11895.full.pdf

Microbiology

Highly localized tracks

of

human

immunodeficiency

virus type 1

Nef

in

the nucleus of

cells of a

human

CD4+

T-cell line

K. G. MURTI*tt, P. S. BROWN*, LEERATNER§, AND J. VICTOR GARCIA*

*DepamentofVirologyandMolecularBiology, St. Jude Children'sResearch Hospital, 332North Lauderdale, P.O. Box 318, Memphis, TN 38101-0318;

tDepartmentofPathology, University ofTennessee, TheHealth ScienceCenter,800Madison Avenue, Memphis, TN 38163; and§Divisionof

Hematology-Oncology, WashingtonUniversitySchool ofMedicine,660SouthEuclid Avenue,St.Louis, MO63110

CommunicatedbyDavid M. Prescott, September7, 1993 (receivedforreviewJuly6, 1993)

ABSTRACT A humanT-cell line constitutively expressing thenef gene from the humanimmunodeficiencyvirus

type

1 SF2 isolate wasused to examie the distribution of the Nef protein in the nucleus. High-resolution immunogold

label-ing/electronmicroscopic studies with polydonal anti-Nef an-tibodies onnef+ and nef- cells revealed that asmallfraction of Nef is in thenucleusanditislocalizedinspecificcurvilinear tracks that extend between the nuclear envelope and the nucleoplasm. An examination of the sequence of the SF2 nef gene revealed aputative nuclear targeting sequence that was previously found in several other eukaryotic nucleoplasmic proteins. The nuclear localizationofNef suggests a potential nuclear functionfor this protein. The presence of Nef in distinct nuclear tracks suggests that Nef is transported along aspecific

pathway that extends from the nuclear envelope into the nucleoplasm. A previous study [Meier, U. T. & Blobel, G. (1992)Cell 70, 127-138] has shown that the nucleolar protein ofrat livercells (Noppl40)

shutties

from thenucleolustothe nuclear envelope on distinct tracks. The present study has suggested that the transport of anucleoplasmic protein may also occur ondistinctnuclearpathways.

The human immunodeficiency virus (HIV) is a complex

retrovirus that contains several genes that regulate virus

replicationand geneexpression(1-3).Theroleofoneofthese genes,nef,hasbeenthesubjectof a great deal of controversy

(4). Nef is encoded by a single open reading frame that

overlapswith the 3'longterminal repeatofHIVandsimian immunodeficiencyvirus (SIV) (4-6). Nefisa27- to32-kDa proteinthatismyristoylatedatits amino terminus(7-9). In some instances, a25-kDaproduct thatis notmyristoylated

has beenreported; however, thisform of Nef isnotalways

detectable(10).nefmRNAisdetectedalongwith thatcoding for tat and rev early after infection (11). The presence of

antibodies reactive with Nef inpatientsinfectedwithHIV or in macaquesinfectedwithSIV is evidence ofnef expression

in vivo(12-15).AlthoughNef is notrequired forreplication

in vitro, it is present in HIV-1, HIV-2, and SIV (4). The conservation of this gene in all three viruses canbe consid-ered an indication that nefmight have an effect on virus

replicationordiseaseprogression. Indeed, experiments con-ductedusingSIV(mac239isolate) indicatethatnef plays an

important role

in

the

development of disease in vivo (16).

A knowledge of the subcellular distribution of Nef may

provide clues to its function. Previous light microscopic

studies using immunofluorescence or immunoperoxidase techniques have found Nefprimarily in the cytoplasm (7, 17-19)associated with theplasmamembrane,

Golgi

complex,

andendoplasmicreticulum.A recentbiochemical

study

has demonstrated that a substantial fraction of Nef of HIV-1 associates with the cytoskeletal fraction ofT

lymphocytes

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and that theassociation is enhanced bymyristoylation ofNef (20). Thedistribution of Nef inthe nucleus remains contro-versial. Some recent studies using immunocytochemical techniques at the light microscopic level have described the

possible association ofNefwiththenuclearenvelope andthe nucleoplasm (18, 19, 21). In thisstudy we have attempted to resolve the question ofthe nuclear localization of Nef in human T cells using the high-resolution method of

immu-nogold/electronmicroscopy. The studies done with ahighly specificpolyclonal antiserum to Nef have revealed that a fraction of Nef islocalizedindistinct tracks in the nucleus.

