complexes encoded by the LA HIV-1 molecular clone HXB2 (18). Infected cells grow as colonies in a mycophenolic acid-supplemented selection medium. The virus was incubated with 2 x 105cells in standard conditions for 2 h at

37°C.

At the low multiplicities of infection that occur by this assay (multi-plicity of infection of approximately 0.005) (38), the numbers of colonies were directly proportional to virus concentrations (Fig. 3A) and were the same, within experimental error, for HeLa-CD4 cells with widely differing amounts of CD4 (Fig. 3A and Table 1). Thus, cells with trace amounts of CD4 were infected as efficiently as cells with large amounts.

If the virus was completely adsorbed even by cells with little CD4, equal infections of the high- and low-expresser cell clones would be anticipated. However, evidence indicated that adsorption of HIV-gpt onto HeLa-CD4 cells was inefficient under the conditions of our assays. First, the number of infections was directly proportional to the number of HeLa-CD4 cells plated in the cultures and was the same for high (clone HI-L)- and low (clone HI-B)-CD4 expressers (Fig.3B). If 2 x 105cells of either clone were able to completely adsorb the virus, addition of more cells would not give more infec-tions. Thus, the equal infection of clone HI-L and HI-B cells was not a consequence of using a limiting amount of HIV-gpt. Second,virus-containing culture medium that had been prein-cubated for 2 h with HeLa-CD4 cultures gave a titer that was approximately equal to 60% of the initial titer when assayed on a second culture (Table 2). Furthermore, the approximately 40%loss of infectious HIV-gpt during the first incubations was causedby spontaneous inactivation rather than byadsorption, because it occurred in the absence of cells or in the presence of CD4-negative control HeLa cells (Table 2). These results

imply that an undetectably small percentage of the HIV-gpt

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FIG. 1. CD4expressionbyHeLa-CD4cells. HeLa cellswereinfectedwith the amphotropic pseudotypeofthe SFF-CD4retrovirus,and the cells were thenpassaged into fresh cultures. Thephotographsshow small colonies ofthe cells inphasecontrast(leftpanels)andcorresponding CD4-specificimmunofluorescence (rightpanels). (Aanda)Cell clone with mediumexpressionofCD4;(Bandb)twocellclones,onewithmedium expressionof CD4 and theother with low butsignificant expressionofCD4;(Candc)control HeLa cellslackinganyCD4;(Dandd)clonedHI-L

cells,which express ahigh amountof CD4. CD4expression appeared uniform within cells of each clone.

was adsorbed onto HeLa-CD4 cells during our standard 2-h incubations at 37°C. Third, HIV-gpt titers reproducibly in-creasedwhen adsorption timeswereextendedbeyond2 h. For example, in oneexperiment, the titers ofHI-Lcellswere598, 1,360, and 1,620 colonies after2-, 4-,and8-hadsorption times, respectively.

The results described above suggest that HIV-gpt infected HeLa-CD4 cells by an inefficient adsorption-limited process under the conditions of our assays. Paradoxically, however, raising CD4 levels above a low threshold had no effect on infections. To stringently test this conclusion, we mixed low

(HI-B)- and high (HI-L)-expresser HeLa-CD4 cells in a 1:1 proportion, infected them with HIV-gpt under our standard

conditions, and obtained approximately 500infected colonies afterselection withmycophenolic acid-containingculture me-dium. The infected cellswerethentrypsinizedinapool,plated

on coverslips, and labeled for CD4-specific immunofluores-cence.The ratioof HI-BcellstoHI-Lcellswasthesamewithin experimental error for the infected and selected (0.87 + 0.085), uninfected (0.96 ± 0.10), and mock-infected (0.94 + 0.10) cell populations. The errors in these numbers are the standarddeviations of the ratios. Therefore,HeLa-CD4cells

expressing low or high levels of CD4 were equally infected within the same culture. All of these results imply that the limiting step in infection of our HeLa-CD4 cell clones by

HIV-gptwasindependentof the level of CD4 expression.

