©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical and Genetic Definition of the Cellular Protease Required for HIV-1 gp160 Processing (*)

(Received for publication, October 5, 1994)

Alex Franzusoff (1)(§) Alison M. Volpe (1)(¶) Denise Josse (1) Sergio Pichuantes (2)(**) Joseph R. Wolf (1)

From the  (1)Department of Cellular and Structural Biology, University of Colorado Medical School, Denver, Colorado 80262 and the (2)Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The surface glycoproteins of enveloped viruses bind to target cell receptors and trigger membrane fusion for infection. The human immunodeficiency virus 1 (HIV-1) envelope glycoproteins gp120 (CD4 binding protein) and gp41 (transmembrane fusion protein) are initially synthesized as a gp160 precursor. The intracellular cleavage of gp160 by a host cell protease during transit through the secretory pathway is essential for viral activities such as infectivity, membrane fusion, and T-cell syncytium formation. We report that gp160 biogenesis, protein processing, and cell-surface expression have been successfully reproduced in the yeast Saccharomyces cerevisiae. Genetic and biochemical approaches are used for defining that the unique cellular protease, Kex2p, is directly responsible for HIV-gp160 processing in yeast, in vivo and in vitro. The yeast system described in this report represents a powerful strategy for identifying, characterizing and inhibiting the host T-cell protease essential for HIV infectivity and AIDS.


INTRODUCTION

The env gene of the human immunodeficiency retrovirus HIV-1 (^1)encodes gp160, the viral surface (envelope) glycoprotein precursor. gp160 is directed into the host cell secretory pathway for transport to the cell surface. During its transit through the pathway, gp160 is modified by glycosylation, and a fraction of the molecules are proteolytically processed to the mature gp120 and membrane-bound gp41 forms (Haseltine, 1991; Willey et al., 1988; Earl et al., 1991b). At the surface of infected human T-cells, gp120 and gp41 remain non-covalently associated for incorporation into budding virions and for the capture of uninfected CD4 cells into syncytium formation (Willey et al., 1988; Bosch and Pawlita, 1990). The fusion of HIV-infected with uninfected cells promotes the virion-independent spread of infection and contributes to the depletion of the T-cell repertoire. By interfering with the gp160 processing event, the viral surface glycoprotein is incapable of mediating membrane fusion, thereby abolishing the spread of infectious virions (Kowalski et al., 1987; Haseltine, 1991; McCune et al., 1988; Bosch and Pawlita, 1990; Renneisen et al., 1990; Walker et al., 1987; Hallenberger et al., 1992).

The host protease responsible for processing HIV-gp160 precursors in the human T-cell secretory pathway has not been unequivocally determined. Examination of the amino acid sequences at the junction between subunits of the envelope glycoprotein precursors in a number of enveloped retroviruses revealed strong pressure to retain a dibasic amino acid sequence (Lys-Arg or Arg-Arg) proximal to the cleavage site (McCune et al., 1988). In yeast, the unique Kex2 protease is responsible for processing pro-protein precursors bearing this dibasic amino acid sequence in transit from distal Golgi compartments of the secretory pathway (Franzusoff et al., 1991; Fuller et al., 1989a; Julius et al., 1984; Mizuno et al., 1988; Redding et al., 1991; Wilcox and Fuller, 1991). Several members of the mammalian, subtilisin-like processing proteases (referred to as subtilisin-like protein convertases or SPC enzymes) were discovered by their similarity to the yeast Kex2 enzyme (for review, see Barr, 1991; Seidah et al., 1991; Steiner et al., 1992 and references therein). To date, five different human SPC proteases have been identified, yet by comparison with gene products identified in other mammalian and insect cells, additional human SPC proteases are predicted to exist. Specific SPC enzymes have been implicated in proprotein processing within either the constitutive or regulated secretory pathways. This means that the individual enzymes may function to cleave protein precursors in trans-Golgi or post-Golgi intracellular compartments, depending on their intracellular localization. However, with the exception of human furin, the distribution of the other SPC enzymes within the cells of different organs has been mostly analyzed by Northern or in situ hybridizations (Barr, 1991; Seidah et al., 1991; Steiner et al., 1992; Schäfer et al., 1993; Molloy et al., 1994; Bosshart et al., 1994).

The high degree of similarity between yeast and mammalian cells in general secretory pathway function and in pro-protein processing by the yeast Kex2 protease and the mammalian SPC enzymes suggested that HIV-1 envelope glycoprotein biosynthesis, processing, and cell-surface expression could be precisely reproduced in yeast. In this report, we describe the characterization of HIV-1 env gene expression in yeast. Both genetic and biochemical approaches were used to define Kex2p as the cellular protease directly involved in HIV-1 gp160 cleavage. The use of yeast mutants defective in Kex2 protease function provides the system for identifying and characterizing the host protease responsible for gp160 processing required for infectious virion production and multi-nucleated cell syncytium formation in HIV-infected human T-cells.


