(Received for publication, October 5, 1994)
From the
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.
The env gene of the human immunodeficiency retrovirus
HIV-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.
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).
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 -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
-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
-factor-gp160 construct (160), and the truncated
-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
-factor-gp160 processing in yeast
are revealed by treatment of the immunoprecipitates with
endoglycosidase H (EndoH) before second round
immunoprecipitation with gp120-, gp41-, or
-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
-factor leader
sequence were more abundant than the fully processed forms (i.e.
F-gp160 and
F-gp120, Fig. 1C, lanes2-4). However, the first two amino acids of the
mature gp160 at the junction between
-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.
To test the cleavage of HIV-1 gp160 expressed in yeast, cultures
were grown in derepression media overnight for expression of the
-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
-factor antisera and protein A-Sepharose to isolate
-factor-gp160 and
-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.
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 -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
-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.
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 factor-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
-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
factor-gp160 processing (see Fig. 1A) are noted on
the right.
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 kex2 (deleted for the KEX2 gene,
1 and
2)
yeast strains expressing
-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.
Figure 4:
Pre-pro--factor processing by
purified Kex2 protease in vitro. Radiolabeled
pre-pro-
-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-
-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-
mature
'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 -factor-gp160 precursors were isolated
under non-denaturing conditions from radioimmune precipitation
buffer-solubilized yeast spheroplast lysates by immunoprecipitation
with
-factor antisera. The precipitates were treated with purified
Kex2p (Fig. 5). The observation of gp120- and gp41-related
cleavage products was contingent on Ca
dependent 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 -factor-related
polypeptides were isolated under non-denaturing conditions from
radioimmune precipitation buffer-solubilized yeast spheroplasts by
immunoprecipitation with
-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.
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 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.