(Received for publication, December 20, 1995; and in revised form, January 10, 1996)
From the
The hepatitis delta virus large antigen (lHDAg) is a virally
encoded protein that contains a prenylation signal sequence at its
carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln.
Although the presence of the Gln as the COOH-terminal residue generally
specifies addition of the 15-carbon farnesyl isoprenoid, earlier
reports had suggested that the protein is modified by the 20-carbon
geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase
and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When
recombinant GST-lHDAg was incubated with bovine brain cytosol in the
presence of either farnesyl diphosphate or geranylgeranyl diphosphate,
GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the
fusion protein was also observed, although at a rate considerably less
than that of farnesylation. Using purified recombinant protein
prenyltransferases, GST-lHDAg was found to be an excellent substrate
(apparent K = 0.8 µM)
for protein farnesyltransferase (FTase), while modification by protein
geranylgeranyltransferase I (GGTase I) was not detected. FTase was also
able to catalyze geranylgeranylation of GST-lHDAg at a very low rate,
suggesting that the low level of geranylgeranylation of GST-lHDAg
observed in cytosolic preparations was mediated by FTase. Consistent
with our observations on the in vitro prenylation of the
GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg
expressed in COS cells demonstrated that the protein was farnesylated.
Geranylgeranylation of lHDAg expressed in COS cells was not observed.
As prenylation of lHDAg is required for the assembly of the hepatitis
delta viral particle, these results suggest that inhibitors of FTase
may be useful therapeutic agents for treatment of delta virus
infection.
Hepatitis delta virus (HDV) ()is a satellite virus of
hepatitis B virus that can cause an increase in the incidence and
severity of liver disease in individuals infected with both
viruses(1, 2) . HDV consists of the HDV RNA genome and
two HDV-encoded proteins, designated as the small (sHDAg) and large
(lHDAg) HDV antigens, encapsulated in an envelope composed of hepatitis
B surface antigens(2) . sHDAg and lHDAg contain identical
deduced amino acid sequences for their first 195 amino acids, with
lHDAg containing an additional 19-amino acid COOH-terminal
tail(2) . Despite their sequence identity, sHDAg and lHDAg have
very different functions. Whereas sHDAg is essential for HDV
replication(3) , lHDAg is a dominant inhibitor of HDV
replication (4) and is required for assembly of the HDV
particle(5, 6) . As lHDAg can package into
pseudo-viral particles with hepatitis B surface antigens in the absence
of both HDV RNA and sHDAg(6, 7) , the current model of
HDV assembly indicates that lHDAg functions by directly interacting
with hepatitis B surface antigens that form the envelope of the viral
particle(2) .
Interestingly, lHDAg is modified by an isoprenoid lipid on a cysteine located near its COOH terminus(8) , and this modification is necessary for lHDAg to facilitate HDV assembly(8, 9) . The prenylation motif contained in lHDAg is the COOH-terminal tetrapeptide Cys-Arg-Pro-Gln (CRPQ)(8) . This sequence is similar to the conventional prenylation motif which consists of the COOH-terminal tetrapeptide CAAX, where the cysteine residue is the prenylation site, ``A'' are generally aliphatic residues, and X can be one of several amino acids. Two distinct protein prenyltransferases modify proteins which contain a CAAX motif; farnesyltransferase (FTase), which modifies proteins with the 15-carbon farnesyl isoprenoid, and geranylgeranyltransferase I (GGTase I), which modifies proteins with the 20-carbon geranylgeranyl group(10, 11) . The COOH-terminal amino acid (i.e., X) in general determines which of the two isoprenoid lipids modify a CAAX motif. If X is Met, Ser, or Gln, the sequence is a substrate for FTase(12) , while Leu at this position directs modification by GGTase I(12, 13, 14) . A third protein prenyltransferase, GGTase II, recognizes a different class of COOH-terminal motifs present in GTP-binding proteins of the Rab family(15) . Following the prenylation of the CAAX motif, two additional processing events occur: proteolytic cleavage of the -AAX tripeptide and carboxymethylation of the new COOH-terminal prenylcysteine(10, 11) .