EXPERIMENTAL PROCEDURES

Strains and Media

Several Saccharomyces cerevisiae yeast strains used in this study were derived from strain AFY16 (MAT alpha, leu2-3,-112, his4-519, trp1-289, pep4::URA3, prb1, gal2). Strain pG5-KEX2DeltaC3/CB023, which secretes a truncated form of Kex2p (ssKex2p), has been previously described (Brenner et al., 1992). The strains were maintained on media containing 0.67% yeast nitrogen base (Difco), relevant metabolic supplements, and 2% glucose. Derepression of transformants bearing a glucose-repressible promoter was achieved for 2 h to overnight by culturing the cells in the same media, with the carbon source modified to 0.1% glucose, 1% raffinose.

Plasmid Constructions

The sequence encoding the mature product of the envelope (env) gene was amplified by polymerase chain reactions, using as template the cloned HIV-1 genome (Sanchez-Pescador et al., 1985). The gp128 sequence, representing a premature truncation of HIV-1 env gene at Trp-678 just before the transmembrane domain sequence of the mature gp41, was also generated by polymerase chain reaction amplification of the HIV-1 genome. The gp160 and gp128 sequences were ligated in frame to the yeast alpha-factor signal and leader (pre-pro-) sequences (Kurjan and Herskowitz, 1982). A putative Lys-Arg protease recognition sequence separated the alpha-factor and HIV-1 env sequences (see text for further details). The construct was placed under the control of the ADH2-GAP promoter and GAP terminator sequences. The 2-µm high copy heterologous expression vector pAF425 included selectable markers for ampicillin resistance, as well as URA3 and leu2-d selection in yeast, as described (Pichuantes et al., 1994).

Expression of gp160 Construct in Yeast

gp160 and its cleavage products were examined by either immunoprecipitation of lysates from radiolabeled yeast or by immunoblot analysis of lysates from unlabeled yeast. For immunoprecipitation analysis, yeast were metabolically radiolabeled with S-Translabel (methionine-cysteine, Amersham Corp.) for 30 min at 30 °C, as previously described (Franzusoff et al., 1991). Cells were lysed with glass beads in the presence of 5 times SDS sample buffer (250 mM Tris, pH 6.8, 50% glycerol, 10% SDS, 12.5% beta-mercaptoethanol, 0.01% bromphenol blue), heated at 70 °C, and then diluted with buffer for immunoprecipitation with rabbit gp120, gp41, or alpha-factor antibodies (Franzusoff and Schekman, 1989). The antibody-antigen complexes were incubated with protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). Endoglycosidase H treatment (Boehringer Mannheim) was performed as previously described (Böhni et al., 1988). The precipitates were solubilized in SDS sample buffer and then resolved on 8% polyacrylamide SDS gels as described before (Franzusoff and Schekman, 1989). The gels were fixed, and the radioactive polypeptide species were enhanced for fluorography with Amplify (Amersham).

For immunoblot analysis, cells were ruptured by glass bead lysis with 8 M urea, 1% SDS, 1 mM dithiothreitol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture. Lysates were heated at 55 °C for 30 min and then diluted with SDS sample buffer. The samples were concentrated by chloroform/methanol extraction (Wessel and Flugge, 1984). The pellets were resuspended in urea/SDS dissociation buffer at 70 °C for 10 min, and the gp120- or gp41-related polypeptides were resolved on 8 or 10% polyacrylamide SDS gels, respectively. The proteins were transferred to nitrocellulose overnight at 0.2 A, 4 °C in Tris-glycine buffer containing 0.02% SDS, 20% methanol as described (Towbin et al., 1979). Antibody binding to the nitrocellulose was detected with I-protein A (ICN), and the blots were exposed to film at -80 °C. The specificities of the rabbit anti-gp120 and anti-gp41 antisera have been previously reported (Barr et al., 1987).

Cell-surface Labeling

Cultures of yeast harboring vector alone or plasmid pAF425 containing the alpha-factor-gp160 construct (see legend to Fig. 1) were shifted to derepressing media for 2 h at 30 °C and then incubated 5 min with 10 mM sodium azide to block subsequent secretory traffic. Approximately 5 times 10^8 yeast cells were harvested, washed 3 times with phosphate-buffered saline, and then resuspended in 1 ml of phosphate-buffered saline. Sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce) was added at 50 µg/ml to the yeast suspension and incubated for 30 min at 25 °C; then, excess reagent was quenched by addition of Tris, pH 7.5, to 50 mM. The cells were sedimented, washed twice with phosphate-buffered saline at 4 °C, and lysed with glass beads and SDS sample buffer. Lysates were incubated overnight with concanavalin A-Sepharose (Pharmacia) or activated monomeric avidin-agarose (Pierce). The beads were washed, and the precipitated proteins were treated with endoglycosidase H and then resolved on SDS gels for immunoblot analysis, as described above.