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[image:3.612.132.473.81.530.2]

100

0 r-K Hn-M rn-P

C2C200tft

2100-0

Wj , < , A Wr-L

200 lo 102 103 10° 10f102 103 10° lo, 103

CW4LEVEL(FL units)

FIG. 2. FACS analysis of CD4 expression in HeLa-CD4 clones. Solid lineswereobtained with anti-CD4 monoclonalantibodySim.4as

the firstantibody. Dashed lines Werenegative controls withnofirst

antibody. The second antibody in both cases was FITC-conj'ugated

goatanti-mouse immunoglobulin.FL, fluorescence.

Infection ofHeLa-CD4 cell clones

by

replication-competent

IA and

'primary

HIV-1 isolates. A

previouis study

of three HeLa-CD4 cell clones had

suggested

that HIV-1 titers

might

correlate

positively

with

C1D4 expression

levels

(9).

Toresolve differences that

might

deperid

on HIV-1 strains or on assay

conditions,

we used the

quantitative

focal

infectivity

assay to

study

infectionof all ofourHeLa-CD4 cell clones

by

various

primary

and IA HIV-1 isolates

(Fig.

4).

The LA HIV-1 isolatesNILA-3 and Ala behaved in a manneridenticalto that of the

HIV-gpt

described above.

Only

the lowest C1D4 ex-presser, clone

iC,

showed a reduced titer with

NL4A-3,

as described

previously (9).

Above this low threshold of CD4

expression,

titersdidnot

significantly depend

onCD4levels in the HeLa-CD4 clones. In

cont'rast,

titers of the

primary

HIV-1 isolates

13956,

208K10,

14558,

208K2,

a'nd208K4 differed

by

2 to3 orders of

magnitude

thatwere

highly

correlated withC1D4

expression

levels. The data with ohe

primary

isolate

(13231)

wereintermnediate betw'een thesetwo

patterns

andwere

c'on-sequently

difficultto

interpret.

Itis conceivable that thisisolate contained

nmultiple

HIV-1 clones with both LA and

primary

isolate

phenotypes.

We also studied

adsorption

of

replication-competent

ILA

(NL4A-3)

and

primary

(208K2,

208K4, and

208K10)

HIV-1 isolates onto cells

by

using

the methods described above in Table 2 for

HIV-gpt.

The results

clearly

indicated that virus

adsorptions

in culture 'were

inefficient,

and no

CD4-specific

depletion

of viral

infectivity

fromthe

supernatant

fluid could

be detected

(data

notshown). Lack of sufficientcellnumbers in these

absorption experiments

might

explain

ourfailure tosee

significant depletion

of virus from the culture media.

There-fore,

weused 10- and 100-foldmore

CD4-positive

clone 1022

[image:4.612.65.307.81.324.2]

cellsa's wellasPHA-stimulated

CD4-positive peripheral

blood mononuclear cells

(PBMC)

inanadditional

absorption

exper-iment.

Again,

however, no

depletion

of

infectivity

could be

TABLE 1. Equal susceptibilities of HeLa-CD4 clones toinfectionby HIV-gpt

Celllinea CD4level' HIV-gptinfections

(colonies/dish)c

Assay 1 Assay2

HeLa 0 0 0

HI-R 14 702 341

HI-Q 25 403 276

HI-F 30 936 503

HI-B 34 545 454

HI-A 52 838 318

HI-K 71 873 458

HI-G 71 852 592

HI-Md 75 492 295

HI-P 128 716 487

HI-J 234 662 371

HI-L 300 737, 598, ands51e 256

aThe cellswere either controlHeLa cells, which lack CD4, or clones of HeLa-CD4cells.

bMeanfluorescence intensity inarbitraryrelative units was determined by

FACS analyses (9) for each population.Abackgroundvalue of 11 was found for

normal HeLa cells, and thisnumberwassubtracted from thevalue foreach populationtogive the valuesshown. By aradioactivityassay for cell surface CD4

(50), theH9line of standard CD4-positive Tlymphoblasts (40),which have approximately1.7 x I04CD4 moleculesper cell(27),wasfound to have an amountofCD4 percell similartothat of our cloneHI-Qcells.

