U84:ZJOBS195XA SIBY

m ~a/n~of

Biomedical Science

Original Paper

J Biomed Sci 1998;5:203-210 Received: December 24, 1997 Accepted: February 19, 1998

Expression of HIV- 1 n e f i n Yeast ...... ........................................................................ C a u s e s M e m b r a n e P e r t u r b a t i o n iiiii~ii~it~iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iiiiiiiiii i i iiiiii iiii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii; and Release of the Myristylated Nef

Protein a Biomolecular Research Institute and b CSIRO Molecular Science,

Parkville, Vic., Australia

m Key Words Acquired immunodeficiency

syndrome Human immunodeficiency virus Nef protein Myristylation Membrane permeabilisation Saccharomyces cerevisiae Yeast

Abstract The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV- 1 infected cells. **********************

m

Genomes of primate lentiviruses contain nef, a highly conserved gene that overlaps the 3' LTR. Recent evidence suggests that the nefgene product, a myristylated protein of 205 amino acids, is involved in AIDS pathogenesis since mutations in HIV-1 nefleading to the loss of Nef correlate with a lack of disease progression, even though HIV-1 infection persists [11, 30]. Genetically engineered point and deletion mutations in molecularly cloned SIV- mac nefalso implicate Nef in AIDS pathogenesis in rhe- sus monkeys [27].

It is well recognised that Nef has a diverse range of activities within the cell [reviewed in ref. 18, 19, 23, 32] and much of its activity occurs through Nef's association with cell membranes. Nef can have multiple locations within the cell, including localisation to nuclear [28, 29, 31], plasma [13, 25, 26] and various cytoplasmic mem-

branes [22, 39, 45]. During the biogenesis of the HIV-1, assembly of the virion takes place at the plasma mem- brane and full-length myristylated Nef, along with the plasma membrane, becomes incorporated in the envelope of the virion where it enhances viral replication [7, 40, 43]. In addition, Nef has an association with various membrane proteins such as protein kinase C [8], [~-COP [5], and serine-threonine kinase [6, 42, 44] where its mem- brane localisation appears important for these interac- tions and subsequent effects.

In a yeast expression system, we reported that Nef, although lacking a recognisable secretion signal, was re- leased from the cells under conditions of stress [35]. While yeast may appear to be an unusual experimental system for the investigation of the mechanism of action of HIV- 1, the viral proteins may act in a similar manner in yeast and

KARG E R Fax+41 61 306 12 34 E-Mail karger@karger, ch www.karger, corn

© 1998 National Science Council, ROC S. Karger AG, Basel 1021-7770/98/0053-0203515.00/0 This article is also accessible online at: http://BioMedNet.com/karger

Ian G. Macreadie, PhD Biomolecular Research Institute 343 Royal Parade, Parkville, Vic. 3052 (Australia) Tel. +61 3 9662 7299, Fax +61 3 9662 7301 E-Mail Ian'Macreadie@m°lsci'csir°'au

U84:ZJOBS195XA SIBY

m

m

m

h u m a n cells. I n d e e d , t he re is e v i d e n c e s u p p o r t i n g re lease

o f N e f in H I V - 1- infec ted pa t i en t s . F o r e x a m p l e , the re a re

n u m e r o u s r epor t s o f a n t i b o d i e s to N e f a p p e a r i n g ear ly

a f te r HIV-1 in fec t ion [ r ev iewed in ref. 35], a n d the C-

t e r m i n u s to N e f has been o b s e r v e d on the sur face o f

HIV-1 in fec t ed cel ls [15]. Also, in a v a c c i n i a exp re s s ion

sys tem, N e f was re l eased in to the cu l tu re m e d i u m [21].

There fo re , in th is s tudy we have fu r the r e x a m i n e d the

effects o f N e f on yeas t cells. W e have d e t e r m i n e d tha t N e f

causes m e m b r a n e d i s r u p t i o n wh ich resul ts in loss o f

m e m b r a n e in to the ex t r ace l lu l a r m e d i a . T h e re l eased N e f

is m y r i s t y l a t e d a n d can be r ead i l y i so la ted f r o m the cul-

tu re b y d i f fe ren t i a l c en t r i fuga t ion a n d gel f i l t ra t ion .

A l ike ly r eason for the m e m b r a n e - p e r t u r b i n g ac t i v i t y

o f N e f is a s equence a n d s t ruc tu ra l s im i l a r i t y be tw e e n bee

v e n o m m e l i t t i n wh ich has these p r o p e r t i e s [4, 9] a n d the

19 a m i n o ac id r e s idues o f Nef . Syn the t i c p e p t i d e s con 'e -

s p o n d i n g to th is reg ion o f N e f h a v e m e m b r a n e p e r t u r b i n g

ac t i v i t y in h u m a n cells, yeas t cells, bac t e r i a l cells a n d ar t i -

f ic ia l m e m b r a n e s [9, 10, 36]. Th i s a c t i v i t y for N e f m a y be

i n v o l v e d in t he d e a t h o f b y s t a n d e r cells, o r in the v i ra l l i fe cycle.

