Experimental Pathology Unit1 and Clinical Virology Unit2, The Hebrew University, Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel
National Public Health Laboratories, Ministry of Health, Israel3
Author for correspondence: Moshe Kotler. Fax +972 2 6758190. e-mail MKOTLER{at}cc.huji.ac.il
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Abstract |
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Introduction |
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Previously it was suggested that the autoprocessing of HIV-1 Gag by PR is an ordered process; the p2/NC junction is the first to be cleaved from Gag, while the CA/p2 is the last site processed (Tritch et al., 1991 ; Pettit et al., 1994
; Krausslich et al., 1995
). Cleavage of Gag at p2/NC yields a fusion intermediate product consisting of membrane-associated protein (MA), capsid protein (CA) and p2 peptide (Tritch et al., 1991
; Pettit et al., 1994
; Krausslich et al., 1995
; Vogt, 1996
; Accola et al., 1998
). If the cleavage at the p2/NC junction is indeed the first to take place in both polyproteins, then the release of PR from its precursor is not a prerequisite for the initiation of GagPol precursor processing. Previously, we and others showed that p6PolPR and PRreverse transcriptase (RT) fusion proteins are enzymatically active (Kotler et al., 1992
; Zybarth et al., 1994
; Louis et al., 1994
; Tessmer & Krausslich, 1998
; Cherry et al., 1998
). Here we support and extend these findings and report that HIV-1 PR is enzymatically active as an integral part of a larger fusion protein which includes NC*p6PolPR.
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Methods |
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Bacterial cells.
E. coli strain BL21 was used as a host for vectors expressing wild-type and mutated MACANCp6PolPR (GagPR) constructs (Fig. 1C). E. coli strain DH5
was used to propagate and maintain all plasmids.
Plasmids and mutagenesis.
CANCp6PolPR constructs were generated by PCR as previously described (Almog et al., 1996 ; Kotler et al., 1992
). Briefly, an NcoI site was introduced into the DNA segments of HIV-1 BH10 strain that encode the N terminus of CA. The NcoI site contains an ATG that serves as an initiator for protein synthesis. A termination codon was introduced just downstream of Phe, the last codon of PR, at position 2125. To permit efficient translation through the Gag/Pol junction, frameshift mutations were introduced: either downstream of the A nucleotide (nt 1634) by inserting an additional base A (FS1), or downstream of the T nucleotide (nt 1631) by inserting an additional base T (FS2) (Fig. 1B
). These single-base insertions put Gag and Pol into the same reading frame. The amplified sequences of HIV-1 containing the CAPR were cloned into pUC12N (Kotler et al., 1992
; Vieira & Messing, 1982
; Norrander et al., 1985
) for propagation and sub-cloned into the pT5 plasmid (Studier et al., 1990
) for expression, as previously described (Kotler et al., 1992
).
A cleavage site mutation (cs) was introduced at the NC*/p6Pol scissile site by a PCR reaction that replaced Phe with Ile, thus preventing hydrolysis at this site (Fig. 1C). The amplified DNA was cut with BglII and BamHI and inserted into pUC12N (CAPR). The double cleavage-site-mutated HIV-1 sequence (CANCcsp6PolcsPR) was constructed by amplification of the same DNA using pUC12N (CANCp6PolcsPR) as the template. The viral fragments were rescued from the pUC12N plasmids by cleavage with NcoI and SpeI and transferred to a pT5 vector cleaved with NcoI and XbaI restriction enzymes, respectively.
The MA-encoding fragment was generated by a PCR reaction and coupled to the wild-type and mutated pT5 (CAPR) to form the wild-type pT5 (GagPR) and the cleavage site mutants cs1 (NC*csp6Pol), cs2 (p6PolcsPR) and cs1+2 (NC*csp6PolcsPR).
Expression of HIV-1 proteins in bacterial cells.
The pT5 plasmids containing wild-type and mutated GagPol fragments were used to transform E. coli BL21 cells, which contain an inducible T7 polymerase (Fuerst et al., 1986 ). Bacterial cells were grown to saturation in LB medium at 37 °C overnight, cultures were diluted 1:20 and grown at 37 °C to a density of 0·40·6 OD600 units before IPTG was added to a final concentration of 0·4 mM to induce the T7 polymerase. Bacteria were harvested 2 h post-induction by centrifugation (3 min, 10000 r.p.m.), and pellets were re-suspended in 200 µl of Laemmli loading buffer.
Expression of HIV-1 GagPol fragments in CV-1 cells.
Monolayer cultures of 2x106 CV-1 cells in 100 mm tissue culture dishes (Nunc) were infected with vaccinia virus expressing the T7 polymerase (vTF7-3) (Fuerst et al., 1986 ; Karacostas et al., 1993
) at an m.o.i. of 10. Two hours post-infection cells were transfected by the CaPO4 method (Demetrios & Welkie, 1984
) with pT5 pDNAs containing wild-type HIV-1, or mutated DNA.
