Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells

VIROLOGY

186, 25-39 (1992)

Analysis

of HIV Particle Formation Using Transient Expression of Subviral Constructs in Mammalian Cells

KLAUS MERGENER,* MICHAEL F;iCKE,* REINHOLD WELKER,* VOLKER BRINKMANN,t HANS R. GELDERBLOM,t AND HANS-GEORG KRP;USSLICH*,’ *Angewandte

Tumorvirologie. Deutsches Krebsforschungszentrum, fm Neuenheimer Fe/d 506, D-6900 Heidelberg, tRobert Koch lnstitut des Bundesgesundheitsamtes, Nordufer 20, D- 1000 Berlin 65, Germany Received July 17, 199 1; accepted

September

Germany; and

10, 199 1

Segments of the human immunodeficiency virus (HIV) type 1 gag and polgenes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pal open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pal open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pal open reading frame did not yield particles. o 1992 Academic Press.

Inc.

INTRODUCTION

RNA associated with the nucleocapsid [NC] protein and probably also with the viral enzymes reverse transcriptase [RT] and integrase [IN]; for nomenclature of retroviral proteins see Leis er a/., 1988) encased in a capsid (CA) shell (reviewed in Gelderblom et al., 1989; Gelderblom, 1991). The structural components of the viral core (coded for by the gag gene) and the replication enzymes (derived from the pal gene) are translated as two polyproteins (pr55g”g and pr160gag-Po’ in the case of human immunodeficiency virus [HIV] type 1) whereby synthesis of the pal products is achieved by translational frameshifting in the 3’ terminal part of the gag region (Jacks et al., 1988). Both polyproteins are cotranslationally myristoylated at their N-terminus and myristoylation has been shown to be essential for particle release (Bryant and Ratner, 1990; GUttlinger et al., 1989). Production of infectious virions occurs only at locations on the plasma membrane where the viral glycoproteins and the gag and gag-pal precursors congregate. However, expression of gag proteins in the absence of glycoproteins appears sufficient for the formation of noninfectious particles (Shields et al., 1978), and the mechanism directing the incorporation of gly-

Retroviruses are released from the host cell as immature, concentrically organized particles via a budding process involving the envelopment of the prospective viral core by a lipid bilayer. Bud formation occurs either from preformed cores (type B and D oncoviruses, spumaviruses) or concomitant with the assembly of the viral core (type C oncoviruses, lentiviruses; reviewed in Gelderblom et a/., 1989; Gelderblom, 1991). Early immature retrovirus cores are spherical, about 80 nM in diameter, and are formed by the assembly of gag- and gag-pal precursor proteins. Condensation of the core, typical of mature virus, is necessary for the virion to become infectious and occurs only after proteolytic processing of the precursor proteins by the viral proteinase (PR; reviewed in Kr2usslich and Wimmer, 1988), leading to either isometric or cone-shaped cores typical of the C-type oncoviruses and lentiviruses, respectively. The cores consist of a ribonucleoprotein complex (two identical molecules of genomic ’ To whom reprint requests should be addressed. 25

0042-6822/92

$3.00

Copynght 0 1992 by Academic Press. inc. All rights of reproduction I” any form reserved.

26

MERGENER

coproteins into budding particles is currently not known. While the genomic structure, protein expression, and molecular architecture of HIV have been analyzed in great detail in recent years, the molecular processes involved in intracellular transport of polyproteins and the molecular interactions driving assembly, bud formation, and maturation are only poorly understood. Given the complex nature of the HIV genome and the considerable hazard of working with infectious HIV, it seems highly desirable to develop a simplified in vitro recombinant expression system leading to the formation of noninfectious HIV-like particles. High level expression of these particles should allow a detailed analysis of intracellular targeting, assembly, and processing of viral polyproteins and, on the other hand, should yield sufficient quantities of HIV-like particles to use for biochemical analysis and immunization trials. Thevalidity of this model system will depend, however, on experimental evidence proving that specific mutations yield the same phenotype when present in the context of the provirus or of the subviral expression vector. Several groups have recently reported HIV polyprotein synthesis and release of virus-like particles after infection of different host cell lines with recombinant vacciniaand baculoviruses (Karacostas et al., 1989; Haffar et a/., 1990; Shioda and Shibuta, 1990; Hu et a/., 1990; Gheysen et al., 1989). A detailed analysis of virus-host cell interactions in these systems is hampered by the pleiotropic effects of the viral vector on the host cell (e.g., transcriptional and translational shut-off, syncytia formation, and ultimately cell lysis) and, in the case of baculovirus, by the restricted host range. Formation of virus-like particles has also been shown recently after transient expression of the HIV-1 gag-polgene using a simian virus 40 late replacement vector which yields high levels of protein expression due to replication of the vector DNA (Smith et a/., 1990). In this case, expression of HIV structural proteins was dependent on the presence of the rev protein in tram which is also required for the expression of structural genes in infected cells (Malim et al., 1989; Felber et a/., 1989). In the present study we report rev-dependent expression of HIV-l gag and gag-pol segments under the control of the cytomegalovirus immediate early promoter/enhancer element (Boshart et al., 1985). Efficient synthesis and processing of HIV polyproteins as well as release of particles with lentiviral morphology was observed after transient transfection of wild-type constructs. In addition, we analyzed the effects of mutations with regard to polyprotein synthesis and particle production.