CVirusfor the HIV-gptassays waspreparedfromCos-7cells cotransfected

with pHIV-gpt and pHXB2-env in the presence of 10 R1M chloroquine as

described in Materials and Methods. The results are the numbers of drug-resistant coloniesonthe 100-mm-diameterdishes.

dHI-M grew moreslowlythan HeLa cells and the otherCD4-HeLaclones.

'The numbers listed inseries werereplicateassays.

demonstrated (Fig.5). These results indicated that as a result

of the inefficient

absorption

process, the amounts of HIV-1 infectivity detected on both HeLa-CD4 cells and PBMC were verylow percentagesof the totalinfectivity in the viral stock.

DISCUSSION

Theresults of the presentexperimentsindicatethat primary and LA HIV-1isolates differ intheir interactionswithcellular CD4duringthe infection process. Infectivitytiters ofprimary

isolates weredirectly influenced by the level of CD4 expressed on HeLa cell clones (Fig. 4). This result implies that the

virus-CD4

interaction isacritical limiting step for infection of

thesecellsby primaryHIV-1 isolates,which is consistent with

the

important

role of CD4as areceptorfor HIV-1.Incontrast,

infectivity titers of LA HIV-1 isolates were not significantly

influencedby the level of CD4 expressed on thesesametarget cell populations, and it was unclear from this result what

exactlywas the limiting step for infection by the LA HIV-1 isolates. However, several lines of evidence suggest that

ad-sorption ofLAviruses from the medium onto the cells is a

limitingprocess ininfection. Duringthe infection step under the conditions of our assays, infectious HIV-1 was removed very inefficientlyfromthe medium (Table 2andFig. 5). This

couldnotbe dueto asaturation of receptorsbyvirusor free

gpl20, because infections occurred at low multiplicities and virustitersincreaseddirectlyin

proportion

tovirus concentra-tions(e.g., Fig. 3).Themost

likely

meansfor theconcentration

of virus in the mediumtohavea

directly

proportional

influence oninfectionofHeLa-CD4cellsis

during

the step of

adsorption

onto the cells

(1,

5,24,

29,

53).

Ifinfectionswere limited

by

saturation or depletion of factors atany

postadsorptive

step, increases in the virusconcentrations would not increase the titers obtainedinourassays.

Thus,

we

interpret

ourresultsto suggestthat

adsorption

of HIV-1 onto the cells increases in

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[image:4.612.325.564.100.245.2]

1200

a

0 0

IL

800

400

0 0.2 0.6 1

B

* HI-L

* --U--Hi-B ,

A HeLa

0,

A 2 3 eeAe A A

1 2 3 4 5 Relative Virus Concentratlon Cell Number (10 5)

FIG. 3. HIV-gpt infection is proportionaltovirusconcentration and cell concentration. (A) Virus-containingmediumwasseriallydiluted 1:2 with mock-transfected Cos-7-conditioned medium,andthe dilutionwasusedtoinfect cells inourHIV-gptassayasdescribedinMaterialsand Methods. The number of drug-resistant colonies ofhigh-expressing CD4-HeLa HI-L clone and oflow-expressing CD4-HeLa clonesHI-Band HI-Ris proportionaltothe relative virus concentrationand is 0intheCD4-negativeHeLa cells. Each datumpointis theaverageofduplicate

assays.(B) HeLa cells and CD4-HeLa clonesHI-Land HI-Bwereplatedatvarious concentrationsin25-cm2 dishesandwereinfectedwithour

HIV-gpt. The infected culturesweretrypsinized48 hlater,andcellsequaltothe number of cellsatinfectionwere platedin100-mm-diameter dishes andweresubjectedtoourmycophenolicacid-supplementedselection medium(seeMaterials andMethods). Drug-resistantcolonieswere

stainedand counted after 10days.