Figure 1: Analysis of HIV-1 gp160 expression in yeast. A, the cartoon summarizing the construct and potential products from expression of the HIV-1 env gene fused to the yeast alpha-factor signal leader sequence. The predicted mass of the different (deglycosylated) protein intermediates is shown on the right. The verticalbars signify the expected cleavage junctions in the alpha-factor-gp160 fusion protein, while the asterisks denote the distribution of the seven potential dibasic amino acid cleavage sequences in the entire polypeptide. B, immunoblot analysis of lysates from yeast strains harboring vector alone (V), the full-length alpha-factor-gp160 construct (160), and the truncated alpha-factor-gp128 construct (128). Lanes1-3 were probed with gp120-specific antisera; lanes4-6 were probed with gp41-specific antisera. The arrow points to a novel polypeptide migrating at 41 kDa, specifically recognized by the gp41 antisera. An additional 100-kDa polypeptide observed in lysates from gp128-expressing yeast that cross-reacts with both antisera may result from latent processing of a dibasic amino acid sequence not normally protease accessible in the full-length polypeptide. C, the gp120-related intermediates of alpha-factor-gp160 processing in yeast are revealed by treatment of the immunoprecipitates with endoglycosidase H (EndoH) before second round immunoprecipitation with gp120-, gp41-, or alpha-factor-specific antisera. The pattern of observed gp120-related polypeptide products are consistent with the expected pattern of specific in vivo cleavage products illustrated in Fig. 1A. Surprisingly, the products still harboring the alpha-factor leader sequence were more abundant than the fully processed forms (i.e. alphaF-gp160 and alphaF-gp120, Fig. 1C, lanes2-4). However, the first two amino acids of the mature gp160 at the junction between alpha-factor and gp120 (Thr-Arg) are apparently incompatible with efficient protease recognition. This observation also indicates that sequences downstream of the dibasic amino acid processing site may profoundly influence the catalytic efficiency of the host cell protease. Ab, antibody.



KEX2 Gene Disruption in Yeast

The isogenic kex2Delta strains were generated by gene replacement of the KEX2 gene in wild-type yeast strain AFY16 harboring the alpha-factor-gp160 plasmid pAF425 (for details of the KEX2 gene replacement construct for linear DNA transformation, see Fuller et al., 1989a). Gene disruption in the kex2Delta strains was confirmed by Kex2p immunoblot analysis and halo assays of alpha-factor mating pheromone maturation (Fuller et al., 1989a). The gp41 immunoblot analysis was performed as described above.

Biochemical Assays of Kex2p Protease Activity

Secreted soluble Kex2 protease (ssKex2p) was purified from the culture supernatant of pG5-KEX2DeltaC3/CB023 yeast as described (Brenner and Fuller, 1992). To test alpha-factor processing in vitro, nuclease-treated, gel-filtered yeast cytosol was programmed with in vitro transcribed alpha-factor RNA for translation in the presence of [S]methionine (Amersham) as described (Franzusoff et al., 1992). Pre-pro-alpha-factor cleavage experiments were performed by dilution of gel-filtered translation extracts into protease reaction buffer (0.2 M BisTris buffer, pH 7.4, 1 mM CaCl(2), 0.01% Triton X-100) and incubated with ssKex2p for 1 h at 37 °C in the absence or presence of 4 mM EGTA (instead of calcium in the reaction buffer). The reaction was terminated by heating at 95 °C with SDS-gel dissociation buffer, and the proteins were resolved on 15% SDS-polyacrylamide gels. The gels were fixed and then treated with Amplify before drying and exposure to film for autoradiography.

To test the cleavage of HIV-1 gp160 expressed in yeast, cultures were grown in derepression media overnight for expression of the alpha-factor-gp160 construct. The harvested yeast cells were converted into spheroplasts by treatment with oxalyticase (Enzogenetics, Corvallis, OR) as described (Franzusoff et al., 1992) and then lysed by solubilization under non-denaturing conditions with detergent-containing radioimmune precipitation buffer (50 mM Tris-Cl, pH 8, 0.15 M NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.5 mg/ml bovine serum albumin) in the presence of a mixture of proteinase inhibitors. Yeast lysates were incubated with alpha-factor antisera and protein A-Sepharose to isolate alpha-factor-gp160 and alpha-factor-gp120 polypeptide precursors, as described above. The immunoprecipitates were resuspended in protease reaction buffer with or without ssKex2p in the absence or presence of 5 mM EGTA for 1 h at 37 °C. The reaction was terminated by heating with 0.5% SDS at 95 °C for 5 min prior to endoglycosidase H treatment as above. The reaction products were resolved on SDS gels and then transferred to nitrocellulose for immunoblot analysis as before.