ET AL

MATERIAL AND METHODS Cells and transfections COS 7 cells were maintained in DMEM supplemented with 109/o heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2 mll/l glutamine. For transfection, approximately 5 X lo6 subconfluent cells that had been plated 24 hr before transfection were suspended in 0.1 ml PBS at room temperature and electrotransfected with 20 pg DNA using a BioRad gene pulser equipped with a capacitance extender and pulse controller. The electrical parameters were 150 V, 960 /IF, and 100 Ohms resistance. Five minutes after transfection, cells were diluted into 20 ml of fresh media and plated on two 1O-cm dishes. Cells were harvested by scraping off the plate and lysed in 2% SDS.

Expression plasmids The relevant HIV sequences present in the different expression vectors are depicted in Fig. 1. The BarnHI-Bglll fragment (nt 7198-8052; containing the rev responsive element [RRE]; Malim et al., 1989; Felber et a/., 1989) of the BHlO strain of HIV-1 (Ratner et al., 1985) was inserted into the BamHl site of pBluescript SK+ (Stratagene) to give PBS-RRE (orientation T7-7 198-8052-T3). Plasmids pHIV-gpll and pHIVFSII (renamed pBR-gpll and pBR-FSII, Krausslich, 1991) have been described previously (Krausslich et a/., 1988) and contain a short stretch (1 12 nt) of 5’ untranslated sequence followed by the entire gag and PR regions of HIV-1 (BHl 0; nt 221-2078) followed by two stop codons and flanked by EcoRl sites. pBR-FSII contains a 4 base pair insertion at nt 1640 leading to a shift from the gag to the pal open reading frame (Krausslich et a/., 1988). The EcoRl fragments of pBRgpll and pBR-FSII were inserted into the EcoRl site of PBS-RRE to give PBS-R-gpll and -FSII (orientation: T7-221-2078-RRE-T3). PBS-R-gplla and PBS-R-FSlla were generated by oligonucleotide-directed mutagenesis using the method of Kunkel (1985) to change the Asp25 codon of the PR active site (nt 1905-l 907) to Ala25 (GAT-GCT). Plasmids PBS-R-gpV, PBS-R-FSV, PBS-R-gpVa, and PBS-R-FSVa were constructed by inserting the &/I-/Vdel fragment (nt 2009-4702) of the BHlO strain of HIV-1 into the respective plasmids of the II-series that had been opened with Bell and Smal (2009 of HIV to polylinker between the coding sequence and the RRE). PBS-R-gpllAmy was made by oligonucleotide-directed mutagenesis (Kunkel, 1985) changing the Gly2 codon of the gag open reading frame (nt 337-339) to Ala2 (GGT-GCC), thereby de-

HIV PARTICLE FORMATION

stroying the signal for N-myristoylation. In addition, PBS-R-gpllAmy’ contains an A-G mutation at nt 345 which does not change the coding sequence but generates a new Bgll site. The described PBS-R plasmids were digested with SalI and NotI (cutting in the Bluescript polylinker 5’ and 3’ of the HIV inserts) and the Sall-NotI fragments were inserted into the eukaryotic expression vector pKEX-XR (Rittner and Sczakiel, 1991) that had been opened with SalI and NotI, to give plasmids pK-R-gpll, pK-R-FSII, and so on. pKEX-XR contains the human cytomegalovirus immediate early promoter/enhancer element (CMV-IE; Boshart et a/., 1985) upstream of a multiple cloning region followed by the SV40 t-antigen splice and polyadenylation signals (see Fig. 1) on a pUC-derived vector. It also contains the gene conferring resistance to hygromycin B driven by the herpes simplex virus thymidine kinase promoter (Bernard eta/., 1985) but does not contain an SV40 origin of replication. For expression of the rev protein, plasmids pCMVrev (Mermer et a/., 1990) and pMTcrev (Hadzopoulou-Cladaras et a/., 1989) were used which contain the rev cDNA under control of the cytomegalovirus and metallothionin promoters, respectively. Indirect

immunofluorescence

Transfected cells were grown on sterile glass cover slips for the time indicated, washed in PBS, and fixed in methanol for 5 min at -20” followed by acetone for 5 min at -20”. For detection of gag gene products, fixed cells were reacted with a polyclonal rabbit antiserum against HIV-1 CA at a dilution of 1:200 in PBS/O.5% bovine serum albumin. This antiserum had been raised against purified recombinant CA protein (Ehrlich et al., 1990) and detected CA antigen in immunofluorescence up to a dilution of 1:500 and in Western blots up to a dilution of 1:2000. For detection of rev protein, cover slips were reacted with a monoclonal antibody against rev (gift of B. Fleckenstein) at a dilution of 1 :lOO. Immune complexes on cover slips were detected with fluorescein isothiocyanate-labeled antirabbit or anti-mouse sera (Dianova, Hamburg), both at a dilution of 1: 100. Electron

microscopy

Transfected cells were washed with PBS at the time indicated, fixed with 2.5% glutaraldehyde in PBS for 30 min, and carefully scraped off the plate and centrifuged at 200 g for 5 min at 20”. Cell sediments were postfixed in 1% OsO,, embedded in agar, treated with 1% tannic acid, and processed and embedded into Epon as described elsewhere (Gelderblom et a/., 1987). Of

27

each specimen sections of 40 to 60 nm in thickness were prepared from at least two replicate blocks, poststained with lead citrate, and evaluated using a Zeiss electron microscope 10 A at 60 kV.

UTP incorporation COS 7 cells were cotransfected with plasmids pK-RgpV and pMTcrev and plated on two lo-cm dishes. Twelve hours after transfection, cells were washed twice with PBS and were labeled for 36 hr with [3H]UTP (50 &i per dish; sp act: 40-60 Ci/mmol; Amersham). To detect particle-associated RNA, released particles were purified by centrifugation through a sucrose cushion and banding on a sucrose density gradient as described below.