Expression of HIV-1 GagPol fragments in COS-7 cells.
Monolayer cultures of 2x106 cells in 100 mm tissue culture dishes (Nunc) were co-transfected by the DEAE-dextran method with plasmids pSVGAGPOL-RRE-RFS5T (pSVGAGPOL) or pSVGAGPOL-RRE-R (codes for HIV-1 GagPol with and without frameshift mutation) and pCMVrev (codes for the Rev protein) (Smith et al., 1993 ). These plasmids were kindly provided by Professor David Rekosh (University of Virginia, Charlottesville, VA, USA). Forty-eight hours post-transfection the cells were washed twice with ice-cold PBS+0·1 mM PMSF, harvested and re-suspended in Laemmli buffer.
Virus production and cell infection.
H9 cells were infected with HIV-1IIIB. Viral spread in the culture was monitored by p24 assay (Organon Teknika). To obtain virus particles, medium of infected cells was centrifuged for 15 min at 10000 g, and supernatant was centrifuged through a 20% sucrose cushion (100000 g for 60 min). Pellets were re-suspended in PBS and stored in aliquots at -70 °C.
Detection of HIV-1 proteins.
Bacterial or CV-1 cell extracts were run electrophoretically on polyacrylamide gels, blotted onto nitrocellulose filters (0·2 µm, Gelman Sciences), which were treated with rabbit anti-PR, monoclonal anti-CA or anti-MA (kindly supplied by Dr S. M. Nigida, SAIC, NCI, Frederick, MD, USA), and developed by alkaline phosphatase (Sigma) or enhanced chemiluminescence (Sigma). For the detection of NC and NC* proteins, cell lysates (bacteria, COS-7) and HIV-1IIIB virions were run on 16·5% or 20% Tricinepolyacrylamide gels, blotted onto PVDF filters and treated with monoclonal anti-NC (SAIC, NCI, Frederick, MD). To prevent loss of NC molecules during blotting, bovine haemoglobin (5 µg) was added to each slot and to the transfer buffer (0·25 mg/ml) as previously described (Gillespie & Gillespie, 1997 ).
Peptide cleavage assay.
Oligopeptides were synthesized and characterized as described before (Baraz et al., 1998 ; Friedler et al., 1999
). Assays were performed in 10 µl reactions containing 50 mM sodium phosphate buffer, pH 5·6, 0·1 M NaCl, 10 nmol of decapeptide, and 10 pmol of purified HIV-1 PR. The assay mixtures were incubated for 60 min at 37 °C, and the reactions were stopped by addition of guanidine hydrochloride (6 M final concentration). The cleavage products were analysed by reverse-phase HPLC on a C18 column (Vydac, 250x4 mm), using a gradient of 050% (v/v) acetonitrile in 0·1% aqueous trifluoroacetic acid at a flow rate of 1 ml/min, and absorbance at 220 nm was recorded.
HIV-1 protease inhibitor Ro 31-5989 (Roberts et al., 1990 ) was kindly supplied by Roche Products.
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Results |
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The autoprocessing results were further investigated by biochemical experiments. As previously described (Jacks et al., 1988 ), ribosomal frameshift occurs either as FS1 or FS2 (Fig. 1B
). We synthesized decapeptides presenting the sequence at the frameshift vicinity (NC/FS1, NC/FS2) and peptides homologous to the wild-type and mutated NC/p6Pol junction (Fig. 3
). The decapeptides NC/FS1 and NC/FS2 were not cleaved by purified HIV-1 PR in a cell-free system (Fig. 3 A
D
). These peptides were not hydrolysed even in high concentrations of PR and after a longer incubation time, conditions which allow almost complete hydrolysis of the accessible NC*/p6Pol peptide (data not shown). In contrast, the decapeptide consisting of the NC*/p6Pol junction was cleaved by HIV-1 PR under the same conditions (Fig. 3E
and F
).
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NC*p6PolPR fusion protein is enzymatically active
Expression of cs1+2 mutant in E. coli BL21 cells has shown that PR could release the NC*p6PolPR fusion protein from the GagPR precursor (Fig. 2A). We were therefore interested to assess whether this fusion protein accurately cleaves at the other sites in the polyprotein. To this end we expressed the GagPR constructs in bacterial and mammalian cells and analysed the cleavage products by Western blots. Fig. 2(B
, C
) shows that prevention of the cleavage at NC*/p6Pol, p6Pol/PR or at both junctions does not interfere with the autocatalysis of the polyproteins, since CA and MA proteins were released. The p41 (MACAp2) protein is the first to be cleaved from the polyprotein. This intermediate product was completely processed in the cs1 and cs1+2 mutants but only partially cleaved in the wild-type and cs2 mutant. We therefore concluded that PR is enzymatically active as part of the NC*p6PolPR (24 kDa) fusion protein, since other sites in the polyprotein were cleaved normally. Addition of 100 µg/ml of Ro 31-5989 inhibitor during IPTG induction (as described in Fig. 5
) verified that the fused viral PR exclusively processed the GagPR polyprotein (not shown).