Materials and Methods

Expression ofHIV-1 nef in Yeast The nefgene of HIV-1 was derived from the pNL4-3 infectious

molecular clone [1]. The Saccharomyces cerevisiae transformants producing Nef originate from host strains DY150 (MATa ura3-52 leu2-3,112 trpl- 1 ade2-1 his3-11) and DMY 1 O0 (hJA Ta/MA Ta ura3- 52/ura3-52 leu2-3,112/leu2-3,112 trpl-1/+ ade2-1/+ his3-11/+) and have been described previously [33-35]. The yeast expression vectors, pYEX-BX and pYEX-S1 (AMRAD, Melbourne), employed for expression of nefutilised the copper-inducible CUP1 promoter and the constitutive PGKpromoter. Yeast cells were grown in YEPD (1% Difco yeast extract, 2% peptone, 2% glucose) or minimal medium (0.67% Difco yeast nitrogen base without amino acids, 2% dextrose) at the stated temperatures.

For copper induction of cells grown in minimal media, CuSO4 was added to the level of 0.1-0.2 raM. A higher level of 1.0 mMwas used for cells grown in YEPD. To heat stress cells, cultures were pre- grown in YEPD at 28°C and cell cultures were shifted to 37°C for 4 days. In one construct, pYEX-BX.Nef27, Nef was produced in its full length form from its own start codon, while in the second con- struct, pYEX-BX.Nef25, Nef20-206 was produced with translation commencing from the second AUG codon of nef

All yeast cultures were batch cultures. After the first day in rich medium, or the second day in minimal medium, cultures were in the stationary phase of growth.

Nef Purification and Western Blotting Following growth of yeast cells in YEPD, the oily layer on the

centrifuged yeast cells was selectively washed off the cells into water. The suspension was centrifuged again and the procedure repeated to

minimise cellular contamination. The oily pellet was then dissolved in 5 ml of 8 M urea in phosphate-buffered saline (PBS) containing 10 mM dithiothreitol (DTT) and 10 mM Pefabloc TM (Boehringer Mannheim, Germany). The solution was mixed at 4 °C for 2 h then centrifuged. The supernatant was run on a column (26 x 600 mm) of Superdex TM 75 (Pharmacia Biotech, Uppsala, Sweden), equilibrated in 8 gturea/PBS/2 rm'~l DTT/1 m ~ Pefabloc TM, at 2.0 ml/min. Frac- tions were analysed by SDS-PAGE and the gels stained with Coo- massie blue. Samples were also blotted on to nitrocellulose mem- branes and probed with a monoclonal antibody that was specific for the C-terminus of Nef (Intracell, Cambridge, Mass., USA) and used for detection of Nef (including Ne~:)0_206) described here. Antibody binding to the Nef was reacted with sheep anti-mouse Ig conjugated to horseradish peroxidase (Silenus, Melbourne, Australia) and the complex visualised by reaction with the substrate, 3,3',5,5'-tetrame- thylbenzidine [38].

The Nef-containing fractions were pooled and dialysed succes- sively against 4 Mm-ea in PBS, 2 Murea in PBS and finally, 50 mM Tris, pH 8.0 all at 4 o C. 30 gl of fractions 7-42 were denatured with 10/.tl gel sample buffer containing 10% SDS and 15% 13-mercapto- ethanol at 100°C for 5 min and then run on a 12% polyacrylamide gel in SDS running buffer. The gel was stained with Coomassie blue. For Western analysis, 30 gl of the fractions from the Superdex TM col- umn were treated as above and run on a 12% polyacrylamide gel as described above. The proteins were electroblotted on to nitrocellu- lose, the blot blocked with 1% casein in PBS and probed with a monoclonal antibody to the C-terminus of Nef as described above.

Myristylation of Nef To incorporate tritiated myristic acid into yeast cells, transfor-

mants were grown in 10 ml minimal selective media containing 0.1 n ~ l CuSO4 and 100 gCi [9,10(n)-3H] myristic acid (Amersham, UK). Culture supernatants were run on duplicate SDS-polyacryl- amide gels, one of which was stained with Coomassie Brilliant Blue, and the other electrophoretically transferred to nitrocellulose. West- ern blots were probed with a mouse mAb specific to the C-terminal region of Nef. Myristylation was assessed after fluorography of the same filter. Fluorography was performed by spraying the filter with ENH3ANCE (Du Pont NEN, Boston, Mass., USA) followed by expo- sure to Kodak XAR5 film for 3 weeks.