Purification

of extracellular

particles

At the time indicated, media were cleared bycentrifugation at 200 g for 10 min and virus-like particles were either precipitated with polyethylene glycol (PEG) or centrifuged through a sucrose cushion. For PEG precipitation, supernatants were made 1 M NaCl and 7.5% PEG 6000 and incubated for 60 min at 0”. Precipitates were collected by centrifugation at 12,000 g for 10 min and lysed in gel loading buffer. Alternatively, supernatants were centrifuged through a cushion of 20% (w/v) sucrose at 120,000 g for 2 hr at 4’. For density gradient analysis, pellets were resuspended in PBS, overlaid on a continuous sucrose gradient (20 to 60% [w/v] sucrose in PBS), and centrifuged for 18 hr at 100,000 g at 4”. Fractions of 500 ~1 were collected from the bottom of the tube and analyzed by ELISA and scintillation counting as applicable. For immunoblot analysis and RT assay, particles were sedimented from gradient fractions by centrifugation at 100,000 g for 60 min at 4’.

Reverse transcriptase

assay

Particles were precipitated from the medium of transfected cells with PEG and disrupted in 50 ~1 20 mMTris-Cl, pH 8.0/O. 1% Triton X-l 00 at 4” for 10 min. Fifty microliters of 50 mM Tris-Cl pH 8.0/10 mM MgClJ60 mM KCl/2 mM dithiothreitol were added and polymerization was performed for 1 hr at 37” using 4 pg poly(rA)-oligo(dT) as template and 10 &i [3H]TTP (sp act: 90-l 30 Ci/mmol, Amersham). After precipitation with 10% trichloroacetic acid, the products were collected on GF/C filters (Whatman) and counted in a scintillation counter.

MERGENER

28

Analysis

of expression

products

For detection of HIV antigens, media were cleared and appropriate dilutions were analyzed using a commercial ELISA kit (Organon Teknika) detecting the cleaved CA protein. Dilutions were chosen to be in the linear response range of the ELISA. For quantitative determination of CA antigen, appropriate dilutions of cleared media were analyzed using a commercial ELISA kit (Cellular Products, Inc., Buffalo, NY) and were plotted against a CA standard curve. For Western blot analysis, cell or particle extracts were separated on SDS-polyacrylamide gels containing 17.5% polyacrylamide (200: 1 ratio acrylamide:N,Nmethylenebisacrylamide) in the resolving gel and transferred to nitrocellulose membranes (Schleicher and Schuell) by electroblotting. Blots were reacted with polyclonal antisera against different HIV proteins as indicated and with alkaline phosphatase conjugated second antiserum (Jackson lmmunochemicals Inc.) and were developed as described (Krausslich et a/., 1987). RESULTS Construction

of expression

vectors

Previously, it had been shown that efficient expression of HIV structural genes in mammalian cells (products of the gag, PO/,and env genes) requires the presence of a highly structured RNA element (rev responsive element; RRE) on the mRNA and the coexpression of the HIV rev protein in tram (Felber et a/., 1989; Malim et al., 1989). In order to construct a large number of expression vectors for different segments of the HIV-1 gag-pol region we wanted to generate a simple vector system that would allow rapid recloning of many different wild type and mutant sequences for expression in mammalian cells. To this end, we constructed a plasmid containing the HIV-1 RRE in the polylinker of pBluescript (PBS-RRE). Using unique restriction enzyme cleavage sites, different segments of the HIV gag-pal region were inserted in the correct orientation. Subsequently, the entire expression cassette was excised using flanking restriction sites that do not occur in HIV-l gag, PO/, or RRE and was inserted into the eukaryotic expression vector pKEX-XR (Rittner and Sczakiel, 1991). In this vector, expression of the HIV segments is driven by the human cytomegalovirus immediate early promoter/enhancer element (CMV-IE; Fig. 1) which has been shown to give strong expression after transient transfection and in stable cell lines. In addition, the vector contains a dominant selectable marker gene. For this study, nine different plasmids were constructed (Fig. 1). Plasmids pK-R-gpll and pK-R-FSII (all

ET AL.

plasmids were named pK-R- to indicate derivatives of pKEX-XR containing theRRE but will be referred to only as gpll etc.) were derived from previously described in vitro transcription/translation vectors (Krausslich et al., 1988) and contain a short segment of the HIV-1 nontranslated sequence (5’NTR), the entire gag gene, and the 5’ terminal part of the pal gene including PR, followed by two termination codons. Plasmids gpV and derivatives contain the entirepol reading frame in addition to the 5’NTR and gag. In all gp plasmids (for gagPO/), translation of the pal reading frame requires ribosomal frameshifting in the 3’terminal part of gag (Jacks et a/., 1988) whereas in the FS constructs (for frameshift) gag-pal fusion proteins are translated due to a 4-bp insertion close to the normal frameshift site (Krausslich et al., 1988). Plasmids gplla, FSlla, gpVa, and FSVa are identical to gpll, FSII, gpV, and FSV except for a point mutation changing the Asp25 of the PR active site to Ala25, thereby completely inactivating the enzyme (LeGrice et al., 1988). Plasmid gpllAmyr is identical to gpll except for a point mutation changing the Gly2 of thegag polyprotein to Ala2 therebyeliminating the signal for myristoylation of the gag and gag-pal precursor polyproteins (Bryant et a/., 1990, Gottlinger et a/., 1989). Analysis

