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Previous Article | Next Article 
Journal of Virology, April 2000, p. 3418-3422, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Assembly and Processing of Human Immunodeficiency
Virus Gag Mutants Containing a Partial Replacement of the Matrix Domain
by the Viral Protease Domain
Chin-Tien
Wang,*
Yen-Chiou
Chou, and
Chien-Cheng
Chiang
Institute of Clinical Medicine, National
Yang-Ming University School of Medicine, and Department of Medical
Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan, Republic of China
Received 4 August 1999/Accepted 20 December 1999
 |
ABSTRACT |
We constructed human immunodeficiency virus (HIV) mutants by
replacing the matrix domain with sequences encoding the viral protease
or p6* and protease. The chimeras retaining matrix myristylation and
processing signals underwent efficient autoprocessing with severely
defective particle budding. The budding defects of the chimeras were
rescued by suppressing the chimera protease activity either through
addition of an HIV protease inhibitor or through inactivating the
chimera protease via a substitution mutation of the catalytic aspartic
acid residue. This resulted in the release of chimeric virus-like
particles with the density of a wild-type retrovirus particle. In
addition, the assembly-competent but processing-defective chimeras
produced proteolytically processed particles with significant reverse
transcriptase activity when a downstream native pol gene was present. These results suggest that HIV has the potential to adapt
heterologous sequences in place of the matrix sequence without major
effects on virus-like particle budding. In addition, the positions of
the protease and substrate accessibility may contribute significantly
toward avoiding a premature Gag or Gag-Pol process, which leads to
severe defects in both particle budding and incorporation.
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TEXT |
The structural proteins of all
retroviruses, including human immunodeficiency virus (HIV), are encoded
by the gag genes (2, 3, 5, 23). During or shortly
after virus budding, the HIV Gag precursor Pr55 is cleaved by the
pol-encoded protease (PR) into four major products: the
matrix (p17; MA), capsid (p24; CA), nucleocapsid (p7; NC), and
C-terminal p6 protein (4, 7, 9, 11). The pol
product is translated as a Pr160gag-pol fusion
protein by a ribosomal frameshifting mechanism that occurs at a
frequency of about 5% during translation of Gag (6). The relatively low level of Gag-Pol is thought to avoid premature Gag
processing so that Gag assembly can proceed. Mechanisms of PR
activation are unclear; it is proposed that PR dimerization, a
prerequisite for PR activation, is promoted by the Gag domains (10, 20, 26). PR, once activated, autocleaves from Gag-Pol and subsequently processes Gag and Pol into mature products. Within Pr160gag-pol, C-terminal p6 is truncated and
replaced by a domain referred to as p6* (13). p6*, adjacent
to PR, separates NC from Pol. A number of studies suggest that p6* may
be functionally involved in the regulation of PR activity (14, 17,
27).
It has been demonstrated that chimeras derived from a replacement of
the HIV or Rous sarcoma virus C-terminal gag sequences by
foreign protein sequences can still direct virus-like particle assembly
and release (21, 22, 25). In this study, we substituted the
HIV-1 PR coding sequence for MA and analyzed the assembly and
processing of the resultant chimeric proteins. The p6*-PR and PR
sequence fragments were amplified by PCR using primers containing a
ClaI and SalI restriction site in the 5' and 3'
primers, respectively. The PCR-generated fragments then were treated
with ClaI and SalI and used to replace the
fragment from ClaI (HIV nucleotide [nt] 831) to
SalI (nt 1147) of an HIV gag mutant that contained a SalI linker at nt 1147 (18). To
assess the proteolytic activities of the inserted PR domains, the
chimeric constructs were subcloned into an HIV PR-defective mutant,
D25, of which the PR catalytic residue Asp was replaced with Asn. The
resultant construct was designated MA(p6*-PR)D25 or MA(PR)D25 (Fig.
1A). The backbone of all mutant
constructs was HIV gpt, which carries simian virus 40 ori
and gpt genes in the env region (12).
Wild-type (WT) and mutant HIVgpt plasmids were transfected into 293T
cells. Expression and release of HIV Gag proteins were probed by
immunoblotting using an anti-p24gag monoclonal
antibody (1, 19). As shown in Fig.