Immuno-Electron Microscopy of Yeast Cells For expression of nefin yeast fbr electron microscopic analysis,

CuSO4 was added to cells in log phase growth and cultures were maintained for the times indicated. Yeast were prepared for resin embedding as follows. Yeast were fixed in 0.04 M KPO4, pH 6.7, 0.8 M sorbitol, 1 mM MgC12, 1 mM CaC12, 3 % formaldehyde (fresh- ly prepared) for 60 min before being recovered by gentle centrifuga- tion. Cells were then washed and gently re-centrifuged in 0.5 Msorbi- tol, 0 .04gi K P O 4 , pH6.7, then 0.257vl sorbitol, 0 .04gf KPO4, pH 6.7, then 0.04MKPO4, pH 6.7, before being resuspended in 1% NaIO4 for 10 rain at room temperature. Cells were then washed at room temperature in Milli-Q water, followed by treatment with 50 mM NH4C1 for 15 min, and then Milli-Q water. The cells were then dehydrated in increasing ethanol concentrations, finishing with 100% ethanol. Pelleted cells were then resuspended in 2:1 ethanol/ LR White (Alltech, Homebush, Australia) for 1 h at room tempera- ture with agitation. This was repeated with a 1 : 1 mix, then finally two changes of 100% LR White. The sample was cured in LR White at 47 o C overnight before sectioning.

204 J Biomed Sci 1998;5:203-210 Macreadie/Fernley/Castelli/Lucantoni/ White/Azad

U84:ZJOBS195XA SIBY

m Immuno-gold labelling was performed on sections of embedded yeast of 70 nm thickness as follows. The grids were floated on to water for 30 s, then floated on to saturated sodium metaperiodate for 5 rain, rinsed in water, and placed on to 0.1 Mglycine for 10 rain. The grids were transferred to a solution of washing buffer (PBS containing 0.1% low-fat dry milk powder and 0.1% ovalbumin) for 45 rain. The grids were then placed on to a 1:100 dilution of the primary mouse mAb specific to the C-terminal region of Nef diluted in washing buffer overnight at 4°C in a moist chamber, washed 6 times, each 4 min in washing buffer, and reacted with 10 nm gold labelled goat anti-mouse IgG/IgM (Amersham, UK) diluted 1:30 in washingbutTer for 1 h. The grids were rinsed as above, then washed 3 times in water, and placed on to a solution of 1% glutaraldehyde for 5 rain to fix the antibody complex. The grids were counterstained with 2% aqueous uranyl ace- tate (ProSciTech, Thuringowa Central, Qld.) for 5 rain, washed, and followed with 5 min in Reynold's lead citrate. Samples were photo- graphed on a Jeol 100B electron microscope at 80 kV.

Results

Fig. 1. Release of membranous material by high-level Nef pro- ducers. DMY100 [pYEX-BX] and DMY100 [pYEX-BX.Nef27] were grown in 500 ml of YEPD with 1.0 rm'v/CuSO4 for 4 days. Cul- tures were then centrifuged and the pellets transferred to 50-ml tubes for re-centrifugation and photography. Labels indicate the foreign protein produced. Note the oily layer on top of the pelleted Nef- producing cells.

m

m

Released Nef Forms Extracellular Membranous Bodies' and Can Be Purified We previously observed that Nef can be released from

cells in exponential growth phase to the culture media in a stress-dependent manner [35]. In a study on production of extracellular Nef by DMY100 [pYEX-BX.Nef27] during copper stress, there was additional material in the culture supernatant after 4 days that was absent after 2 days cul- ture. This material was also produced by DMY100 [pYEX-S1.Nef27] transformants when grown with heat stress (data not shown), but was absent from cultures of DMY100 [pYEX-BX] grown under those conditions (fig. 1). This material appeared as membranous cell debris that settled as a layer above the cells upon centrifugation. The membranous material could be purified by physical means involving flushing the layer off the cell pellet and centrifugation.

Analysis of the recovered layer by SDS-PAGE and Western blotting revealed that the predominant protein in the layer was Ne fand this led us to devise a purification strategy, exploiting the properties of the membranous material. Because of its abundance in the layer, the proce- dure was relatively straightforward, involving solubilisa- tion in urea followed by column fractionation as de- scribed in 'Materials and Methods'. Fractions from the Superdex TM column (fig. 2A) were subsequently analysed by SDS-PAGE and Western blotting. The main peak emerging from the column showed a major protein band on SDS gels at about 27 kD (fig. 2B). Analysis in Western blots showed that this material was reactive with mono- clonal antibody to the C-terminus of Nef(fig. 2C) and was likely to be full-length Nef.