MATERIALS ANDMETHODS

Cells and Antibodies. HPBALL human CD4+ T cells ex-pressing nef(HPBALL/LnefSN-S1) ortransduced with a control vector (HPBALL/LN) have been described (22). Cells were grown in RPMImedium containing25 mMHepes and supplementedwith 10% fetal bovine serum, penicillin, and streptomycin. A rabbit anti-Nef polyclonal antiserum (23) whosespecificitywasestablishedbyWestern blot

anal-ysis (see Fig. 1) was used in immunogold labeling studies. Monoclonal anti-vimentin (no. 814318) and anti-actin (no. 1378996) antibodies were obtained from Boehringer Mann-heim. Monoclonal anti-tubulin (no. MABO65) antibodies were purchased from Chemicon. The specificity of three

antibodies was established by immunofluorescence and/or

Western blot analysis.

Western BlotAnalysis. Todeterminethe specificity of the rabbit anti-Nef polyclonalantiserum,HPBALL/LnefSNor LNcellswerelysedin1%Nonidet P-40bufferandproteins

wereseparated bySDS/PAGEon a12.5%gel.Proteins were

transferredto anitrocellulosefllter, probedwith theanti-Nef antiserum (1:500 dilution), and developed with goat anti-rabbitalkaline phosphatase secondary antibody essentially

asdescribed (24).

Immunofluorescence and Immunogold Labeling.

Immuno-fluorescencewasperformedasdescribed(25). Briefly,cells were spun (Cytospin) onto glass slides, fixed with 3.7% paraformaldehyde, and permeabilized with acetone. Cells

werethenincubatedwith theprimaryantibodies [diluted 10-to20-foldwithphosphate-buffered saline (PBS)]at37°Cfor 1 hr. Afterthoroughrinsing, cells wereincubated asabove

withfluorescein-conjugatedgoatanti-rabbitantibodies(ICN; diluted 10-fold with PBS). The slides were viewed and

photographed inaZeiss IM35microscope.

Immunogoldlabelingwas doneas described(26,27)with

slightmodifications. Cellswerefixed in2.0% paraformalde-hyde/0.05% glutaraldehyde in 0.01 M sodium

cacodylate

buffer,dehydratedin

15%, 30%,

and50%ethanol for 15 min

each, and stained with2%uranylacetatein50%ethanol for 30 min. They were further

dehydrated

in 70% and 100%

Abbreviations: HIV, humanimmunodeficiency virus; SIV, simian

immunodeficiencyvirus.

ITo whomreprintrequests should beaddressed.

Proc. Natl. Acad. Sci. USA 90(1993)

ethanol for 15 min each and immersed in 1:1 ethanol/LR White resin (Polyscience) for 1 hr. Finally, theywere

em-bedded in LRWhite resin for24hrat50°C. Sectionswerecut

withadiamondknifeon aSorvallMT6000 ultramicrotome

andpicked up onnickelgrids. Forantibody labeling, grids bearingsections were floated ondropsofprimaryantibodies

(anti-Neforanti-cytoskeleton) diluted10-to20-fold in

Tris-buffered saline (TBS, 500 mM

NaCl/25

mM Tris, pH 7.6) containing 0.1%fishgelatin and0.5% bovineserumalbumin.

Incubation was carried out at 10°C for 16 hr. Grids were

rinsed with TBS and floated on gold-conjugated second

antibodies (Amersham) diluted 20-fold with TBScontaining fishgelatin and bovine serumalbumin. Incubation withthe

gold-conjugated antibodywas carried out at21°C for 3 hr.

After thorough rinsing, grids were stained with Reynold's lead citrate before electron microscopic examination. All

sampleswereexamined inaPhilips EM301 electron

micro-scopeoperatedat80kV.