of expression

products

Expression of HIV proteins was analyzed 48 hr after cotransfection of COS 7 cells with plasmids pK-R-gpll and pCMVrev by indirect immunofluorescence using antisera against HIV-1 CA and rev proteins (Fig. 2). With both antisera, abundant strongly fluorescent cells were seen. CA fluorescence was entirely cytoplasmic with some enhanced fluorescence in the perinuclear region and brightly fluorescent “granules.” The rev protein, on the other hand, showed a very strong nucleolar fluorescence (Fig. 2) as has been described previously (Felber et a/., 1989). Immunofluorescencepositive cells could be detected as early as 8 hr after transfection and were still found 72 hr after transfection and 5-l 5% of cells were fluorescence-positive at 48 hr after transfection. The intensity of staining with antiserum against CA was independent of whether pCMVrev or pMTcrev was used for cotransfection but expression of rev from pMTcrev could not be detected by immunofluorescence (data not shown). Transfection of pK-R-gpll in the absence of a rev expression vector did not yield any fluorescence-positive cells (data not shown). Synthesis and proteolytic processing of HIV antigens was analyzed by immunoblot of transfected cells using an antiserum against CA (Fig. 3a) or against RT (Fig. 4, left panel). Expression of gpll yielded the pr55g”g

HIV PARTICLE

FORMATION

5’NTR

PO1

NC p6

I

4

gPV

I

29

I1; I

i~:.~.:.;,:,~ ,l,iiIj. :,

,, ,i,,,,

gPVa FSV FSVa

----I

CMVIE

H

7 Asp25 -Ala

~HRRE

V Gly2 +

Ala

-

4 base pair

insertion

FIG. 1. Schematic diagram of the HIV-1 (BH 10) sequences contained in the expression plasmids. At the top, the relevant segment of the HIV-1 genome is depicted. The gag region (MA, CA, NC, p6; Leis ef a/., 1988) is shown as an open box, the pal region (p6*, PR, RT, IN) is shown as a shaded box, and the 5’ nontranslated region (8NTR) is shown as a black bar. In the middle part, the HIV segments inserted into the expression vector are depicted. Plasmid names are given to the left (plasmids are called pK-R-gpll. etc.). An Asp to Ala mutation in the PR active site (black triangle), a Gly to Ala mutation at the N-terminus of gag (open triangle), and the 4-bp insertion leading to gag-pal fusion proteins (-) are indicated. In the bottom part, the eukaryotic expression cassette of plasmid pKEX-XR (Rittner and Sczakiel, 1991) containing the human cytomegalovirus immediate early promoter/enhancer element (CMV-IE), the rev responsive element (RRE), and the SV40 t-antigen splice and polyadenylation signals (SV40 PA) is shown. For clarity, this diagram is not to scale.

precursor, an intermediate containing the matrix protein (MA) and CA (this intermediate did not react with antiserum against NC which detected primarily a protein of apparent M, 20 kDa; data not shown) and approximately equal quantities of two species of CA (Fig. 3a). This heterogeneity of CA is due to utilization of at least three different processing sites at its C-terminus giving rise to CA species that vary by a total of 14 amino acids (Henderson et a/., 1988; Mervis et al., 1988). Mutation of the PR active site in this construct (gplla) completely abolished proteolytic processing of the gag precursor and large amounts of pr55gag were observed (Fig. 3a). No additional immunoreactive pro-

teins were found, indicating that, in contrast to in vitro translation experiments (Krausslich et a/., 1988) translational initiation commences primarily at the authentic initiation codon and not at internal sites. Mutation of the signal for N-myristoylation (gpllAmyr) yielded a similar pattern of uncleaved and cleaved gag proteins as observed for wild type (Fig. 3a). However, in the case of gpl Wr, only the larger species of CA (variously called CA+ or ~25) was found and no processing to the smaller CA species (~24) occurred. Extending plasmids gpll and derivatives by the entire pal reading frame yielded very similar results after transfection. Expression of gpV and gpVa gave a pat-

MERGENER

30 anti

-

CA

anti - rev

ET AL.

pose that protein d is an intermediate processing product containing RT and IN (predicted M, 98 kDa) whereas proteins c and a correspond to PR-RT-IN and p6*-PR-RT-IN (M, 109 kDa and 1 15 kDa), respectively. No processing intermediates reacting with both, RT and CA, antisera were detected suggesting that separation of gag and pal segments of prl 60gagmpo’occurs very efficiently followed by separate processing of the gag and pal domains. The amount of cell-associated gag antigens was markedly reduced when gag and PR were translated in the same reading frame (FSII; Fig. 3a). Small amounts of CA and MA-CA but no pr55gag were observed. A mutation of the PR active site in this construct (FSlla) restored accumulation of cell-associated gag antigen yielding the expected gag-PR precursor (pr66; Fig. 3a). Similar results were obtained for FSV and FSVa. In the case of the PR mutant (FSVa), only the full-length prl 6OWg-Pol was detected with antiserum against CA (Fig. 3a) or against RT (Fig. 4, left panel). Translation of active PR as component of the gag-pol fusion protein all but abolished accumulation of gag proteins (Fig. 3a, FSV), whereas several species of RT products, closely related to those observed in the case of gpV (protein b probably corresponds to an alternative cleavage product in the p6* region), were observed with antiserum against RT (Fig. 4, left panel). Analysis

FIG. 2. Indirect immunofluorescence of COS 7 cells cotransfected with plasmids pK-R-gpll and pCMVrev. Cells were fixed 48 hr after transfection and were probed with a polyclonal antiserum against HIV-1 CA (top) or a monoclonal antibody against HIV-1 rev (bottom), followed by fluorescein isothiocyanate-labeled second antiserum.