RESULTS

HIV-1 gp160 Expression in Yeast

For expression in yeast, the env gene (from HIV-1 viral isolate) lacking its signal sequence was fused to the signal and leader sequences of the yeast gene encoding the secreted alpha-factor pheromone under the control of an inducible ADH2-GAP promoter (pAF425, Fig. 1A). The alpha-factor signal-leader sequences facilitate the trafficking of heterologous proteins through the yeast secretory pathway (Pichuantes et al., 1994). The amino acids at the junction between the alpha-factor leader and the N terminus of HIV-1 gp160 would normally be processed by the yeast Kex2 protease during transit through the Golgi apparatus (Franzusoff et al., 1991; Redding et al., 1991). Growth of pAF425-transformed yeast on media containing raffinose resulted in the production of novel polypeptides recognized on immunoblots by both gp120 and gp41 antisera (Fig. 1B, lanes2 and 5). The high molecular weight gp160 polypeptides represented heterogeneously glycosylated species, as shown by precipitation with concanavalin A-Sepharose and by antibodies specific for the yeast Golgi-specific carbohydrate modifications (Franzusoff and Schekman, 1989; data not shown). These results indicate that HIV-gp160 expressed in yeast had been properly directed into the secretory pathway and that the quality of intra- and inter-molecular folding of the heterologous membrane protein was apparently sufficient for egress to post-endoplasmic reticulum destinations (Gething et al., 1986; Hurtley and Helenius, 1989; Earl et al., 1991a). Processing of gp160 precursor protein to a discrete polypeptide of 41-kDa mobility on SDS gels was detected by gp41-specific antisera (Fig. 1B, lane5versuslane3). The 41-kDa polypeptide was not observed in yeast transformed with vector alone (lane4) or gp128-expressing plasmid, which encodes a C-terminal truncation of the env gene (lane6). This result confirmed that the gp41 species observed in yeast transformed with the entire gp160 construct resulted from a discrete proteolytic processing event rather than a random fragment of the gene product.

The expected gp120-related products were not distinguishable by immunoblots due to the overlap in mobility on SDS gels with the more abundant, heterogeneously glycosylated gp160 precursor (Fig. 1B, lanes1-3). Endoglycosidase H digestion of the N-linked carbohydrates following immunoprecipitation with gp120-specific antisera from radiolabeled yeast lysates simplified the polypeptide pattern. Second round immunoprecipitations with gp120-, gp41-, or alpha-factor-specific antisera revealed the predicted number of gp120-related polypeptides (Fig. 1, A and C). Approximately 10-25% of gp160 is cleaved in yeast, consistent with the 5-25% efficiency of processing in virally infected human T-cells and lymphoid cell lines expressing the HIV-1 env gene (Willey et al., 1988; Earl et al., 1991b). Furthermore, despite the presence of seven dibasic amino acid sequences (Lys-Arg or Arg-Arg) distributed throughout the alpha-factor-gp160 polypeptide (marked by asterisks in Fig. 1A), the observed pattern of gp120 and gp41 cleavage products in Fig. 1, B and C, implied a restricted availability of protease recognition sites. Hence, these results demonstrated the presence of in vivo host cell protease activity in the yeast secretory pathway with gp160 precursor cleavage specificities similar to that observed in HIV-1-infected human T-cells.

Cell-surface Expression of the Viral Glycoproteins

The presence of the envelope glycoproteins on the yeast cell surface was evaluated by two separate approaches. Indirect immunofluorescence and confocal microscopy of yeast expressing HIV-1 gp160 showed intracellular labeling with both gp120 and gp41 antisera and abundant, distinct staining evenly distributed on the cell surface (data not shown).

The immunofluorescent results do not distinguish whether intact gp160 or the processed products were exhibited on the yeast cell surface. To resolve this question, biotin labeling of intact yeast, followed by precipitation with either avidin-agarose or concanavalin A-Sepharose of the proteins from cell lysates, was performed. The precipitates were treated with endoglycosidase H to distinguish the gp120 products from the intact gp160 forms. Immunoblotting with antibodies to the plasma membrane ATPase showed that the protein was efficiently labeled by the membrane-impermeant biotin labeling reagent (Fig. 2, lanes7 and 8). Both the intact and processed products of gp160 were labeled in yeast harboring the gp160 expression plasmid but not vector alone (Fig. 2, lanes1-6). A vacuolar enzyme, carboxypeptidase Y, was not precipitated with avidin-agarose, confirming that the biotin labeling reagent had not penetrated the membrane (data not shown). The combined results demonstrated that the HIV envelope glycoprotein traffic through the secretory pathway has been reproduced in yeast and that proteolytic processing is not essential for transit to the cell surface.