As prenylation of lHDAg is required for HDV assembly, inhibition of the enzyme responsible for its prenylation is a potential treatment of HDV infection. Due to the presence of Gln at its COOH terminus, lHDAg would be predicted to be a FTase substrate(11, 12) . However, two reports have appeared indicating that lHDAg is modified with the geranylgeranyl isoprenoid(8, 9) . Thus, it is not clear which protein prenyltransferase is responsible for the prenylation of lHDAg. In this report, we have examined both the nature of the isoprenoid group attached to lHDAg and the identity of the enzyme responsible for its prenylation. Our results indicate that the predominant modification of lHDAg is in fact farnesylation, and the protein is exclusively a substrate for FTase.
For kinetic determinations,
the concentrations of prenylation-competent GST-lHDAg, Ha-Ras, and
Ha-Ras-CVLL were determined by driving the prenylation reactions to
completion in the presence of excess FTase or GGTase I and the
respective prenyldiphosphate, and determining the amount of protein
prenylated by the filter binding assay described below. Final
concentrations of substrates ranging from 0.1 to 20 µM were utilized in kinetic assays. Assay conditions were identical
to those described above, with the exception that the specific
activities of [H]FPP and
[
H]GGPP were 3 Ci/mmol. Reactions were initiated
by the addition of 75 ng of FTase or GGTase I and incubated for 15 min
at 37 °C. The reactions were stopped by addition of 4% SDS,
proteins precipitated by addition of trichloroacetic acid, and
prenylated proteins separated from free isoprenoid by filtration
through nitrocellulose filters as described (21) . The amount
of prenylated protein retained on the filters was quantitated by liquid
scintillation spectroscopy.
Figure 1:
Prenylation of GST-lHDAg by bovine
brain cytosol. GST-lHDAg, Ha-Ras, and Ha-Ras-CVLL were incubated with
bovine brain cytosol in the presence of either 2 µM [H]FPP or 2 µM [
H]GGPP (as indicated under each panel) for
1 h at 37 °C. Prenylated proteins were resolved by SDS-PAGE and
visualized by fluorography. The gel was exposed for either 2 days (panel A) or 14 days (panel B). Samples processed in
the various lanes are as follows: lanes 1 and 5, no
added substrate protein; lanes 2 and 6, Ha-Ras; lanes 3 and 7, Ha-Ras-CVLL; lanes 4 and 8, GST-lHDAg. Data shown are from a single experiment, which
is representative of several such
experiments.
Figure 2:
Prenylation of GST-lHDAg by purified
recombinant protein prenyltransferases. GST-lHDAg, Ha-Ras, or
Ha-Ras-CVLL were incubated with FTase (panel A) or GGTase I (panel B) in the presence of either 2 µM [H]FPP or 2 µM [
H]GGPP (as indicated under each panel) for
1 h at 37 °C. Prenylated proteins were resolved by SDS-PAGE and
visualized by fluorography. Exposure time was 4 days. Samples processed
in the various lanes are as follows: lanes 1 and 5,
no added substrate protein; lanes 2 and 6, Ha-Ras; lanes 3 and 7, Ha-Ras-CVLL; lanes 4 and 8, GST-lHDAg. Data shown are from a single experiment, which
is representative of two such experiments.
Figure 3: Kinetics of protein prenyltransferase modification of GST-lHDAg. Saturation curves for modification of GST-lHDAg, Ha-Ras, and Ha-Ras-CVLL by both FTase and GGTase I were determined. Assays were conducted with either FTase and FPP (panel A) or GGTase I and GGPP (panel B) as described under ``Experimental Procedures.'' Reactions were stopped by addition of 2% SDS and prenylated proteins produced determined by filter binding assays. Data shown represent the mean of duplicate determinations from a single experiment, which is representative of several such experiments.