RESULTS

NefExpression in HPB-ALLCells.We haveestablisheda

series ofcell populationsthatconstitutivelyexpressthenef

geneofHIV-1(SF2isolate;refs. 6 and 22). The human T-cell

line

HPBALL/LnefSN-S1

was chosen to determine the

intracellular distributionof Nef because it is a human CD4+

T-cellline susceptibleto HIVinfection. Asshown inFig. 1,

HPBALL/LnefSN-S1 cells express a 27- to 29-kDaprotein

that reacts specifically with a rabbit anti-Nef antiserum. These cellswereoriginally isolated by fluorescence-activated

cellsortingonthebasis of their low CD4cell surface levels (22). Alow levelof surface CD4 expression correlates with Nefexpression andserves as anindicator of thepresenceof

afunctionalnefgene (22, 24).

Lcalizationof Nef by Immunofluorescence. Asapreludeto

the immunogold/electron microscopy studies to map the

subcellular localization ofNef, we performed

immunofluo-rescence studies with a polyclonal anti-Nef antiserum on

nef-

(HPBALL/LN)

and

nef+

(HPBALL/LnefSN-Sl)

cells. As showninFig. 2, thecellsaregenerallyround and

containedalargekidney-shaped nucleusthatfilledmostof the volume ofthecell. Thecytoplasmwaspolar andwas most

abundantnear anindentation ofthenucleus, typicallyseenin T cells. In nef+ cells, the anti-Nef antibody revealed a

z

C,)

4)

z z

-J -1

dom

FIG. 1. Specificityofanti-Nef rabbit polyclonalantiserum. Extracts from HP-BALLcellstransduced with LnefSN or

acontrol vector,LN,wereseparatedby

32.5

SDS/PAGE

on12.5%gels,and Nefwas

detectedby

Western

blotanalysiswitha -.--Nef rabbit anti-Nef

specific

antiserumusing

=27.5

alkaline

phosphatase-labeled

goat anti-rabbit antibodies (heavychain specific)

asdescribed in the text. Thepositionof Nef onthe gel is indicated. Positions of prestained molecular mass standards are indicatedin kDa.

homogeneous labeling of the cytoplasm (Fig. 2A). Ingeneral, thedistribution of Nef seemspolar butthisevidently isdue tothepolar distribution of the cytoplasm; the thin ring of the cytoplasm surrounding the nucleus, when visible, also showedlabeling. Thenuclei, ingeneral, showed littleorno

fluorescence (Fig. 2A). However, focusing of the nucleiat different planes revealed narrow bands of fluorescence in somenuclei (Fig. 2B). These bands were seenin8ofthe 89 nuclei examined and each nucleus showed only one band. The bands are very faint and required long exposures to

photograph them. No fluorescence was observed in nefr cellsincubated withtheanti-Nefserum(Fig. 2C) orinnef+ cellsincubated withnormalrabbitserum(Fig.2D),although severalhundred of thesecells wereexamined.

NuclearLocalizaton of Nef byImmunogold Electron Micros-copy. Thetechnique used for immunogold labelingwas the

post-embeddingmethod(27). Fixed anddehydrated cellsare

embedded inawater-soluble embeddingresin (LRWhite)and

sectioned, andsectionsareincubated with primary and

sec-ondary (gold-conjugated) antibodies. We have maintained threesetsof controlstoensurethespecificity of the antibodies used. First, we performed the immunogold labeling with

anti-Nefantibodiesusing sections ofHPBALL/LN control cells(nef-). As shown in Fig. 3A, the nonspecific binding of theanti-Nefantibodies inthesecells wasnegligible. Second,

as a control for nonspecific binding of primary and

gold-conjugated secondary antibodies,weperformed immunogold labeling onnef+

(HPBALL/LnefSN-S1)

cells using normal

FIG. 2. Immunofluorescence analy-sis of Nef+(HPBALL/LnefSN-S1)and Nef- (HPBALL/LN)cells with antise-rumagainstNef.(A) Nef+ cells labeled with the anti-Nef antiserum show label in thecytoplasm. (B)TheNefW cells labeled

asin Awerefocusedonthe interior of the nucleus. Noteafluorescentbandacross

the nucleus.(C) Nef- cells labeledwith the anti-Nef antiserumwerenotlabeled. (D)Nef+ cellslabeled with normal rabbit

serum werealsonotlabeled.(x800.)