Figure 2: Cell-surface detection of HIV-1 envelope glycoproteins expressed in yeast. Biotinylation with the membrane impermeant sulfosuccinimidyl-6-(biotinamido)hexanoate reagent was used to label cell-surface proteins of intact yeast harboring vector alone (v) or alphafactor-gp160 constructs (160). Yeast lysates were incubated with concanavalin A-Sepharose (CA) to precipitate all yeast glycoproteins or with monomeric avidin-agarose (AA) to precipitate biotinylated proteins. The washed precipitates were treated with endoglycosidase H and then resolved on SDS gels for immunoblot analysis with antisera directed against gp41 (lanes1-3), gp120 (lanes4-6), or the yeast plasma membrane proton-ATPase (lanes 7 and 8). Note that both uncleaved forms and processed intermediates of the alpha-factor-gp160 construct were modified at the cell surface by biotinylation. A soluble, lumenal marker protein of the yeast lysosome-like vacuole, carboxypeptidase Y, was not precipitated by avidin-agarose (data not shown). The mobility of the various deglycosylated polypeptides derived from alphafactor-gp160 processing (see Fig. 1A) are noted on the right.



Abolishing HIV-1 gp160 Processing Activity in Yeast kex2 Mutants in Vivo

Precursor processing at dibasic amino acid sequences in the secretory pathway has been assigned to a growing family of eukaryotic subtilisin-like serine proteases, or SPC enzymes (Barr, 1991; Steiner et al., 1992). The prototype used for cloning and characterizing the mammalian members of this family was the yeast Kex2 protease (Kex2p), which was isolated by its involvement in the processing of alpha-factor-mating pheromone and killer toxin precursors (Fuller et al., 1989b; Julius et al., 1984). This transmembrane protease has been localized to the trans-Golgi apparatus (Franzusoff et al., 1991; Redding et al., 1991), which is suggested to be the intracellular localization of gp160 processing activity in human T-cells (Willey et al., 1988). Yeast strains harboring deletions of the KEX2 gene (kex2Delta strains) are viable, yet alpha-factor processing activity is completely eliminated, implicating the action of a unique protease encoded by a single gene (Julius et al., 1984). We used a genetic approach to determine whether kex2Delta strains expressing the HIV-1 env gene retained the ability to process the gp160 precursor. As shown by gp41 immunoblot analysis of lysates from KEX2 and kex2Delta yeast strains (Fig. 3), eliminating Kex2 protease activity resulted in complete abrogation of gp160 cleavage to gp41 in vivo without affecting alpha-factor-gp160 expression in the mutant kex2Delta yeast.


Figure 3: Kex2 protease is required for HIV-1 gp160 processing in vivo. Lysates were prepared from colonies of wild-type (W1 and W2) or isogenic kex2Delta (deleted for the KEX2 gene, Delta1 and Delta2) yeast strains expressing alpha-factor-gp160 constructs. Immunoblot analysis was performed with gp41-specific antisera as described in the legend to Fig. 1B. Note the presence of the intact gp160 polypeptides in all lanes, yet gp41 is detected only in wild-type yeast lysates.



Biochemical Analysis of HIV-1 gp160 Processing by the Kex2 Protease

To test whether the Kex2p was directly responsible for gp160 processing, the protease was purified from the media of a yeast strain expressing a truncated soluble secreted gene product first described by Fuller and his colleague (Brenner and Fuller, 1992). The activity of the purified enzyme was measured by fluorogenic peptide assays (Brenner and Fuller, 1992; data not shown) and by alpha-factor processing experiments (Fig. 4). Incubation of in vitro translated, radiolabeled alpha-factor precursor with the purified enzyme resulted in the detection of the expected discrete digestion products. Trypsin addition resulted in the rapid degradation of the precursor alpha-factor protein to small peptides (data not shown). These results showed that the biochemical activity of the purified Ca-dependent Kex2 enzyme retained precise sequence recognition yet, by comparison with other serine proteases such as trypsin, exhibits low catalytic efficiency in vitro.


Figure 4: Pre-pro-alpha-factor processing by purified Kex2 protease in vitro. Radiolabeled pre-pro-alpha-factor was prepared by in vitro translation experiments using yeast lysates (Franzusoff et al., 1992). Kex2 protease, a Ca-dependent serine protease, was purified from the media of yeast cultures expressing the soluble, secreted form of the enzyme (ssKex2p) (Brenner and Fuller, 1992). Incubation of the translation extracts with the purified Kex2 protease resulted in several processing intermediates of the radiolabeled pre-pro-alpha-factor substrate at the expected specific cleavage sites. Each precursor contains four copies of the Kex2 recognition sequence resulting in four mature peptide copies of the mating pheromone, in vivo (pro-box box box box mature box's). Note that ssKex2p activity is sensitive to EGTA but not to N-p-tosyl-L-lysine chloromethyl ketone (TLCK), an effective trypsin inhibitor.