direct proportiontotheconcentration of virus in the medium and that this results in a corresponding increase in virus titers in our infectivity assays. In this circumstance of

adsorption-limitedinfection, we anticipated that infection woulddepend

on the concentration ofadsorptive sites on the cell surfaces. For primary HIV-1 isolates, these limiting adsorptive sites appeared to be CD4. However, for LA HIV-1 isolates,

infec-tionrequired CD4-positivetarget cells butwas not limitedby

CD4.

These results areconsistent withamultistagemodelof virus attachment and entry into cells. First, virusmustdiffusetothe cellsurfaces prior topotential encounter with CD4. Diffusion oflarge substances suchasviruses is slow andcanbelimiting

for some infections (1, 5, 24, 29, 43). Other steps preceding

potential interaction with CD4 could involve barrier or pre-requisite reactions such as passage through the exoskeletal meshwork, release ofhydration, and the overcoming of elec-trostaticrepulsion(53).Inaddition,theremightbe features of thecell surfaces such as weakbindinginteractions that entrap HIV-1 andhold it long enough for subsequent efficient asso-ciation with CD4. Analogously, attachment onto accessory cell surface components facilitates adsorption of herpes simplex

TABLE 2. Inefficientadsorptionof HIV-gpt onto HeLa-CD4cellsat37°C

Cells used for Firstinfection Cells used for Second infection first infectiona colonies/dish infection colonies/dish

None HI-L 887

HeLa 0 HI-L 812

HI-B 1,401 HI-L 860

HI-R 2,210 HI-L 1,000

HI-L 1,770 HI-L 923

aA2-mlvolumeofvirus-containing mediumwas incubatedwiththecellsfor

2h at37°CasdescribedinMaterialsand Methods.Afterthefirst infections,the samemediumwastransferred for2 hontoculturesthat containedHI-Lcellsthat

hadbeenplated and treated in a manner identicalto thatused forthefirst

infections.The same medium was usedforeachround.Eachrow indicatesthe samemedium.Thus,themedium preincubatedinanemptyflaskandlaterused toinfectHI-Lcellsresultedin887drug-resistantcolonies afterselection.

virustype1 (19, 44). Second, the virusmust encounterCD4in

asterically appropriatemannerand formareversibleunivalent encounter complex (29, 43). Third, other CD4 molecules presumably interact with the virus to form a multivalent

irreversiblecapturecomplex (29,43) that is committedto enter postadsorptionsteps ofthe infectionpathway.

From theperspective ofourresults withLA HIV-1isolates,

the stepspreceding CD4 encounterwould obviously be inde-pendent of CD4 expression levels. Moreover, if the CD4 receptordensitywereaboveacertainlowthreshold, formation ofencountercomplexes and their conversion into irreversible capture complexes could also behighly efficient, as has been demonstrated experimentally for phage lambda adsorption ontoEscherichia coli (5, 43). Efficient bindingto CD4 could alsoensue ifthevirus were held evenbriefly byan accessory

factoron the cell surfaces. Thus,our resultswith LA HIV-1 isolates suggest that the preencounter reactions are slowbut

that capturecomplexes thenform efficientlyeven atvery low

cell surface densities of CD4. In contrast, our results with

primary isolates of HIV-1 suggest that their attachment to CD4must be relatively inefficient. Consequently,we propose

thatprimary HIV-1 isolates formencountercomplexes with a

lowefficiency that is highly dependenton CD4concentration

and that these encounter complexes might also frequently dissociatewithout conversiontocapturecomplexes.