Nef Released from Yeast Is Myristylated It was of interest to determine whether Nef in the cul-

ture supernatant was myristylated, because we previous- ly showed that intracellular yeast Nef was myristylated [33]. Two Nef producers, DMY100 [pYEX-BX.Nef27] and DMY100 [pYEX-S1.Nef27], were utilised for this study. For copper-inducible Nef production, DMY100 [pYEX-BX.Ne[27] cells as grown in minimal medium plus 0.1 m M copper sulphate and labelled myristic acid for three days. For constitutive Nef production, DMY 100 [pYEX.S1.Net27] was grown in mimimal medium at the permissive growth temperature, labelled myristic acid was added and cell cultures were shifted to 37°C for 4 days. Because of the small scale of this experiment we did not seek to isolate the membranous layer but used the total culture supernatant. Cells were spun down, and the culture supernatant was harvested and analysed for the production and labelling of Nef. As shown in figure 3, lanes 2 and 3, under copper or heat stress Nef is released into the culture media as a 27-kD protein that reacts with antibody to its C-terminus. In lanes 5 and 6 it can be seen that the 27-kD protein, unique to the Nef producers and readily identified as Nef, is also myristylated. The release of full-length myristylated Nef into culture media repre- sents a more convenient source of Nef than intracellular myristylated Nef that we have obtained previously [33].

Membrane Perturbation in Yeast Cells Producing HIV- 1 Nef

J Biomed Sci 1998;5:203-210 205

U84:ZJOBS195XA SIBY

m

1.5'-I

A

i ~.o-I & o.s-!

o.ol - - J ' ~o 2o ~o ,;o

Fraction Nurrber

B C S E M23 25 27 29 31 33 23 25 27 29 31 33

iiiiiiii••iiiiiiiii•iiiiii•iii!ii!•iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii -94- ~i.:'..!}.~iii:̀.~i~iiiiiiiiiiii~iiiiiiiiiii~:.ii.:̀.:iiiiiiiiiiiii~iiiiiiiiii~ii.:.::iii~i£~ii~ ilii!iil ~i !~tl

!:i~i~:!:~:~:?~iiiii:iiiiiiiiiii.~iiii~iiiiiiiiiiiiii -67- : ..'..:: i: i.~: ~i:i~ i:N:.'.'i:i : ::::::::::::::::::::::::::::::::::::::::::::::::: ~,.'~:, .:..':z:,.'~:~ ~: i~ '~: i~,~ :~? .~:~ ,.~: .,.': : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

4 3- ............................ .,~..~.~...~ . . . . . . . . . . . . : : . ~ . ~ : : ~ . . . : , ~ ~ : ~::..::$::-.,...~@ ,~ .. • . :+: ::::::::::::::::::::::::::::::::::::::::::::::: ~. ~;, :~,,.N~'" . , ~ / ~ ~ . . . . . . . . . . . . . . : : . . . . . . . . . . . . . . . , . , , . . + . . : : . . . . . . . . .

..... .,v~.~.,., ........... ~e..~,x,:.:,,x.:..:..~..-30- ~ - - ~ . -~ . : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :

iiiiiiiiii~!:~:!ii~!iiiiiiiii~iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii..'i i~i:iiiii -20- ~i:.".i~:.. .::.x." ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ~ : : ~ . : . ~ . - - : ~ " . ~ . . '

:.,." ~ ,: ~i; : : ~ ~ ~ . : ' ~ :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

m

Fig. 2. Analysis of release of Nef in yeast culture supernatants. Following centrifugation, the oily layer was scooped off and 5 ml of 8 M urea/PBS/10 mM DTT/10 mM Pefabloc T M added. After incubation at 4 ° C for 2 h with occasional mixing, the suspension was centrifuged at 12,000 rpm for 10 min and the supernatant decanted. The supernatant was centrifuged again and 3.0 ml of the supernatant loaded on to a 2.5 cm x 60 cm column of Super- dex T M 75 equilibrated in 8 M urea/PBS/2 mM DTT. The column was run at 2.0 ml/min and the A280 monitored (A). Fractions were analysed by SDS-PAGE (B) and by Western analysis with mAb directed to the Nef C-terminus (C). Lanes are: S = crude supernatant; E = E. coli-derived Nef [3]; M = molecular weight markers whose sizes are shown in kilodaltons. Numbers refer to fraction numbers.

m

:i:,i:,i:,i:,i:,i:,i:,iiiiiiiii:,i:,iiiii:,i

i!iiiiiliiiiii iiiiiiiiii!iii

Fig. 3. Analysis of myristylation of extracellular Nef. Transfor- mants of strain DMY100 were grown in minimal synthetic medium (YNB) as 10-ml batch cultures in 50-ml culture tubes for 3 days. The DMY100 [pYEX-BX] and DMY100 [pYEX-BX.Nef27] transfor- mants were grown at 28°C in media containing 0.1 mM CuSO4, while DMY100 [pYEX-S 1 .Nef27] transformants were grown in the absence of added copper but the incubation temperature was 37 o C. Culture supernatants were fractionated on 0.1% SDS/4-20% poly- acrylamide gradient gels (Novex) that were Western blotted with mAb directed to the C-terminus of Nef. Lanes containing 500 gl of the respective culture supernatants are as follows: 1, 4. DMY100 [pYEX-BX]; 2, 5. DMY100 [pYEX-BX.Nef27]; 3, 6. DMY100 [pYEX-S1.Nef27]. A Western blot, while B is the fluorograph. The positions of molecular weight markers are shown: the arrow points to Nef.