FIG. 3. Electron micrographs of controls for the immunogold labeling technique. (A)Nef- cells wereincubated with polyclonal

anti-Nefantiserum followedby gold-conjugated anti-rabbit antibody.

Labelingofcytoplasmic and nuclear componentsisnegligible. (B)

Nef+cellswereincubated withnormal rabbitantiserum followed by

goatanti-rabbitantibody conjugatedwithgold particles.No

nonspe-cifilc labelingof eithercytoplasmornucleus is evident.(C) Nef+ cells

were incubated with a monoclonal anti-intermediate filament

(vi-mentin) antibodyfollowedby gold-conjugated (anti-mouse) second

antibodies. The label is seen overintermediate filaments (IF). M,

mitochondrion; C, centriole; N, nucleus; Cy, cytoplasm. (A, x8100; B, x8550; C, x19,800.)

rabbit antiserumfollowedby gold-conjugated anti-rabbit

an-tibody.The resultsillustrated inFig.3B show that neitherthe rabbit serum proteins nor the secondary antibodies bind nonspecifically tonef+ cells. Finally, tocheck for the speci-ficity of labeling of subcellular structures by immunogold labeling method used here, nef+ cellswere labeled with an

anti-vimentin (intermediate filament) specific antibody. The intermediate filaments are readily identifiable cytoplasmic structuresthat provide convenient markers to testthe

reso-lution and specificity of immunogold labeling. The results illustrated inFig. 3C show the exclusivedistribution of label

overthe 10-nmintermediate filaments. Littleor nolabelingin the nucleus was observed in any of these controls. These studies suggest that the immunogold technique used here provides specific labelingof subcellular structures.

When T cells expressing Nef (HPBALL/LnefSN-S1)

were examined by the immunogold labeling method, the results were as follows. In afew sections of the nuclei the label due to Nefwasdetected inhighlylocalized tracks that extended between the nuclearenvelopeand thenucleoplasm. Fig. 4 illustrates these tracks in sections of three different nuclei(A-C).Thelongestof the tracks measured about7Am (Fig. 4A)and the tracksappearedtocommence/terminateat

the cytoplasmic side of the nuclearenvelope (Fig. 4B).The

tracks were seen in only4 nuclei among 100 examined and

serial sections revealed only one track per nucleus. It is

possiblethat these tracks mayoccurwithgreater frequency than that observed but that they are not detectable due to

technicalreasons.The tracksoccurin thin bands andoccupy

afraction of the total nuclear volume and, therefore, their detection would depend on their perfect alignment to the plane of sectioning. In addition to the tracks, a small amount of label is alsofound in thenucleoplasm but the nucleoliare totally free from the label. These tracks may correspond to the fluorescence bands observed in a few nuclei by the immunofluorescence method (Fig. 2B). A thorough exami-nation of sections of hundreds of nuclei of nef- cells incu-bated with anti-nef serum and nef+ cells incubated with normalserumhasfailed to reveal any nuclear labeling. In fact there is only one other instance in published literature concerning a nuclear protein that forms tracks in the nucleus. Astudy by Meier and Blobel (28) has shown that a nucleolar phosphoprotein (Noppl40) of rat liver cells shuttles on tracks that extend between the nucleolus and nuclear pore com-plexes. The Nef tracks are different from Noppl40 tracks in thattheytraversethenucleoplasm with no relationship to the nucleolus.

Inthecytoplasm(datanotshown) the label due to Nef was mostabundant near the indentation of the nucleus as was the case withcellsprocessed by immunofluorescence (see Fig.

2A);thisregioncontained mostof the cellorganelles,

includ-ing the microtubuleorganizing center, Golgi complex, and vesicles.