Thebiochemicalexplanationforthesedifferences is unclear; however, some clues maybe provided by previous studies of

interactions of HIV-1 with sCD4.Although sCD4 binds with approximately equal affinitiesto gpl20 purified fromLAand primary virus isolates, recent evidence suggests that sCD4 binds to gpl20 of intact virions with lower affinity in primary isolatesthanin LAisolates(22, 34). Furthermore, LAHIV-1 isolatesareinactivated byexposuretoconcentrations of sCD4 that are 100- to 1,000-fold lower than those required to inactivate primary isolates (14, 21, 33). The kinetics of virus bindingandentryintoCD4-positiveTcellsappearstobemore

rapid forLAthan forprimary isolates (17, 46). These

differ-ences cannot be ascribed to amounts of gpl20 per virion,

because LA viruses spontaneously shed gpl20 and have a

relatively low gpl20 content (52). These observationson the A

1200 a 0 0

o

800

400

0

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[image:5.612.132.485.80.233.2]

5.0-

4.0-i

3.0-cm

-Cs

_ 2.0-0

O

--

5.0-> 4.0-Ic

3.0-2.0- 1 I

1.0

-1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0

CD4

Expression(log mean FL)

FIG. 4. Comparison of HIV-1 titer versus CD4 expression using two LA HIV-1 isolates (NL4-3 and ALA) and six primary AIDS

patient-derivedHIV-1isolateson aseries of HeLa-CD4clones. The individual clones can be identified by their CD4 expression expressed as the

logof themeanfluorescence units(FL)onthe horizontal axis: clone 1C, 0.7; clone 6C, 0.9; HI-R, 1.15; HI-Q, 1.40; HI-F, 1.48; HI-B, 1.53; HI-A,

1.72;HI-G, 1.85; HI-K, 1.85; HI-M, 1.87; HI-P, 2.11; HI-J, 2.37; HI-L, 2.48; clone 1022, 2.68. Error bars indicate standard errors of geometric meansbasedonfoci counted at several different dilutions. The experiment was repeated three times with similar results. The solid lines are

least-squarebest fitstothe datum pointsfor all clones, except the two with the lowest CD4 contents; the latter two datum points are indicated by opencircles.This exclusionwasdone becausethesepoints deviatesomewhat from the curves in a manner consistent with the fact that infections are0for controlHeLacells, and because these cell clones haverelatively heterogeneous CD4 expression on cells of the clonal populations. The

correlation coefficientsfor these computer-drawn best-fit lineswere asfollows:Ala, 0.13; NL4-3,0.25; 208K2, 0.92; 208K4, 0.89; 14588, 0.84;

208K10, 0.81; 13956, 0.84; and 13231, 0.42. Thisstronglysupportsthe conclusionthat titers correlate with CD4 levels for virus strains 208K2, 208K4,

14588,208K10,and 13956.Theslopes ofthebest-fitlines are also close to 1 (0.74 to 1.48) for all of these last viruses, consistent with a direct

relationshipof titers with CD4. The results suggest no relationship of titers with CD4 levels for the LA viruses. The primary virus 13231 data are

scatteredand areof uncertainsignificance (see text).

interactions of sCD4 with LAversus primary HIV-1 virions

might reflect similar differences in the interactions of these viruses with cellsurfaceCD4. If so,thismight account for our

dataon the differences in CD4 dependencefor infectivity of

primaryand LAviruses.Forexample,LAviruses withahigher CD4bindingaffinity andmorerapidkinetics of entry might be lessdependent onthe level of cell surface CD4,andprimary

isolate virions with a lower CD4binding affinity and slower

kineticsof entrymightbemoredependentonthe level of cell

surface CD4.

The HIV-1envelopeproteinis knowntobeimportantin cell

binding and entry and probably is the main viral structure

influencing virus-CD4 interactions of LA andprimaryHIV-1 isolates. HIV-1 envelope is also known to influence cell tropism, leading to differences between

T-cell-tropic

virus

strains,whichreadily infect HeLa-CD4 cells, and macrophage-tropicvirus strains,which do notinfect HeLa-CD4 cells

(12).