Localisation of Ne f in Ne f Producers by Immune Electron Microscopy U n d e r s t a n d i n g the in t racel lular local isat ion o f N e f is

i m p o r t a n t for the e luc ida t ion o f its funct ion. F o r the analysis o f yeast p roduc ing Nef , D M Y 1 0 0 [pYEX- BX.Nef27] was grown in m i n i m a l m e d i a in the p resence o f 0.2 m M CuSO4 for 16 h and cells subjec ted to i m m u n e electron mic roscopy , emp loy ing a p r i m a r y m o u s e anti- b o d y to the C- t e rminus o f Nef , fo l lowed by an a n t i - m o u s e secondary a n t i b o d y coup led to 10 n m gold spheres. Th is a p p r o a c h resul ted in specific labell ing o f Nef , wi th no b ind ing f r o m the first or second a n t i b o d y to n o r m a l yeast p ro te ins (see fig. 4B, D). W i t h this technique , the major i - ty o f the N e f p r o d u c e d was localised in nove l cy top la smic bodies (fig. 4A), a l though m i n o r a m o u n t s were found scat tered th roughou t the cy top lasm. These bodies , t e r m e d N e f bodies , a p p e a r e d un ique to N e f p roduc ing cells and were not p resen t in D M Y 1 0 0 [pYEX-BX] cells (fig. 4D). In cells p roduc ing Nef20-206, the N e f bod ies were absen t and the gold labell ing was cy top la smic (fig. 4C).

T h e N e f bodies were found in m o s t th in sect ions o f Ne f -p roduc ing cells, were usual ly found at one pe r cell and were typical ly large and well rounded , as shown in figures 4 and 5. In s o m e thin sect ions we have found div- iding cells wi th one N e f b o d y in each cell (fig. 5A).

206 J Biomed Sci 1998;5:203-210 Macreadie/Fernley/Castelli/Lucantoni/ White/Azad

U84:ZJOBS195XA SIBY

m A B

m

Fig. 4. Immune electron microscopy of yeast transformants producing Nef. Cells were grown in 0.2 mM CuSO4 overnight, fixed, thin-sectioned and examined by im- mune electron microscopy. NB = Nef body; N = nucleus; size bar is 1 gM. A DMY100 [pYEX-BX.Nef27] diploid cell showing typi- cal extranuclear Nef body that is labelled with 10-nm gold particles. B DMY100 [pYEX-BX.Nef27] diploid cell showing typi- cal extranuclear Nef body with Nef antibody omitted to test the specificity of the gold probe. C DMY100 [pYEX-BX.Nef25] dip- loid cell showing gold labelling in the cyto- plasm, but absent in the nucleus. D DMY100 [pYEX-BX] diploid cell show- ing gold labelling (and Nef bodies) to be totally absent.

D

m

Although the Nef bodies are usually found in the cyto- plasm, we have found numerous examples where they are present in the nucleus (fig. 5B, D). Their appearance is not specific to the yeast strain examined (data not shown) and they can also be found in the nucleus or in the cytoplasm of haploid cells (fig. 5C, D). In rare cases we have found up to three Ne f bodies in a single cell (fig. 5C). Close examination of the bodies reveals the presence of either a single or double membrane surrounding the body and uniform staining for N e f through the body.

Our interpretation of these data is that Ne f causes the formation of the bodies, although we have not excluded the possibility that their formation has a requirement for copper. That they are novel structures is confirmed by their appearance in the cytoplasm, or in the nucleus, and with their inclusion within a single or double membrane. Their source is likely to originate f rom disruption to possi- bly any cellular membrane, including the plasma mem- brane, or the double membranes of the nucleus.

Membrane Perturbation in Yeast Cells Producing HIV- 1 Nef

J Biomed Sci 1998;5:203-210 207

U84:ZJOBS195XA SIBY

m

m

A B

Fig. 5. Immune electron microscopy of yeast transfbrInants producing Nef. Cells were treated as in figure 4. NB = Nef body; N = nucleus; size bar is 1 pM. A DMY100 [pYEX-BX.Nef27] diploid cell dividing and exhibiting two typical extranuclear Nef bod- ies labelled with 10 nm gold particles. B DMY100 [pYEX-BX.Nef27] diploid cell showing an intranuclear Nef body labelled with 10-nm gold particles. C DY150 [pYEX- BX.Nef27] haploid cell showing three extra- nuclear Nef bodies labelled with 10-rim gold particles. D DY150 [pYEX-BX.Nef27] ha- ploid cell showing an intranuclear Nef body labelled with 10-nm gold particles.

c D

mm

D i s c u s s i o n

In the present study, we have examined Ne f localisa- tion under various growth conditions with moderate lev- els of Ne f production. Under such conditions we found that yeast Nef was targeted to, and caused the product ion of, bodies within the cell that appeared to be surrounded by either single or double membranes. These bodies were capable of formation within the nucleus or in the cyto-

plasm. The product ion of these bodies was dependent on determinants within the first 19 amino acids and/or the myristic acid attached to the N-terminal glycine. Sanfrid- son et al. [41 ] recently observed Nef localised in cytoplas- mic bodies of similar appearance which they identified as endosomes and lysosomes. However, in yeast we find that the bodies are novel entities unique to Nef producers and they are not strictly cytoplasmic in localisation.