tern that was virtually identical to expression of gpll and gplla (Fig. 3a). Analysis with an antiserum against RT revealed a series of RT related proteins (Fig. 4, left panel). Transfection of gpVa yielded the pr160gag-po’ precursor which remained completely uncleaved. Translation of prl 60gag-po’depended on an intact HIV frameshifting signal since mutation of this signal abolished its synthesis without effect on pr55gag (M.F. and H.-G.K., unpublished observation). In the case of gpV, we observed residual prl 60gag-po’and five major cleavage products. The two smallest products correspond to the p66 and p51 subunits of the RT heterodimer and did not react with antisera against PR or IN (data not shown). Antiserum against IN detected proteins d (97 kDa), c (108 kDa), and a (1 14 kDa) in addition to mature IN protein (32 kDa; data not shown), whereas only proteins c and a but not protein d were detected by antiserum against PR (data not shown). We therefore pro-

of particle

release

Culture media were collected from COS 7 cells 48 hr after transfection and were analyzed by ELISA. The antigen-capture ELISA used (Organon Teknika) detects only the cleaved CA protein but not the pr55gag precursor or MA-CA intermediate (H.-G.K.; unpublished observation). Accordingly, culture media from cells transfected with PR active site mutants remained completely negative, even in those cases where particle release could be confirmed by Western blotting or density gradient centrifugation (see below). Transfection of wild-type constructs gpll and gpV yielded release of large amounts of CA antigen into the media (Table 1). Using a quantitative ELISA we determined that approximately 400-750 rig/ml CA (8-l 5 pg total per transfection of 5 X 1O6 cells) were released 48 hr after transfection with gpll or gpV. Approximately 50% of this antigen was precipitated with PEG (Table 1) or was sedimented through a sucrose cushion (data not shown) indicating that it was particulate. Release of HIV antigen was completely rev-dependent (Table 1) and even after PEG precipitation of culture media from cells transfected with gpll in the absence of rev, no CA antigen could be detected in an undiluted sample whereas PEG precipitates from rev-cotransfected cells

HIV PARTICLE

FORMATION

b

66 -

31

PEG pellets

- pr160

- pr160

- ~166 - pr55

- ~166

45 -

- MA-CA

30 -

- CA/CA+

- pr55

- CA/CA+

FIG. 3. Western blot analysis of gag gene products after transient transfections. COS 7 cells transfected with the constructs indicated (in all cases, cotransfection with pMTcrev was performed) and media were harvested 48 hr after transfection. (a) Lysates of transfected cells were resolved by SDS-PAGE and Western blots were stained with rabbit polyclonal antiserum against CA. (b) Virus-like particles were precipitated from cleared media with PEG and particle lysates were analyzed as in (a). Molecular mass standards (in kDa) are indicated on the left, HIV-specific precursor proteins and cleaved gag products are identified on the right. CA+ indicates heterogeneity in C-terminal processing giving rise to several species of CA with slightly different electrophoretic mobilities (Krausslich et al., 1989).

were positive for CA antigen at a dilution of 1:5000. Release of CA antigen was also observed after transfection of FSII and gpllAmyr but in these cases no antigen could be precipitated with PEG. These results were confirmed by Western blotting of PEG precipitates with antisera against CA (Fig. 3b) and RT (Fig. 4, right panel). The major product detected in the case of gpll and gpV was the cleaved CA protein with little precursor remaining whereas in the case of gplla and gpVa pr55 gagand small amounts of pr66gag-PR (gplla) or prl 60gag-po’(gpVa) were detected (Fig. 3b and Fig. 4). In addition, PEG precipitates from gpv-transfected cells contained approximately equimolar amounts of both subunits of the heterodimeric RT (Fig. 4, right panel) but none of the intermediates observed in transfected cells (Fig. 4, left panel). No immunoreactive antigen could be found in PEG precipitates of culture media from cells transfected with FSII, FSV, or FSVa. Conversely, transfection with FSlla, which contains a short insertion at the frameshift signal and therefore lacks the C-terminus of gag, yielded release of a pr66gagmPR precursor protein that could be precipitated by PEG (Fig. 3b) and could be sedimented through a sucrose cushion (data not shown). For analysis of particle-associated RT activity, cells were transfected with plasmids gpll, gpV, or gpVa (cotransfection with pMTcrev in all cases) and PEG precipitates from culture media 48 hr after transfection were tested for RT activity. Only background levels of RT

activity were found in media from mock-transfected or gpll-transfected (not containing the RT gene) cells whereas significant levels of RT were found after transfection with gpV (Fig. 5). Culture media from gpVatransfected cells reproducibly yielded RT levels that were lo- to 20-fold lower than in the case of gpV but were significantly higher than background levels (Fig. 5). Characterization

of virus-like

particles

In order to characterize the physical nature and content of particles released from transfected cells, extracellular particles were sedimented through a sucrose cushion followed by a 20-600/o sucrose gradient. Fractions were analyzed for CA content and RT activity. A sharp peak of RT activity and CA antigen coinciding at a density of 1.16 g/ml was observed for particles derived from gpV-transfected cells (Fig. 6a). In addition, these particles contained RNA since a peak of radioactivity at the same density was observed when gpVtransfected cells were labeled with [3H]UTP and extracellular particles were analyzed on a sucrose gradient (Fig. 6b). Density gradient analysis of particles released from cells transfected with gpll or gplla showed virtually identical profiles, again giving a peak of CA antigen at approximately 1.16 g/ml. Conversely, sedimentation of particulate material from media of FSlla-transfected cells did not give a sharp peak but a diffuse