The intact alpha-factor-gp160 precursors were isolated under non-denaturing conditions from radioimmune precipitation buffer-solubilized yeast spheroplast lysates by immunoprecipitation with alpha-factor antisera. The precipitates were treated with purified Kex2p (Fig. 5). The observation of gp120- and gp41-related cleavage products was contingent on Cadependent Kex2 protease activity, as processing was reduced or abolished in the presence of EGTA, or in the absence of enzyme. Despite the mild detergent solubilization of gp160 from yeast spheroplasts, additional gp41 cleavage products were observed. This probably reflects processing at upstream dibasic amino acid cleavage sites (see asterisks in Fig. 1A). These additional cleavage sites may highlight subtle differences in the gp160 protein folding state and the accessibility of latent recognition sequences for Kex2 processing in vivo and in the detergent solubilized protein. However, together with the genetic experiments, the biochemical results demonstrate that the Kex2 protease is capable and directly responsible for HIV-1 gp160 processing in yeast, both in vivo and in vitro.


Figure 5: HIV-1 envelope glycoprotein processing by purified Kex2 protease in vitro. The alpha-factor-related polypeptides were isolated under non-denaturing conditions from radioimmune precipitation buffer-solubilized yeast spheroplasts by immunoprecipitation with alpha-factor antibodies and protein A-Sepharose. The immunoprecipitates were incubated with ssKex2p in the absence or presence of EGTA. The reaction products were treated with endoglycosidase H and then resolved on SDS gels for immunoblot analysis with gp120- (A) or gp41-specific (B) antisera.




DISCUSSION

In this report, we have shown that heterologous expression of HIV-1 envelope glycoproteins and trafficking through the secretory pathway is reproduced in yeast. The processing of gp160 into the gp120 and gp41 forms is dependent on the activity of the Kex2 protease in yeast, since cleavage is abolished in strains harboring deletions in the KEX2 gene and because the purified protease cleaves the gp160 precursor polypeptide in vitro. This is the first genetic and biochemical demonstration that the requisite cleavage of gp160 precursors for HIV-1 infectivity in AIDS is directly dependent on the activity of Kex2p-like enzymes. Because of differences observed in the extent and precision of gp160 processing in vitroversusin vivo (cf.Fig. 5B and Fig. 1B), the results from this study emphasize the importance of examining the role of the host protease under conditions that most reflect those existing in the constitutive secretory pathway. By demonstrating that the Kex2 protease and, by extension, its mammalian homologs are directly responsible for the HIV-1 gp160 processing activity, these results secure the probability that inhibitors of this class of host cell proteases will have an impact on viral infectivity. However, the efficacy of specific protease inhibitors may critically depend on the proper identification of the host cell enzyme.

The identity of the gp160 processing protease in HIV-sensitive human T-cells and macrophages has not been unequivocally determined. Previous studies have circumstantially implicated furin/SPC1 protease to be required for gp160 processing in human cells (Hallenberger et al., 1992; Anderson et al., 1993; Decroly et al., 1994). However, several observations challenge this assertion as to the identity of the true gp160 processing protease. HIV-1 gp160 should principally represent an ideal substrate for furin/SPC1, whose specificity favors the REKR sequence found at the cleavage junction (Molloy et al., 1992). However, the very low efficiency of gp160 cleavage in HIV-infected human T-cells is not consistent with the 55-80% processing activity measured in tissues known to express furin or in cells transfected with furin cDNA (Earl et al., 1991b; Hallenberger et al., 1992). The lower degree of gp160 processing in HIV-1-infected human T-cells and macrophages may be due to very low levels of furin in lymphoid cells, to the activity of uncharacterized SPC enzymes of the lymphoid cell lineage, to cleavage by SPC enzymes that normally function in the regulated secretory pathway, or perhaps to a protease activity unrelated to SPC enzymes (Kido et al., 1993). Studies implicating an unrelated protease were based on biochemical enrichment of a 26-kDa polypeptide (or protein fragment) from T-cell lysates capable of Ca-independent gp160 processing activity in vitro.

Several other lines of evidence favor an alternate SPC protease to furin/SPC1 as the enzyme responsible for gp160 processing. Other members of the SPC enzyme family besides SPC1, such as human SPC3 and yeast Kex2, are capable of correctly processing gp160 precursors in vitro (Decroly et al., 1994; this study). Hence, the inhibitors used to implicate SPC1 as the gp160 processing enzyme could have inadvertently affected other host cell SPC proteases (Hallenberger et al., 1992; Anderson et al., 1993). The level of SPC1 mRNA in human T-cells is not very abundant, being about 10-fold lower than SPC3, which itself is a very low abundant message (Decroly et al., 1994). Most importantly, Ohnishi et al.(1994) conclusively demonstrated in vivo that, in human LoVo cells lacking functional SPC1 protease activity, cleavage of the Newcastle disease virus F(o) precursor protein is eliminated, yet HIV-1 gp160 processing was observed in this cell line. The current view that SPC3 does not apparently function in the constitutive secretory pathway through which HIV-1 gp160 is transiting implies that further work is needed to pinpoint the identity of the true host enzyme required for gp160 processing activity in HIV-infected human cells. Experiments aimed at identifying this enzyme are in progress in our laboratory.