208 J Biomed Sci 1998;5:203-210 Macreadie/Fernley/Castelli/Lucantoni/ White/Azad

U84:ZJOBS195XA SIBY

m

m

Nef was also released from yeast cells into the culture medium raising the possibility that it could be detected on the surface of cells by immune electron microscopy. While occasional labelling concentrated in patches at the cell membrane is observed (data not shown), its infre- quent appearance suggests that it might be an artefact. Nef expressed from a baculovirus expression system and from T cells acutely and persistently infected with HIV-1 was found clustered at the cell surface as well as within the cytoplasm [15, 16], and insect cells decorated with Nef on their surface could kill bystander cells [17]. Peculiarities in the surface of a yeast cell may contribute to our difficul- ty to detect Nef on the cell surface or it may be released from the yeast very rapidly. We have also looked for Nef bodies in the culture supernatant by electron microscopy but our results have been inconclusive.

The release of Nef from yeast is dependent upon myris- tylation [35] which occurs through an amide linkage of the N-terminal glycine [2]. Myristylation of Nef is a post- translational modification of proteins having specific N- terminal sequences. The Nef myristylation sequence is highly conserved and causes myristylation in various expression systems including mammalian [2, 20, 21, 45], insect [37], and yeast cells [33]. It is clear from our studies with myristylated Nef peptides that the N-terminal por- tion of Nefcan perturb membranes [9, 10, 36]. Membrane alterations have been previously noted in HIV-1 infection [ 12] and our data indicate a potential involvement of Nef. Foti et al. [14] have noted that Nef can act on the plasma membrane causing a doubling of the plasma membrane occupied by clathrin-coated pits. What is clear from our studies and various other reports on Nef [13, 22, 25, 26, 28, 29, 31, 39, 45] is that Nef has affinity for membranes

and its localisation to membranes can be disruptive. It is of interest that another myristylated retroviral protein, HTLV-1 Gag, can also be released from yeast cells [24], presumably in a manner similar to what we have observed here. These properties of Nef have been suggested from previous studies with myristylated peptides [9, 10, 36], however, this appears to be the first demonstration of an effect from the myristylated Nef protein on cellular mem- branes. It is of interest that Fujii et al. [17] have indicated that cell surface Nef could kill bystander cells [17], how- ever, the mechanism by which this occurred was not described. In view of this and our data here, we suggest that Nef, like other extracellular HIV-1 proteins Tat, Vpr and gpl20, should be also considered as a candidate for extracellular effects, including bystander killing in AIDS. Our data also suggest that there should be closer analysis of extracellular Nef in HIV-1 infected patients to define and determine its properties. For example, there are recent reports on Nef in virions [7, 40, 43], but could it be that this material is actually in membranous material that co-purifies with virions?

Acknowledgments

We thank Dr. Jayne Brookman (University of Manchester School of Biological Sciences) for providing methods on embedding yeast for immune electron microscopy, and Dr. Eileen O'Toole and the High Voltage Electron Microscopy Laboratory (University of Colorado, Boulder, Colo., USA) for their invaluable assistance in providing teaching and facilities for the high-pressure freezing and freeze-sub- stitution of yeast cells. We acknowledge the helpful support of Dr. John Ramshaw throughout this project. This work has been support- ed by grants from Commonwealth AIDS Research Grant and the Commonwealth Department of Industry Science and Technology.

mm

o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o

References

1 Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA. Production of acquired immunodeficiency syndrome-asso- ciated retrovirus in human and nonhuman cells transfected with an infectious clone. J Virol 59: 284-291;1986.

2 Allan JS, Coligan JE, Lee T-H, McLane MF, Kanki PJ, Groopman JE, Essex M. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science 230: 810-813;1985.

3 Azad AA, Failla P, Lucantoni A, Bentley J, Mardon C, Wolfe A, Fuller K, Hewish D, Sen- gupta S, Sankovich S, Grgacic E, McPhee D, Macreadie I. Large-scale production and char- acterization of recombinant human immuno-

deficiency virus type 1 Nef. J Gen Virol 75: 651-655;1994.

4 Barnham KJ, Monks SA, Hinds MG, Azad AA, Norton RS. Solution structure of a polypeptide from the N terminus of the HIV protein Nef. Biochemistry 36:5970-5980; 1997.