Dothe Nuclear TracksRepresent CytoskeletalFilaments? It has beenhypothesizedthat thetransportofRNA(29, 30) and

proteins (28)occurs onspecific tracksin the nucleus and that thesetracks maybecomposed of cytoskeletal filaments (e.g.,

microfilaments, microtubules,orintermediate filaments). To determine if any of these filaments form tracks within the nucleus ofTcells, wehaveconductedimmunogold labeling studies withanti-tubulin, anti-vimentin, and actin anti-bodies. The nuclei remained largely unlabeled with either anti-vimentin (Fig. 3C) oranti-tubulin antibodies (data not

shown).However, anti-actinantibodies showed some

label-ingof the nuclei(Fig.5). Although notasclear as Nef tracks, the label duetoactin showedapreferentialalignment in the nucleus. Additionally, short tracks of actin were seen

ex-tendingbetween thenuclearenvelope and the nucleoplasm

(Fig. 5).These resultsareconsistent withprevious biochem-ical evidence for the presence of actin in the nucleus (for

references, seeref. 28)anditsproposedrole in theshuttling ofproteinsbetween thenucleus and cytoplasm(28).

DISCUSSION

Thenefgeneis present inHIVand SIV and severalfunctions have beenascribedtoit. Thesefunctions include a negative effectonHIVreplicationin vitro(31-34),down-regulationof CD4 from the cell surface (8, 22, 24), and binding and

hydrolysis of GTP (35). Conflictingreports havealso been

published questioningthe role of Nef in each of the above functions(9, 36). Theissue of thebiologicalfunction of Nef is furthercomplicated bythefact thatisoformsof Nef exist thatdiffer either in the primaryamino acid sequence orin theirposttranslational modification (37)and these isoforms may havedifferent functions.

In an attempt to gain insights into the function of Nef,

earlier studies havefocusedonthe intracellularlocalization of Nefusing immunocytochemicalstudiesatthelight

micro-scopiclevel(7, 17-19, 21)andobtained varied results. Initial studies suggested an exclusive cytoplasmic distribution of Nef (7, 17), whereas three recent studies indicated the presence of Nef in the nucleus as well (18, 19, 21). In the present study our objective was to obtain a more precise

intracellular localization ofNef

using immunogold

labeling

techniquesat the electron microscopic level.

Thestudies described here have

important

implications

for thefunction of Nef in HIV-1 life

cycle

and for the

general

question

of the

import

of

proteins

into the nucleus.

They

have

y

Proc. Natl. Acad. Sci. USA 90(1993)

11898 Microbiology: Murtietal.

shownthatafraction of Nef ispresentin thenucleusand that it occurs in localized tracks within the nucleoplasm. The precise nuclear role of nuclear Nef remains unknown but

someprevious studies have suggested aregulatory rolefor

Nef in theHIVgeneexpression (31-34). Thelocalizationof

Nefindistinct tracksinthenucleoplasmissignificantinview of transportofproteinsintothenucleus. The firstexample of

a nuclear protein that occurs in tracks is the nucleolar

FIG. 5. Electronmicrographshowingimmunogold labelingofthe

nucleus ofNef+ cellswithamonoclonal anti-actin antibody.Inthe

nucleus the actin label shows preferentialalignment along tracks.

(Inset) Shorttrack of actin labelnearthe nuclearenvelope(NE). (x27,000;Inset, x46,800.)

FIG. 4. Electron

micrograph

showing immunogold

labeling

of thenuclei of three different Nef+ cell.Noe

te

tracks ofgold

par-~~. ~ ~ 5~E.

tidles

extendingfrom the nuclear

pt, -Tf

envelope

tothenucleoplasmin A

s andB.(A)Arrows markthetrack

~~~ ~~~~

~Of

Nef label.(B)The tracks ofNef ~~~~

~~~appear

to originate in the cyto-plasm(arrowhead).(C)Partof the Nef track at high magnification.

NE, nuclear

envelope;

Cy,

cyto-plasm.

(A,

x23,800;

B,

x30,800;

C,

x56,700.)

phosphoprotein

(Noppl40)

of rat liver

cells

(28).