Previous studies ofvirus-CD4 interactions using sCD4

com-pared

macrophage-tropic primary

isolates or clones with

T-cell-tropicLAisolatesorclones

(14).

Inthese

experiments,

the V3 region of the envelope gene from a

macrophage-tropic

clonewas shown to influence both macrophage

tropism

and resistancetosCD4

(36).

The present data withLAand

primary

HIV-1isolates,whichwereall T-cell

tropic,

demonstrate that thephenotypicdifferences between virus-CD4interactions of

LAand primary isolates are not solely due to differences in macrophage or T-cell tropism. Thus, it seems likely that the

envelope regions such as V3, which are involved in the

specificityof celltropism,maybedifferent fromthose involved in acquisition of the LA phenotype. For example, in our

previousstudy, the V3 region of primary isolate 14558 gave the LAphenotype ofhighinfectibility for leukemia T cells when it was inserted in the genome of an LA virus (clone NL4-3),

whereasthis same V3regionwasassociated with the

primary-isolate phenotype when in its original 14558 genome

(12).

Studies are in progress to determine whether these non-V3 differences alsocorrelatewithchangesinsensitivityto neutral-izationbysCD4.

LA isolates differ from primary isolates in their ability to

spread efficientlyinrapidly dividing leukemicT-cell cultures. HeLa-CD4 or T-cell-tropic primary isolates often appear to

enter leukemic T cellsinitially, but subsequently

they

fail to

spread rapidly enough to keep pace with the

dividing

cells

(1Oa).Asaresult,theseinfected cell cultures

usually

clear the infections byoutgrowing the virus. Our resultswould

predict

that in mixed virus infections, more

rapidly

spreading

LA HIV-1variants would beselectedby

growth

in

dividing

human leukemic Tlymphocytes. In contrast, passage in PHA-stimu-latednormalPBMC,which

replicate

more

slowly,

might apply

less pressure selecting for LA HIV-1 variants.

Thus,

PBMC

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[image:6.612.129.492.82.336.2]

4.00

-NL4-3 208K10

3.50

3.00-None 2x104 2xl05 2xl 06 None 2x104 2x105 2x1

Number ofAbsorbingCells

FIG. 5. Failure ofadsorptionwithlargenumbers ofCD4-positive

HeLacellsorPBMC todepleteinfectious HIV-1 fromcell-free virus

stocks. A 0.2-ml volume of a 1/10 dilution of each virus stock was incubated for 2 hat37°Cwith various numbers ofCD4-positiveclone

1022HeLa cells (0) orwith PHA-stimulated PBMC (A).The cells

were thenpelleted, andtitersweredeterminedforsupernatantfluid

forinfectivityon 1022cellsasusual.

cultures might not result in selection of the LA phenotype capable of efficient replicationin leukemic T cells.

Conversely, inseropositive patients,thereappears tobeno selection for the LA phenotype, at least prior to onset of

advanced disease. Inpart,thismaybebecause normal

nonma-lignant CD4-positive lymphocytes are not a rapidly dividing

population. One consequence is that the T-cell-tropicvirus

populationsinvivorequire highamounts ofCD4 forinfection

of cells. Because these viruses probably adsorb onto cells

inefficiently, they might also be prone to disseminate in vivo and to escape local immune responses. It isconceivable that

selection of these viruses might account for the increasing killingof CD4-positive Tlymphocytes duringtheprogression

ofAIDS. The isolatesthatwestudied herewereobtained from

bothsymptomaticandasymptomatic patients. Further studies

will be required to determine howthese viruses are selected

and tounderstandtheir roles in disease.

ACKNOWLEDGMENTS

Supportedinpart byNIHgrantsCA25810 andAI28572.This work

was initiated by D.K. during a sabbatical leave with Robin Weiss

(Instituteof CancerResearch)thatwassupported byaFogartySenior InternationalFellowshipfrom theNIH.

We aregrateful to RobinWeiss and his coworkers forgenerous

warmhospitality and forstimulatingdiscussions. PaulClapham

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