2, the WT Pr55, the p41, and the mature
p24gag proteins were detected in the medium and
in cell samples (lanes 3 and 9). A major band representing
Pr55gag was seen in the medium and cell samples
of D25 (lanes 2 and 8). In contrast, chimeric proteins derived from
processed MA(p6*-PR)D25 or MA(PR)D25 were detected only in the cell
samples (lane 10 or 11, respectively). A faint band corresponding to
Pr55gag observed in the MA(p6*-PR)D25 medium
sample (Fig. 2, lane 4) may have resulted from a spillover from the
adjacent WT sample because it was not seen in any repeat experiments.
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FIG. 1.
Schematic presentation of the WT and mutant HIVgpt
constructs. Mature WT processed Gag proteins and the p6* (black) and
PR (stippled) domains of pol are indicated. The "X"
indicates a PR-defective point mutation (D25 Asn). (A)
The D25 mutant, which contains a substitution of an Asn residue for the
PR catalytic Asp residue, is defective in Gag processing. The
MA(p6*-PR)D25 mutant contains a deletion of 105 codons and a
replacement of the p6*-PR coding sequence in the MA protein. The
myristylation signal residues and a few residues in the C terminus of
MA remain intact (underlined). Changed or added codons (boldfaced) and
residues in the N and C termini of the PR domain are indicated.
Upstream of PR, there are 45 codons (italics) of the p6* domain
starting from the N-terminal 12th codon, K. The MA(PR)D25 mutant is
identical to the MA(p6*-PR)D25 mutant except that it contains only
five C-terminal codons of p6*. Instead of having 132 codons as in the
WT MA protein, the MA(p6*-PR) and the MA(PR) constructs contain a
total of 182 and 142 codons in their MA regions, respectively. (B)
Mutant constructs were derived from the constructs shown in panel A. MA(p6*-D25)D25 and MA(D25)D25 were identical to MA(p6*-PR)D25 and
MA(PR)D25, respectively, except that the former two contain the
PR-defective mutations (D25) in their chimera PR fragments.
Recombination of the WT with MA(p6*-D25)D25 and MA(D25)D25 yielded
MA(p6*-D25) and MA(D25), respectively.
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FIG. 2.
Expression and processing of the chimeric proteins. 293T
cells were transfected with the designated constructs. At 48 h
posttransfection, cells and supernatants were collected for protein
analysis. Supernatant samples (lanes 1 to 5) corresponding to 50% of
the total samples and cell samples (lanes 7 to 11) corresponding to 5%
of the total samples were fractionated by sodium dodecyl sulfate-10%
polyacrylamide gel electrophoresis and electroblotted onto a
nitrocellulose filter. HIV p24gag and
p24gag-associated chimeric proteins were
detected with mouse anti-p24gag monoclonal
antibody at a 1:5,000 dilution, followed by a secondary alkaline
phosphatase-conjugated sheep anti-mouse antibody at a 1:5,000 dilution,
and alkaline phosphatase activity was determined. Positions of standard
(Std.) molecular size markers (lanes 6 and 12) are indicated on the
right, and those of HIV Gag proteins Pr55, p41, and p24 are shown on
the left.
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To test whether the inability of the chimeras to release from cells is
due to PR-mediated premature autoprocessing (8, 24), we
added an HIV-1 PR inhibitor, Ro31-8959 (15), to the WT and
chimera transfectants. Figure 3 shows
that proteolytic Pr55gag processing was
significantly suppressed in the presence of the PR inhibitor (lanes 2 to 3 and 12 to 13) compared with that of untreated samples (lanes 1 and
11). The levels of released chimeric proteins correlated with the
degree of the PR activity suppression (Fig. 3, lane 6 versus lane 5, and lane 9 versus lane 8). The expected chimera intermediates p6*, PR,
and CA are absent in Fig. 2 and 3; instead, a band migrating with WT
p41gag is readily observed. This might result
from altered PR preferential cleavage sites. Alternatively, the
p41gag chimera was derived from the incompletely
cleaved product CA-NC-p6 (11). Further experiments are
required to test this proposition.
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FIG. 3.
Release of the chimeras into the medium in the presence
of an HIV PR inhibitor. 293T cells grown on 10-cm-diameter dish plates
were transfected with the WT, MA(p6*-PR)D25, and MA(PR)D25 HIVgpt
constructs. At 18 h posttransfection, cells were split equally
onto three 10-cm-diameter dishes and treated, respectively, with 0 µM
(lanes 1, 4, 7, 11, 14, and 17), 1.5 µM (lanes 2, 5, 8, 12, 15, and
18), and 7.5 µM (lanes 3, 6, 9, 13, 16, and 19) concentrations of the
HIV PR inhibitor Ro31-8959. Four hours later, the culture supernatants
were removed and replaced with medium plus the designated concentration
of the PR inhibitor. At 48 h after addition of the PR inhibitor,
culture supernatants and cells were collected for protein analysis.