MERGENER

32

PEG pellets

cells

> iii

g &;

> [2

9 f.2

5 8

> 8

116-

9166-

ET AL

structures leading to the release of immature extracellular particles were not observed. Assembly appeared to be arrested at an early stage and large plaques of electron-dense HIV ribonucleoprotein beneath the plasma membrane of an otherwise healthy cell were found (Fig. 9a). Extracellular membrane-bound particles of irregular size and shape were shed from FSllatransfected cells and these particles contained multiple assembly sites that were arrested at an early stage (Fig. 9b). DISCUSSION

45-

24-

FIG. 4. Western blot analysis of RT proteins after transient transfections. Lysates from COS 7 cells transfected with the constructs indicated (cotransfection with pMTcrev in all cases; panel cells) and PEG precipitates of released particles (panel PEG pellets) were resolved by SDS-PAGE. Western blots were stained with rabbit polyclonal antiserum against RT. Molecular mass standards (in kDa) are shown on the left, relevant RT precursor proteins and cleavage products are identified in the middle. a, b. c, and d identify processing intermediates of prl60 gagepo’that are described in the text.

distribution of antigen in many fractions of the gradient suggesting heterogeneity of particles (data not shown). To obtain direct evidence for budding and release of HIV-like particles, transfected cells and sediments of respective supernatants were analyzed by thin section electron microscopy. Typical lentiviral budding structures, consisting of a crescent electron-dense ribonucleoprotein layer directly under the plasma membrane, were found in cells transfected with gpll (Fig. 7a). Immature HIV-like particles of uniform size and morphology containing an electron dense ring structure were released from transfected cells (Fig. 7b) and were found in the extracellular space (Fig. 74. Budding structures were also observed in cells transfected with gplla but, in contrast to gpll, budding occurred mainly intracellularly into cytoplasmic vacuoles and considerably less budding structures were observed at the plasma membrane (Fig. 8a). Particles appeared delayed or arrested in budding and were not completely uniform in size or morphology. The electron-dense immature core did not appear as a closed ring structure as in the case of the wild-type sequence (Figs. 7b and 7c) but had a polar distribution as half-circle (Figs. 8b and 8~). Ultrastructural analysis of cells transfected with FSlla revealed a very different pattern. Regular budding

Although infectious retroviral particles are complex structures consisting of the viral gag, pal, and env proteins in addition to RNA and a host-derived lipid membrane, it appears that only the products of the viral gag gene, and potentially only segments thereof (Weldon et a/., 1990) are required for assembly and budding. This was first suggested by the observation of defective particles apparently consisting only of gag gene products (Shields et al., 1978) and was recently supported by several studies reporting that virus-like particles are released into the culture medium on expression of HIV1 gag and pal genes (Karacostas et al., 1989; Shioda and Shibuta, 1990; Haffar et a/., 1990; Hu et a/., 1990; Gheysen et a/., 1989; Smith et a/., 1990). In the present communication, we studied polypro-

TABLE 1 ANALYSISOF HIV ANTIGEN RELEASEDAFTERTRANSFECTION

Plasmid

0 gpll -rev gpll +rev gpll Amyr +rev pgV +rev FSII +rev FSV +rev

Total antigen (absorbance U/ml)

PEG precipitated/ total antigen, %

0

nd.

0 707 122 956 242 161

50 0 40 0 nd.

Note. Analysis of HIV antigen released from COS 7 cells after transient transfection. Supernatants from COS cells transfected with the constructs indicated were collected 48 hr after transfection and analyzed for HIV antigen by ELISA. Samples of supernatants were also measured after PEG precipitation and the relative amount of precipitated antigen, given as percentage of total antigen in the medium, was calculated. The numbers given represent a single experiment but were reproducible to f 20% in three independent experiments. Antigen concentrations are given as absorbance U/ml, an arbitrary unit derived from the absorbance readings. 1000 absorbance U/ml were approximately equivalent to a CA protein concentration of 800 rig/ml as measured by a quantitative ELISA. Note that the ELISA used in this experiment detects only cleaved CA antigen but not precursor polyproteins. - indicates that no HIV antigen was detected in the PEG precipitate; nd.. not determined.

HIV PARTICLE FORMATION

33

FIT-Assay

wm

ClPll

c!PV

ClpVa

FIG. 5. Reverse transcriptase assay. Particles from 4 ml culture medium of COS 7 cells, 48 hr after transfection with the plasmids Indicated (cotransfection with pMTcrev), were sedimented through a sucrose cushion and analyzed for RT activity. The cpm values represent the mean of two independent experiments

tein synthesis and processing as well as particle assembly and release after transient transfection of expression vectors encoding segments of the HIV-l gag and pal genes and mutants thereof in mammalian cells. The validity of a simplified expression system for the analysis of HIV morphogenesis requires that mutant phenotypes observed in proviral constructs can be reproduced in the subviral system. We therefore analyzed constructs encoding either the wild-type gag-pal region of HIV-1 or mutants destroying the PR active site or the signal for N-myristoylation. Expression of the wild-type sequence in COS cells yielded release of virus-like particles and microgram quantities of particle-associated CA antigen could be obtained from a single transfection. Protein synthesis and particle production were dependent on the presence of the rev protein which had been shown to be required for expression of HIV gag, PO/, and env proteins in infected cells (Felber et al., 1989; Malim et a/., 1989). Extracellular particles consisted primarily of cleaved gag proteins and contained the p66/p51 heterodimeric RT. Particle release occurred by typical lentiviral budding and extracellular particles had the size and morphology of HIV except for the lack of glycoprotein spikes. Analysis of particles on a sucrose density gradient revealed a sharp peak at a density of 1.16 g/ml for CA antigen, RT-activity, and RNA, in good agreement with the density reported for HIV particles (Popovic et al., 1984). Specific packaging of the HIV-derived RNAs