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To demonstrate the release of NC* protein from the GagPol precursor in eukaryotic cells, COS-7 cells were co-transfected with pSVGAGPOL and pCMVrev plasmids (Smith et al., 1993 ). These constructs co-expressed the wild-type and frameshift-mutated GagPol and the Rev proteins. An NCp1 band, which was identified by specific anti-p1 antibodies (data not shown), is clearly distinguishable from the NC protein (Fig. 5C
, lane 2). NC* protein is released from the frameshift-mutated GagPol (lane 3). As anticipated, this NC* protein (63 aa) migrates to midway between NC (55 aa) and NC+p1 (72 aa). NC* protein could not be detected in lane 2 because it is expressed at a ratio of 1:20 of NC and NC-p1, which are expressed by the pSVGAGPOL construct. Taken together, these results demonstrate that the GagPol polyprotein is processed by the viral PR, yielding the NC* protein, which is 8 amino acids longer than the NC released from Gag.
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Discussion |
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This assumption was supported by demonstrating that: (i) abrogation of the NC*/p6Pol and NC/p6Pol/PR cleavage sites causes accumulation of NCp6PolPR fusion protein, indicating that a single cleavage site separates NC from p6Pol; (ii) synthetic decapeptides homologous to the junctions at the C terminus of NC* (NC fs1 and NC fs2) are not cleaved by HIV-1 PR, while peptide identical to the NC*/p6Pol junction is efficiently hydrolysed by the viral PR. These results strongly suggest that HIV-1 PR is unable to cleave at the C terminus of NC when it is part of the GagPol precursor; (iii) NC released from Gag polyprotein, extracted from mature virions, was found to be of lower molecular mass than NC* released from the GagPR expressed in bacterial cells; and (iv) NC* protein cleaved from the frameshift-mutated GagPol polyprotein expressed in COS-7 cells migrates in SDSPAGE between NC and NCp1. Based on the biochemical experiments and the analysis of the viral proteins cleaved from the polyproteins in bacterial and eukaryotic cells, we claim that during autoprocessing, the HIV-1 GagPol precursor releases an extended protein, NC*. The NC* protein could not be detected in the virions released from HIV-1-infected cells. One explanation may be that our test is not sufficiently sensitive to detect NC*, which is only about 5% of the NC. However, it also possible that NC* is not packed in the virions or that it is degraded before particle assembly. Elongated proteins released from GagPol in the frameshift vicinity were previously described in avian leukaemia sarcoma virus, where the PR is 12 amino acid residues longer than the PR translated in the Gag frame (Arad et al., 1995 ; Sedlacek et al., 1988
).
The function of NC* is not yet known, nor is it clear whether NC* and NC fulfil the same functions. Computerized prediction of the secondary structure of the NC* protein reveals that the additional 8 amino acid residues elongate an -helix structure from the Gag into the Pol domain. The
-helix structure provides flexibility and elasticity for the PR-bearing polyprotein. This prediction is in agreement with the model in which the p6Pol region is employed as a flexible hinge between the Gag and Pol domains (Tessmer & Krausslich, 1998
; Beissinger et al., 1996
). Thus, it is plausible that elongation of NC enables the initiation of autoprocessing before the release of PR from GagPol.
In accord with this model, we demonstrate that NC*p6PolPR is enzymatically active and thus the release of PR is not a prerequisite for the autoprocessing. Previous studies show that PR is enzymatically active as part of the p6PolPR or PRRT fusion proteins (Kotler et al., 1992 ; Zybarth & Carter, 1995
; Cherry et al., 1998
; Tessmer & Krausslich, 1998
). The enzymatic activity of NC*p6PolPR could not be compared to that of mature PR, since it was assessed in the truncated GagPol context only. It could be that this fusion protein is less active than the mature enzyme, as previously shown (Zybarth et al., 1994
; Zybarth & Carter, 1995
; Louis et al., 1994
, 1999
; Cherry et al., 1998
; Tessmer & Krausslich, 1998
). Since the fusion with NC* does not interfere with the autocleavage of the other viral protein, it is plausible that PR is active as part of the GagPol precursor, enabling autoprocessing which leads to virus maturation.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 August 2000;
accepted 22 November 2000.