Samples were fractionated by sodium dodecyl sulfate-10%
polyacrylamide gel electrophoresis and subjected to immunoblot analysis
with anti-p24gag antibody. Std., standards
(lanes 10 and 20). Positions of the molecular size markers are
indicated on the right, and those of the HIV Gag proteins Pr55, p41,
and p24 are shown on the left.
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To further confirm that suppression of the PR activity promotes chimera
release, the PR-inactivating mutation D25 was introduced into the
chimeras MA(p6*-PR)D25 and MA(PR)D25, yielding constructs MA(p6*-D25)D25 and MA(D25)D25, respectively (Fig. 1B). To test whether a normal PR downstream of the chimeric mutations could functionally compensate for the proximal, catalytically inactivated chimera PR, chimeras MA(p6*-D25) and MA(D25) were constructed by
placing the native HIV pol gene downstream of the chimeras (Fig. 1B). As shown in Fig. 4, chimeras
MA(p6*-D25)D25 and MA(D25)D25 were assembled and released efficiently,
at a level at least 1.7-fold higher than that of D25. Interestingly,
chimeras MA(p6*-D25) and MA(D25) exhibited an efficient processing
profile (Fig. 4, lanes 10, 11, 17, and 18) and had, respectively,
three- and sevenfold (lanes 3 to 4 and 10 to 11) higher levels of
release efficiency than the WT (lanes 2 and 9). Sucrose density
gradient fractionation analysis indicated that all the mutants had a WT
retrovirus particle density of 1.16 to 1.18 g/ml (data not shown). To
further assess the particle incorporation of the chimera-Pol fusion
proteins, the particle-associated RT activity of the assembly-competent chimeras was assayed using exogenous templates (19). Because particle processing can affect the RT assay (16), RT
activities of the processing-defective chimeras MA(p6*-D25)D25 and
MA(D25)D25 were compared in parallel with those of D25. As
shown in Table 1, the chimeras
MA(p6*-D25) and MA(D25) possessed significant RT activity at a level
just over 50% of that of the WT. Surprisingly, the
processing-defective chimeras, MA(p6*-D25)D25 and MA(D25)D25, exhibited
relatively low RT activity; all levels were below 20% of the level
shown by D25 in three independent experiments. Because the results
shown in Fig. 4 indicate that the chimeric particles contained
significant levels of chimera-Pol (lanes 6 and 7), the low RT
activities of MA(p6*-D25)D25 and MA(D25)D25 were less likely due to
insufficient Pol incorporation. Inaccessibility of substrates to the
chimera-Pol construct and/or impaired enzymatic activity due to the
chimeric mutations might account for the low RT activity.
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FIG. 4.
Assembly and processing of chimeric particles. 293T
cells were transfected with the designated plasmid. At 48 to 72 h
posttransfection, supernatants and cells were prepared for Western
immunoblotting. HIV-1 CA-associated proteins were detected by an
enhanced-chemiluminescence detection system (Amersham). The primary
antibody was an anti-p24gag monoclonal antibody
used at a 1:5,000 dilution. The secondary antibody was a sheep
anti-mouse horseradish peroxidase-conjugated antibody used at a 1:5,000
dilution. Positions of chimeric protein precursors are indicated on the
right (arrows), and those of the HIV Gag proteins Pr55, p41, and p24
are shown on the left.
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ACKNOWLEDGMENTS |
This work was supported by grant NSC88-2314-B010-075 from the
National Science Council and, in part, by grant DOH88-DC-1020 from the
Ministry of Health, Taipei, Taiwan.
The hybridoma clone 183 H12-5C was a gift provided by the AIDS Research
and Reference Reagent Program, Division of AIDS, NIAID, from Bruce
Chesebro. The HIV-1 protease inhibitor Ro31-8959 was kindly provided by
Hoffmann-LaRoche (Switzerland).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-pai Rd., Shih-pai, Taipei 11217, Taiwan, Republic of
China. Phone: 886-2-2871-2121, ext. 2655. Fax: 886-2-2874-2279. E-mail:
ctwang{at}vghtpe.gov.tw.
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Journal of Virology, April 2000, p. 3418-3422, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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