The development of inhibitors directed against the host cell protease activity holds several exciting prospects for novel anti-viral therapies. Elimination of host SPC activity will bypass the problems associated with HIV-1 or HIV-2 variations and with viral resistance to such inhibitors and is a tangible target for therapy based on observations that yeast and mammalian (mutant) cells remain viable in the absence of Kex2p-like enzymatic activity (Inocencio et al., 1993; Moehring et al., 1993; Ohnishi et al., 1994). The advantage of the heterologous yeast system described in this report is the possibility to identify, clone, and express the gene encoding the human T-cell and macrophage gp160-processing activity in yeast strains lacking the endogenous Kex2 protease. In addition, this system will enable rapid testing and development of specific protease inhibitors that interfere with cleavage activity both in vivo and in vitro. The implications of this therapeutic approach on the cohort of enveloped viruses exhibiting cleaved surface glycoproteins that infect humans, pets, and livestock have been considered.


FOOTNOTES

*
This work was supported by NIAID, National Institutes of Health Grant AI-34747 and American Federation for AIDS Research Grant 1513 (to A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cellular and Structural Biology, University of Colorado Medical School, Box B-111, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-270-6280; Fax: 303-270-4729.

Present address: Dept. of Genetics, University of Washington, Seattle, WA 98195.

**
Present address: Chiron Corp., 4221 Horton St., Emeryville, CA 94608.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus 1; Kex2p, Kex2 protease; ssKex2p, secreted soluble Kex2p.


ACKNOWLEDGEMENTS

The contributions to this work by Chang-You Chen, Jonathan Leighton, Carolina Gonzalez-Aller, and Hilary Chouinard are gratefully acknowledged. We are indebted to Dr. Robert Fuller and Charles Brenner for generously providing several important reagents and protocols used in this study, including the yeast strain expressing the ssKex2 protease. We thank Drs. Dan Kuritzkes, Robert Schooley, and Mary Rosandich for their collaboration with the HIV tissue culture facility. We are grateful to Drs. Paul Melançon, Richard Duke, Kerstin Henkel, Dan Kuritzkes, Fiona Jucker, and members of the laboratory for helpful discussions during the course of this work and for critical reading of the manuscript.


