Assembly and Processing of Human Immunodeficiency Virus Gag Mutants Containing a Partial Replacement of the Matrix Domain by the Viral Protease Domain

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J

OURNAL OF

V

IROLOGY

,

0022-538X/00/$04.00⫹0

Apr. 2000, p. 3418–3422

Vol. 74, No. 7

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

We constructed human immunodeficiency virus (HIV) mutants by replacing the matrix domain with

se-quences 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

pro-duced 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.

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 Pr160

gag-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,

auto-cleaves from Gag-Pol and subsequently processes Gag and Pol

into mature products. Within Pr160

gag-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

re-placement 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

ana-lyzed the assembly and processing of the resultant chimeric

proteins. The p6*-PR and PR sequence fragments were

am-plified by PCR using primers containing a

Cla

I and

Sal

I

re-striction site in the 5

⬘

and 3

⬘

primers, respectively. The

PCR-generated fragments then were treated with

Cla

I and

Sal

I and

used to replace the fragment from

Cla

I (HIV nucleotide [nt]

[image:1.612.311.546.382.630.2]

* 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: [email protected].

TABLE 1. RT activities of HIV mutants

a

Constsruct Expt cpm incorporated Relative activity (%)b

WT

1

29,227

100

2

146,976

100

3

61,654

100

4

97,524

100

MA(p6*-D25)

1

7,715

67

2

66,830

72

3

78,033

95

4

94,678

86

MA(D25)

2

46,275

93

3

77,703

95

4

35,095

42

D25

1

29,589

100

2

131,614

100

3

304,404

100

MA(p6*-D25)D25

1

2,409

19

2

8,522

7

3

57,915

2

MA(D25)D25

1

2,689

9

2

5,892

5

3

21,414

2

a_{Supernatants were prepared and RT assays were performed as described in}

Materials and Methods. For each sample, virus-associated Gag or chimeric protein levels were quantitated by scanning mutant and WT Pr55, p41, and 24 or p24-associated band densities from immunoblots. Results of four separate trans-fection experiments are given.

b_{RT activities of the processing-defective chimeric mutants}

MA(p6*-D25)D25 and MA(MA(p6*-D25)D25 were compared with that of their parental PR-defective mutant D25. Relative activities were determined as percentages of WT or D25 activities (100%) by the equation 100⫻[(mutant counts per minute⫺ background)/mutant Gag protein⫻WT or D25 Gag protein densitometry units/ (WT or D25 counts per minute⫺background)]. All RT activities with counts per minute were at least threefold over the background level (730⫾239).

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FIG. 1. Schematic presentation of the WT and mutant HIVgpt constructs. Mature WT processed Gag proteins and the p6ⴱ(black) and PR (stippled) domains of

polare indicated. The “X” indicates a PR-defective point mutation (D253Asn). (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(p6ⴱ-PR)D25 mutant contains a deletion of 105 codons and a replacement of the p6ⴱ-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 p6ⴱdomain starting from the N-terminal 12th codon, K. The MA(PR)D25 mutant is identical to the MA(p6ⴱ-PR)D25 mutant except that it contains only five C-terminal codons of p6ⴱ. Instead of having 132 codons as in the WT MA protein, the MA(p6ⴱ-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(p6ⴱ-D25)D25 and MA(D25)D25 were identical to MA(p6ⴱ-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(p6ⴱ-D25)D25 and MA(D25)D25 yielded MA(p6ⴱ-D25) and MA(D25), respectively.

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831) to

Sal

I (nt 1147) of an HIV

gag

mutant that contained a

Sal

I linker at nt 1147 (18). To assess the proteolytic activities of

the inserted PR domains, the chimeric constructs were

sub-cloned 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

trans-fected into 293T cells. Expression and release of HIV Gag

proteins were probed by immunoblotting using an anti-p24

gag

monoclonal antibody (1, 19). As shown in Fig. 2, the WT Pr55,

the p41, and the mature p24

gag

proteins were detected in the

medium and in cell samples (lanes 3 and 9). A major band

representing Pr55

gag

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

de-tected only in the cell samples (lane 10 or 11, respectively). A

faint band corresponding to Pr55

gag

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.

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

Pr55

gag

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

re-leased 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 p41

gag

is readily observed. This might result from

altered PR preferential cleavage sites. Alternatively, the p41

gag

chimera was derived from the incompletely cleaved product

CA-NC-p6 (11). Further experiments are required to test this

proposition.

[image:3.612.56.288.72.230.2]

To further confirm that suppression of the PR activity

pro-motes 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

com-pensate 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,

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 nitro-cellulose filter. HIV p24gag_{and p24}gag_{-associated chimeric proteins were}

de-tected with mouse anti-p24gag_{monoclonal antibody at a 1:5,000 dilution,}

[image:3.612.314.547.84.422.2]

fol-lowed 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.

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(p6ⴱ-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␮M (lanes 1, 4, 7, 11, 14, and 17), 1.5␮M (lanes 2, 5, 8, 12, 15, and 18), and 7.5␮M (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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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

enzy-matic activity due to the chimeric mutations might account for

the low RT activity.

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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