was not investigated but appears possible since these RNAs contain a sequence which had been shown to be important in HIV genomic RNA packaging (Lever et a/., 1989). Particle production was not dependent on the presence of an active PR and, similar to what has been observed with proviral mutants (Kohl et a/., 1988; Peng et a/., 1989), morphologically immature particles consisting only of uncleaved gag and gag-pal polyproteins were released from transfected cells. Immature particles banded at the same density as mature particles but contained lo- to 20-fold reduced levels of RT activity, indicating that the uncleaved gag-pal polyprotein contains low RT activity or that undetectable processing of polyproteins by a nonviral proteinase had taken place. These results are in agreement with observations reported for immature ALV virions (Stewart et al., 1990) where RT activity of immature particles was reduced by at least 30-fold and for bacterially expressed HIV-l RT where proteolytic removal of both the PR domain and the IN domain was necessary for full activation of RT (LeGrice et a/., 1988). Conversely, however, Peng eta/. (1991) reported that an HIV mutant containing a PR active site substitution had nearly normal levels of RT activity. This discrepancy is currently unresolved although we cannot completely rule out an effect of other HIV gene products, not present in our or the bacterial expression systems, on RT activity of the gag-pal polyprotein. This appears unlikely, however,

MERGENER

34

a z

1.24

\ 25 h Y s2

1.20

5 3 s b

ET AL.

w

A450

16000

........ .... “k_‘._,

1.50

0 ELISA l RT-Assay 12000

1.00 1.16 8000

1.12

0.50 4000

1.08

bottom

fractions

b cpm

1-5

6-7

3H-UTP

8

9

10

11

12

13

14

incorporation

15

16

17

18

19

20

21 I22

23 25-27 124

FIG. 6. Density gradient analysis of HIV-like particles. (a) Particles sedimented from the medium of COS cells 48 hr after cotransfection with plasmids pK-R-gpV and pMTcrev were analyzed on a continuous 20-60% sucrose density gradient. After centrifugation, 0.5-ml fractions were collected and analyzed for CA protein by ELISA (open circles; left ordinate A450 represents absorbance values of ELISA readings) or for RT activity (solid circles; right ordinate represents cpm values). Refraction was determined for each fraction and the corresponding density of sucrose [g/ml] is depicted as a dotted line. (b) RNA content of HIV-like particles was analyzed by sucrose density gradient centrifugation of extracellular particles from transfected cells that had been grown in the presence of [3H]UTP. Gradient fractions were collected as above and were analyzed for refractive index and for radioactivity. The peak fraction (14) corresponds to a density of 1 .16 g/ml.

given that a specific inhibitor of HIV PR reduced RT activity in the medium of a chronically infected culture to 39'0 while production of particles was almost unchanged (Schatzl et al., 1991).

Ultrastructural analysis of ceils expressing uncleaved mutant polyproteins revealed only few budding structures at the plasma membrane and budding occurred mainly into cytoplasmic vacuoles. While PR

HIV PARTICLE

FORMATION

35

FIG. 7. Thin section electron microscopy of COS cells cotransfected with pK-R-gpll and pMTcrev at 38 hr after transfection. (a) In the representative low magnification view, part of the cell is shown with the plasma membrane involved in particle formation and release. (b) Late stages of particle assembly showing a dense layer of precursor protein underneath the lipid bilayer. (c) Release of particles showing still immature core organization and, as to be expected, the absence of SU protein knobs. Magnifications: (a) 20,000; bar = 1 PM; (b, c) 100,000; bar = 100 nn/l

activity is not required for release of particles, it is conceivable that, at least in some retroviruses, active PR may have a role in targeting of polyproteins to the site of assembly, e.g., by proteolytic cleavage of cytoskeletal components. Peng et al. (1989) reported that transfection of a mutant HIV provirus containing a deletion of the PR active site region gave primarily intracellular budding into cytoplasmic vacuoles, very similar to what was seen in the present study. Moreover, specific inhibition of HIV PR in an infected culture appeared to induce budding arrest and alterations of particle morphology were observed (Schatzl et al., 199 1). Conversely, Stewart et al. (1990) reported that, in the case of ALV, budding was mainly at the plasma membrane, but also into cytoplasmic vacuoles, indepen-

dent of PR activity. These discrepancies may be entirely due to expression levels or cell lines used but may also reflect differences in retroviral PR activation. In contrast to the situation in avian retrovirus infected cells where processing is tightly linked with particle release (Eisenman eta/., 1975; Wills eta/., 1989) significant intracellular processing is seen in lytically, but not in chronically HIV-infected cells (Kaplan and SwanStrom, 199 1), as well as in transiently transfected cells (Gdttlinger et al., 1989; this study). Deletion of the signal for N-myristoylation in the subviral constructs abolished release of particles, as observed for proviral mutants (Bryant and Ratner, 1990; Gdttlinger et a/., 1989). Extracellular antigen was reduced to a level of approximately 10% of wild type but

36

MERGENER

ET AL.