5 Benichou S, Bomsel M, Bodeus M, Durand H, Doute M, Letourneur F, Camonis J, Benarous R. Physical interaction of the HIV-1 Nef pro- tein with beta-COP, a component of non-cla- thrin-coated vesicles essential for membrane traffic. J Biol Chem 269:30073-30076; 1994.

6 Bodeus M, Marie-Cardine A, Bougeret C, Ra- mos-Morales F, Benarous R. In vitro binding and phosphorylation of human immunodefi- ciency virus type 1 Nef protein by serine/

threonine protein kinase. J Gen Viro176:1337- 1344; 1995.

7 Bukovsky AA, Dorfman T, Weimann A, Gott- linger HG. Nef association with human immu- nodeficiency virus type 1 virions and cleavage by the viral protease. J Virol 71:1013-1018; 1997.

8 Coates K, Cooke SJ, Mann DA, Harris MPG. Protein kinase C-mediated phosphorylation of HIV-1 nef in human cell lines. J Biol Chem 272:12289-12294;1997.

9 Curtain CC, Separovic F, Rivett D, Kirkpa- trick A, Waring AJ, Gordon LM, Azad AA. Fusogenic activity of amino-terminal region of HIV type 1 Nefprotein. AIDS Res Hum Retro- viruses 10:1231-1240;1994.

Membrane Perturbation in Yeast Cells Producing HIV- 1 N e f

J Biomed Sci 1998;5:203-210 209

U84:ZJOBS195XA SIBY

m

m

10 Curtain CC, Lowe MG, Arunagiri CK, Mobley PW, Macreadie IG, Azad AA. Cytologic activi- ty of amino-terminal region of HIV type 1 Nef protein. AIDS Res Hum Retroviruses 14: 1213-1220;1997.

11 Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, McPhee D, Greenway AL, Ellett A, Chatfield C, Lawson VA, Crowe S, Maerz A, Sonza S, Learmont J, Sullivan JS, Cunningham A, Dwyer D, Dowton D, Mills J. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfu- sion donor and recipients. Science 270:988- 991;1995.

12 Fermin CD, Garry RF. Membrane alterations linked to early interactions of HIV with the cell surface. Virology 191:941-946; 1992.

13 Franchini G, Robert-Guroff M, Ghrayeb J, Chang NT, Wong-Staal F. Cytoplasmic local- ization of the HTLV-III 3' off protein in cul- tured T ceils. Virology 155:593-599;1986.

14 Foti M, Mangasarian A, Piguet V, Lew DP, Krause K-H, Trono D, Carpentier J-L. Nef- mediated clathrin-coated pit formation. J Cell Biol 139:37-47;1997.

15 Fujii Y, Nishino Y, Nakaya T, Tokunaga K, Ikuta K. Expression of human immunodefi- ciency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells. Vaccine 11:1240-1246; 1993.

16 Fujii Y, Otake K, Fujita Y, Yamamoto N, Nagai Y, Tashiro M, Adachi A. Clustered local- ization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell sur- face. FEBS Left 395:257-261;1996.

17 Fujii Y, Otake K, Tashiro M, Adachi A. In vitro cytocidal effects of human immunodefi- ciency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction. J Gen Viro177:2943-2951; 1996.

18 Geleziunas R, Miller MD, Greene WC. Unrav- elling the function of HIV type 1 Nef. AIDS Res t tum Retroviruses 12:1579-1582;1996.

19 Greenway A, McPhee D. N e f - the Machiavelli of cellular activation. Res Virol 148:58-64; 1997.

20 Guy B, Kieny MP, Riviere Y, Le Peuch C, Dott K, Girad M, Montagnier L, Lecocq J-P. HIV F/3 ~ o~fencodes a phosphorylatod GTP-bind- ing protein resembling an oncogene product. Nature 330:266-269; 1987.

21 Guy B, Rivi6re Y, Dott K, Regnault A, Kieny MP. Mutational analysis of the HIV Nef pro- tein. Virology 176:413-425;1990.

22 I tammes SR, Dixon EP, Malim MH, Cullen BR, Greene WC. Nef protein of human immu- nodeficiency virus type 1 : Evidence against its role as a transcriptional inhibitor. Proc Natl Acad Sci USA 86:9549-9553;1989.

23 Harris M. From negative factor to a critical role in virus pathogenesis: The changing fortunes of Nef. J Gen Virol 77:2379-2392;1996.

24 Hayakawa T, Miyazaki T, Misumi Y, Kobaya- shi M, Fujisawa Y. Myristoylation-dependent membrane targeting and release of the HTLV-I Gag precursor, Pr53gag, in yeast. Gene 119: 273-277;1992.

25 Kaminchik J, Bashan N, Itach A, Sarver N, Gorecki M, Panet A. Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells. J Virol 65:583-588;1991.

26 Kaminchik J, Margalit R, Yaish S, Drummer H, Amit B, Sarver N, Gorecki M, Panet A. Cel- lular distribution of HIV type 1 Nef protein: Identifcation of domains in Nef required for association with membrane and detergent-in- soluble cellular matrix. AIDS Res Hum Retro- viruses 10:1003-1010;1994.