Noppl40

binds

nuclear

localization signal peptides and shuttles be-tween the nucleolus and cytoplasm. Immunogold labeling studies have shown that this protein occurs in tracks that

extendfrom the nuclearenvelopetothe

fibrillar

component ofthenucleolus. These results led theauthors (28)tosuggest that

Noppl40

shuttles on distinct tracks that may be

com-posed of actin with the motive force for transport being

generated

by the putative nuclear myosinmotors.Although details ofthe mechanism ofnucleartransportremain tobe

resolved, it appears that most, if not all,

macromolecular

trafficincludingthatof mRNAmolecules (29, 30)

proceeds

ondistinct nuclearpathways.Toourknowledge, demonstra-tionofanucleoplasmic

protein

ondistinct trackshasnotbeen

reportedpreviously. TheNeftracks inthe nucleus remark-ably resemblethe

Noppl40

tracksand, likethe latter, may

representnucleartransportpathways. The fact that thereis

onlyoneNeftrack pernucleus suggeststhat Nef traversesto thenucleus alongasingle

specific

pathwayinto the nucleo-plasm. Ashas been

suggested

beforefor

Noppl40

(28),the

specificity

mayreside in the nuclear porecomplextowhich

the protein binds and to which the track is

presumably

anchored.

Previous studieshave shownthat the

eukaryotic

nucleus containsan

organized

matrixof

proteinaceous

filament that

providesascaffoldfor the

organization

andfunctionof the

nuclear components (for references, see ref. 38). Recent studies have shownthat various biochemical reactions, in-cluding DNA replication, transcription, and RNA

process-ing,take place ondefined regions ofthe nuclear matrix and the matrix may also provide tracks for the movement of proteins and RNA transcripts (28-30) in and out of the nucleus. Theidentity of proteins that compose the nuclear matrix remains tobe resolved. Some studies have implicated lamins (proteins of the fibrous lamina beneath the inner nuclearmembrane) in forming the nuclear matrix (39); lamins are related toproteins of cytoplasmicintermediate filaments and can formcoiled-coil rods, which, inturn, can associate toform higherorder structures (40). Another protein that has been detected in thenucleus (forreferences, see ref. 28) and isbelieved to composethe matrixis actin. It hasalso been speculated thatactin may provide tracks in the nucleus along whichmacromolecules move in and out of the nucleus (28). A recent study has demonstrated theaffinityof HIV-1 Nef to the cytoskeletalcomponents in vitro and in vivo (20). Allof

these findings led us to conduct apreliminary immunogold labeling study to determine if any of themajor cytoskeletal

proteins occur in tracks within the nucleus. The results

suggested that, among the cytoskeletal proteins, actin ap-pears to formshort trackswithin the nucleus.

Thelocalizationof Nef in the nucleus also led us to search for a nuclearlocalization signalin the nefsequence. Since

Nef is found in thenucleoplasm and not in thenucleolus,we focused on localization sequences on nucleoplasmic pro-teins. Previousstudies with Xenopus oocyte nuclearproteins (nucleoplasminand N1) have shown thatthese proteins share a bipartite nucleartargeting motif(41). Themotif consistsof a 16-amino acid sequence with two basic residues at the amino-terminalend, 10 "spacer"residues,andaclusterof4 basic residues at the carboxyl-terminal end (see below). Aminoacids inboth basic domains arerequired for nuclear

targeting and the transportdefect ofamutationinonedomain

is amplified by a simultaneous mutation in the other. In addition to Xenopus proteins, anumber ofeukaryotic and viral nuclearproteins contain this motif(41). The SF2 clone of Nef used here also shares thismotif(24).

Nucleoplasmin KRpaatkkagqaKKKK Thyroid al KRvakrklieqnReRRR

Nef-SF2 KRsmggwsaireRmRR

The presence of the nuclear targeting sequence may be required but notsufficient forthenuclear localization ofNef. Indeed, asubstantial fraction of Nef appears to be

cytoplas-mic associating with organelles and cytoskeletal elements,

suggesting that it may disrupthost cytoplasmicactivities. It is still possible that a fractionofNef hasanuclearfunction

that is yet to be identified.

We thank Mrs.GlenithNewberryfortypingthemanuscript.This work was funded in part by Grants CA-59195 from the National Cancer Institute and 1405 and 1559fromthe American Foundation for AIDS Research (J.V.G.), Grant FRA-373 from the American Cancer Society and Contract 17-90C-0125 from the U.S. Army (L.R.), Cancer CenterSupportGrant CA-21765 from the National Institutes of Health toSt. Jude Children's ResearchHospital,and the American Lebanese SyrianAssociated Charities.

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