FIG. 8. Thin section electron mrcroscopy of COS cells cotransfected with pK-R-gplla and pMTcrev at 38 hr after transfection. (a) The low magniftcatron shows multiple particle formation into intracellular vacuoles as well as a single bud at the plasma membrane. (b, c) Budding and released particles at plasma membrane and in vacuole, respectively. Magnifications: (a) 50,000; bar = 0.5 p/l; (b, c) 100,000; bar = 100 nM.

was entirely nonparticulate. In the case of Rous sarcoma virus, Wills et a/. (1989) have shown that high level expression of nonmyristoylated gag polyproteins in mammalian cells yields fivefold less extracellular antigen than was observed for a myristoylated polyprotein but these authors did not determine the particulate nature of the nonmyristoylated extracellular gag protein. Normal levels of polyproteins were synthesized and processed in gpllAmyr-transfected cells. In contrast to our results and to those of Gottlinger et al. (1989), Bryant and Ratner (1990) reported significant reduction of polyprotein processing for a myristoylation deficient provirus and when myristoylation was inhibited in HIV-

infected cells (Bryant et a/., 1991). Conceivably, these differences are due to different expression levels. Dimerization of gag-pol polyproteins is presumed to be required for PR activation and higher amounts of mutant HIV polyproteins, which are not concentrated at the assembly site, may be required to initiate processing. The products of the retroviral polgenes are normally produced by ribosomal frameshifting or by readthrough of a termination codon, and in MuLV it has been shown that constitutive expression of gag-pal polyproteins in the absence of gag proteins is incompatible with particle assembly and yields stable intra-

HIV PARTICLE FORMATION

FIG. 9. Thin section electron microscopy of COS cells cotransfected with pK-R-FSlla and pMTcrev at 38 hr after transfection. (a) Assembly of precursor proteins at multiple sites underneath the plasma membrane. (b) Release of membrane bound particles with multiple early budding sites but no release of normally formed immature virions. Magnifications: (A) 25,000; bar = 1 pM; (b) 100,000; bar = 100 nM.

cellular gag-pol polyproteins (Felsenstein and Goff, 1988). Similarly, we observed that expression of HIV gag and pal in the same reading frame (FSV) abolished particle release but, in contrast to the results of Felsenstein and Goff (1988) on MuLV, we observed significant processing of the fused HIV gag-pal polyproteins. Markedly reduced levels of cell-associated gag and pal antigens were observed in FSV-transfected cells. This effect was enhanced when the RT and IN domains were deleted (FSII), thereby obviating the need for cleavage at the C-terminus of PR. Extracellular CA antigen was completely cleaved but was nonparticulate in both cases. Recovery of cell-associated antigens was restored by mutation of the PR active site in these constructs, indicating that the observed phenotype was caused by PR activity. Similar results have recently

been obtained after expression of a single chain dimer of HIV PR as component of the viral gag-pol polyprotein (Krausslich, 1991 a). Overexpression of enzymatitally active HIV PR therefore appears to cause rapid autocatalytic processing of viral polyproteins thereby dissociating the components of the particle from the site of assembly. Increased cytoplasmic concentration of active HIV PR also leads to the rapid killing of the transfected cell presumably by cleavage of essential cellular proteins (e.g., vimentin; Shoeman eta/., 1990). These effects can be completely prevented by specific inhibitors of HIV PR (Krausslich, 1991 b) or by mutation of the PR active site. In a recent study, it was reported that introduction of a stop codon at the N-terminus of p6 (the C-terminal processing product of pr55 gag, released from NC) in an

MERGENER

38

HIV proviral construct abolished particle release with large numbers of crescent particles, apparently arrested in the budding process, lining the plasma membrane of the transfected cell (Gdttlinger et al., 1991). A similar phenotype might be expected after transfection of FSlla which has a short insertion, thereby changing to the pol reading frame, only 11 codons upstream from the N-terminus of p6. Transfection of FSlla yielded the expected precursor of 66 kDa but, contrary to expectation, significant amounts of extracellular particles consisting of the gag-PR polyprotein were found which appeared heterogeneous when analyzed on a sucrose gradient. Ultrastructural analysis of FSlla-transfected cells revealed that budding was arrested even earlier than in the case of the p6 stop mutant and multiple assembly sites underneath the plasma membrane were found. In contrast of the results of Gbttlinger et a/. (1991) who did not find any extracellular particles, we observed shedding of membrane bound particles of irregular size and shape but no release of normally formed virions. Extracellular particles were not found when a full-length gag-polfusion polyprotein with a PR active site mutation was expressed. In the case of Rous sarcoma virus, Weldon et al. (1990) reported the release of pleomorphic particles by a normal budding mechanism after transfection of subviral constructs encoding C-terminal fusions of cytochrome c with gag. Substitutions of foreign polypeptides for the C-terminal regions of gag may disrupt the organized arrangement of gag polyproteins normally found in viral particles and a range of phenotypes might be expected depending on the level of structural alteration. Particle release may only be compatible with a certain length of the gag polyprotein and lack of partcle formation in the case of truncated gag polyproteins (Gi)ttlinger et al., 1991; H.-G.K., unpublished observation) as well as in the case of gag-pal fusion proteins (Felsenstein and Goff, 1988; this study) may conceivably reflect this size requirement. Alternatively, or in addition, the p6 domain may also serve a specific function in HIV budding and further experiments will address this question. ACKNOWLEDGMENTS We are grateful to B. Felber and G. Pavlakis, and to K. Rittner and G. Sczakiel for plasmids, and to B. Fleckenstein, M. Pawlita and T. Restle for antisera. We also thank H. zur Hausen for continued support and interest, and V. Bosch, M. Pawlita. and G. Sczakiel for discussions and suggestions. We are grateful to A.-M. Traenckner for excellent technical assistance and to V. Bosch for critically reading the manuscript. This work was supported in part by grants from the Ministery for Research and Technology to H.R.G. (OlZR8901/8) and to H.G.K. (FG5-1075).

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39

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