REFERENCES

  1. Anderson, E. D., Thomas, L., Hayflick, J. S., and Thomas, G. (1993) J. Biol. Chem. 268, 24887-24891 [Abstract/Free Full Text]
  2. Barr, P. (1991) Cell 66, 1-3 [Medline] [Order article via Infotrieve]
  3. Barr, P. J., Steimer, K. S., Sabin, E. A., Parkes, D., George-Nascimento, C., Stephans, J. C., Powers, M. A., Gyenes, A., Van Nest, G. A., Miller, E. T., Higgins, K. W., and Luciw, P. A. (1987) Vaccine 5, 90-97 [CrossRef][Medline] [Order article via Infotrieve]
  4. Böhni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042 [Abstract]
  5. Bosch, V., and Pawlita, M. (1990) J. Virol. 64, 2337-2344 [Medline] [Order article via Infotrieve]
  6. Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorshot, V., Peters, P. J., and Bonifacino, J. S. (1994) J. Cell Biol. 126, 1157-1172 [Abstract]
  7. Brenner, C., and Fuller, R. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 922-926 [Abstract]
  8. Decroly, E., Vandenbranden, M., Ruysschaert, J-M., Cogniaux, J., Jacob, G. S., Howard, S. C., Marshall, G., Kompelli, A., Basak, A., Jean, F., Lazure, C., Benjannet, S., Chretien, M., Day, R., and Seidah, N. G. (1994) J. Biol. Chem. 269, 12240-12247 [Abstract/Free Full Text]
  9. Earl, P., Koenig, S., and Moss, B. (1991a) J. Virol. 65, 31-41 [Medline] [Order article via Infotrieve]
  10. Earl, P., Moss, B., and Doms, R. W. (1991b) J. Virol. 65, 2047-2055 [Medline] [Order article via Infotrieve]
  11. Franzusoff, A., and Schekman, R. (1989) EMBO J. 8, 2695-2702 [Abstract]
  12. Franzusoff, A., Redding, K., Crosby, J., Fuller, R. S., and Schekman, R. (1991) J. Cell Biol. 112, 27-37 [Abstract]
  13. Franzusoff, A., Lauzé, E., and Howell, K. E. (1992) Nature 355, 173-175 [CrossRef][Medline] [Order article via Infotrieve]
  14. Fuller, R., Brake, A., and Thorner, J. (1989a) Proc. Natl. Acad. Sci. U. S. A. 86, 1434-1438 [Abstract]
  15. Fuller, R. S., Brake, A. J., and Thorner, J. (1989b) Science 246, 482-486 [Medline] [Order article via Infotrieve]
  16. Gething, M.-J., McCammon, K., and Sambrook, J. (1986) Cell 46, 939-950 [Medline] [Order article via Infotrieve]
  17. Hallenberger, S., Bosch, V., Angliker, H., Shaw, E., Klenk, H-D., and Garten, W. (1992) Nature 360, 358-361 [CrossRef][Medline] [Order article via Infotrieve]
  18. Haseltine, W. (1991) FASEB J. 5, 2349-2360 [Abstract/Free Full Text]
  19. Hurtley, S. M., and Helenius, A. (1989) Annu. Rev. Cell Biol. 5, 277-307 [CrossRef]
  20. Inocencio, N. M., Moehring, J. M., and Moehring, T. J. (1993) J. Virol. 67, 593-595 [Abstract]
  21. Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089 [Medline] [Order article via Infotrieve]
  22. Kido, H., Kamoshita, K., Fukutomi, A., and Katunuma, N. (1993) J. Biol. Chem. 268, 13406-13412 [Abstract/Free Full Text]
  23. Kowalski, M., Potz, J., Basiripour, L., Dorfman, T., Goh, W. C., Terwilliger, E., Dayton, A., Rosen, C., Haseltine, W., and Sodroski, J. (1987) Science 237, 1351-1355 [Medline] [Order article via Infotrieve]
  24. Kurjan, J., and Herskowitz, I. (1982) Cell 30, 933-943 [Medline] [Order article via Infotrieve]
  25. McCune, J., Rabin, L. B., Feinberg, M. B., Lieberman, M., Kosek, J. C., Reyes, G. R., and Weissman, I. L. (1988) Cell 53, 55-67 [Medline] [Order article via Infotrieve]
  26. Mizuno, K., Nakamura, T., Ohshima, T., Tanaka, S., and Matsuo, H. (1988) Biochem. Biophys. Res. Commun. 156, 246-254 [Medline] [Order article via Infotrieve]
  27. Moehring, J. M., Inocencio, N. M., Robertson, B. J., and Moehring, T. J. (1993) J. Biol. Chem. 268, 2590-2594 [Abstract/Free Full Text]
  28. Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) J. Biol. Chem. 267, 16396-16402 [Abstract/Free Full Text]
  29. Molloy, S. S., Thomas, L., VanSlyke, J. K., Stenberg, P. E., and Thomas, G. (1994) EMBO J. 13, 18-33 [Abstract]
  30. Ohnishi, Y., Shioda, T., Nakayama, K., Iwata, S., Gotoh, B., Hamaguchi, M., and Nagai, Y. (1994) J. Virol. 68, 4075-4079 [Abstract]
  31. Pichuantes, S., Nguyen, A. T., and Franzusoff, A. (1995) in Principles and Practice of Protein Engineering (Cleland, J. L., and Craik, C. S., eds) John Wiley & Sons, Inc., in press
  32. Redding, K., Holcomb, C., and Fuller, R. S. (1991) J. Cell Biol. 113, 527-538 [Abstract]
  33. Renneisen, K., Leserman, L., Matthes, E., Schroder, H. C., and Muller, W. E. G. (1990) J. Biol. Chem. 265, 16337-16342 [Abstract/Free Full Text]
  34. Sanchez-Pescador, R., Power, M. D., Barr, P. J., Steimer, K. S., Stempien, M. M., Brown-Shimer, S. L., Gee, W. W., Renard, A., Randolph, A., Levy, J. A., Dina, D., and Luciw, P. A. (1985) Science 227, 484-492 [Medline] [Order article via Infotrieve]
  35. Schäfer, M. K.-H., Day, R., Cullinana, W. E., Chrétien, M., Seidah, N. G., and Watson, S. J. (1993) J. Neurosci. 13, 1258-1279 [Abstract]
  36. Seidah, N. G., Day, R., Marcinkiewicz, M., Benjannet, S., and Chrétien, M. (1991) Enzyme 45, 271-284 [Medline] [Order article via Infotrieve]
  37. Steiner, D., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992) J. Biol. Chem. 267, 23435-23438 [Free Full Text]
  38. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  39. Walker, B., Kowalski, M., Goh, W. C., Kozarsky, K., Krieger, M., Rosen, C., Rohrschneider, L., Haseltine, W., and Sodroski, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8120-8124 [Abstract]
  40. Wessel, D., and Flugge, U. I. (1984) Anal. Biochem. 138, 141-143 [Medline] [Order article via Infotrieve]
  41. Wilcox, C., and Fuller, R. S. (1991) J. Cell Biol. 115, 297-307 [Abstract]
  42. Willey, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A., and Klausner, R. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9580-9584 [Abstract]

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