27 Kestler HW III, Ringler DJ, Mori K, Pmaicali DL, Sehgal PK, Daniel MD, Desrosiers RC. Importance of the nefgene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662; 1991.

28 Kienzle N, Bachmann M, Muller WE, Muller- Lantzsch N. Expression and cellular localiza- tion of the Nef protein from human immuno- deficiency virus-I in stably transfected B-cells. Arch Virol 124:123-132;1992.

29 Kienzle N, Enders M, Buck M, Siakkou H, Jahn S, Petzold G, Schneweis KE, Bachmann M, Muller WE, Muller-Lantzsch N. Expression of the HIV-1 Nef protein in the baculovirus system: Investigation of anti-Nef antibodies re- sponse in human sera and subcellular localiza- tion of Nef. Arch Virol 126:293-301;1992.

30 Kirchoff F, Greenough TC, Brettler DB, Sulli- van JL, Desrosiers RC. Brief report: Absence of intact nef sequences in a long-term survivor with progressive HIV-1 infection. N Engl J Med 332:228-232; 1995.

31 Kohleisen B, Neumann M, Herrmann R, Brack-Weruer R, Krolm KJE, Ovod V, Ranki A, Erfle V. Cellular localization of Nef ex- pressed in persistently HIV-1-infected low-pro- ducer astrocytes. AIDS 6:1427-1436; 1992.

32 Luo T, Foster JL, Garcia JV. Molecular deter- minants of Nef function. J Biomed Sci 4:132- 138;1997.

33 Macreadie IG, Ward AC, Failla P, Grgacic E, McPhee D, Azad AA. Expression of HIV-1 nef in yeast: The 27 kDa Nef protein is myristy- lated and fi*actionates with the nucleus. Yeast 9:565-573;1993.

34 Macreadie IG, Castelli LA, Failla P, Azad AA. Yeast-derived HIV-1 Nef protein lacks G-pro- tein activities. Mol Biol (Life Sci Adv)12:99- 105;1993.

35 Macreadie IG, Castelli LA, Lucantoni A, Azad AA. Stress- and sequence-dependent release into the culture medium of HIV-I Nef pro- duced in Saccharomyces cerevisiae. Gene 162: 239-243;1995.

36 Macreadie IG, Lowe MG, Curtain CC, Hewish DR, Azad AA. Cytotoxicity resulting from ad- dition of HIV-1 Nef N-terminal peptides to yeast and bacterial cells. Biochem Biophys Res Commun 232:707-711;1997.

37 Matsuura Y, Maekawa M, Hattori S, Ikegami N, Hayashi A, Yamazaki S, Morita C, Takebe Y. Purification and characterization of human immunodeficiency virus type 1 Nef gene prod- uct expressed by a recombinant baculovirus. Virology 184:580-586; 1991.

38 McKimm-Breschkin JL. The use of tetrame- thylbenzidine for solid phase immunoassays. J Immunol Methods 135:277-280;1990.

39 Ovod V, Lagerstedt A, Ranki A, Gombert FO, Spohn R, Tahtinen M, Jung G, Krolm KJE. Immunological variation and immunohisto- chemical localization of HIV-1 Nef demon- strated with monoclonal antibodies. AIDS 6: 25-34;1992.

40 Pandori MW, Fitch NJ, Craig HM, Richman DD, Spina CA, Guatelli JC. Producer-cell modification of human immunodeficiency vi- rus type 1: Nef i s a virion protein. J Virol 70: 4283-4290; 1996.

41 Sanfridson A, Hester S, Doyle C. Nefproteins encoded by human and simian immunodefi- ciency viruses induce the accumulation of en- dosomes and lysosomes in human T cells. Proc Natl Acad Sci USA 94:873-878;1997.

42 Sawai ET, Baur AS, Peterlin BM, Levy JA, Cheng-Mayer C~ A conserved domain and membrane targeting of Nef from HIV and SIV are required fbr association with a cellular ser- ine kinase activity. J Biol Chem 270:15307- 15314;1995.

43 Welker R, Kotfler H, Kalbitzer HR, Krausslich HG. Human immunodeficiency virus type 1 Nef protein is incol~rated into virus pal~icles and specifically cleaved by the viral proteinase. Virology 219:228-236; 1996.

44 Wiskerchen M, Cheng-Mayer C. HIV-1 Nef association with cellular serine kinase corre- lates with enhanced virion infectivity and effi- cient proviral DNA synthesis. Virology 224: 292-301;1996.

45 Yu G, Felsted R E Effect of myristylation on p27nef subcellular distribution and suppres- sion of HIV-LTR transcription. Virology 187: 46-55;1992.

m

210 J Biomed Sci 1998;5:203-210 Macreadie/Fernley/Castelli/Lucantoni/ W h i t e / A z a d