Figure 4:
Prenylation of lHDAg in animal cells. Panel A, COS cells were transfected with either an expression
vector encoding MEV alone (lanes 1 and 3) or with
expression vectors for both MEV and lHDAg (lanes 2 and 4). [H]Mevalonate labeling of the
transfected cells was conducted as described under ``Experimental
Procedures.'' Detergent-solubilized extracts were prepared and
either analyzed directly (lanes 1 and 2) or after
immunoprecipitation using anti-lHDAg antibody (lanes 3 and 4). Samples were resolved by SDS-PAGE and prenylated proteins
visualized by fluorography. Exposure time was 3 days. Panels
B-D, isoprenoid analysis of metabolically labeled lHDAg.
Samples identical to those in panel A were subjected to
isoprenoid analysis. Solubilized cell extract (panel B) or
samples subjected to immunoprecipitation with anti-lHDAg (panels C and D) were processed by trichloroacetic acid
precipitation and isoprenoid lipids cleaved from prenylated proteins
using methyl iodide. Farnesol (C15) and geranylgeraniol (C20) were added to the samples as internal standards, and
released isoprenoids were resolved by reverse-phase HPLC. Fractions
were analyzed by liquid scintillation spectroscopy. Isoprenoid analysis
was performed on the following samples: panel B,
detergent-solubilized extract of cells expressing both MEV and lHDAg
(corresponding to panel A, lane 2); panel C,
immunoprecipitate from cells expressing both MEV and lHDAg
(corresponding to panel A, lane 4); panel D,
immunoprecipitate from cells expressing only MEV (corresponding to panel A, lane 3). For all panels, data shown are from
a single experiment, which is representative of data obtained from two
different transfections.
The isoprenoid modifying lHDAg was cleaved from the protein by methyliodide cleavage, and the liberated lipid identified by HPLC. Isoprenoid analysis was performed both on the total pool of prenylated proteins present in solubilized extracts from COS cells, and on immunoprecipitated lHDAg. For the total pool of prenylated proteins, approximately 20% of the protein-associated isoprenoid was farnesyl and 80% was geranylgeranyl (Fig. 4, panel B), a ratio consistent with previous studies(26) . Isoprenoid analysis of the immunoprecipitated lHDAg, however, revealed that the protein was exclusively modified by the farnesyl isoprenoid (Fig. 4, panel C); the amount of geranylgeranyl lipid in immunoprecipitated lHDAg was essentially identical to that observed when COS cells not expressing lHDAg were subjected to the same procedure (Fig. 4, compare panels C and D). These results are completely consistent with the in vitro data that indicated that lHDAg was a FTase substrate, and we conclude that lHDAg is farnesylated in cells by FTase. We also predict that the geranylgeranylation of lHDAg observed by others in vitro(9) was mediated by FTase.
Mutational analysis of lHDAg has revealed that while the prenylation motif at the COOH terminus of the protein is not necessary for lHDAg to bind sHDAg or the RNA genome, it is essential for lHDAg to interact with hepatitis B surface antigen in vitro(27) . Mutation or deletion of the prenylation motif also results in lHDAg that is unable to form pseudo-viral particles with hepatitis B surface antigens in transfected cells(8, 9, 28) , providing strong evidence that prenylation of lHDAg is required for HDV particle formation. Although prenylation of lHDAg is necessary for interaction of the protein with hepatitis B surface antigen and for the formation of pseudo-viral particles, it is not sufficient in this regard in that a 15-amino acid cassette immediately upstream of the CAAX motif is also required(9, 27, 28, 29) . This finding parallels those from studies of Ras proteins, in which it was determined that the proteins require either a polybasic domain or palmitoylated cysteine residues immediately upstream of a farnesylation motif for efficient association with membranes(30, 31) . Thus, both in lHDAg and Ras proteins, a second ``signal'' is required for a function that is also dependent upon prenylation.
The requirement of the prenylation motif for HDV particle assembly, coupled with our finding that lHDAg is exclusively a substrate for FTase, indicates that inhibitors of FTase should prevent viral particle formation. A number of laboratories have developed potent FTase inhibitors for use in the treatment of cancers that contain oncogenic Ras proteins(32, 33, 34) . Recent reports have indicated that many of these inhibitors are effective against some tumors containing activated Ras proteins and are relatively non-toxic in animals(35, 36) . Taken together, these findings indicate that FTase inhibitors may be an effective route to blocking the pathological consequences of HDV infections.