1 Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK
2 Institute of Cancer Research, Chester Beatty Laboratories, London, UK
Correspondence
David Griffiths
d.j.griffiths{at}ucl.ac.uk
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ABSTRACT |
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Present address: Proteomika, Parc Científic de Barcelona, 08028 Barcelona, Spain.
The sequence of the RERV-H PR shown in Fig. 1(a) has been deposited in GenBank under accession no. AF515800.
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Introduction |
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The RERV-H family appears to be restricted to the European rabbit because the genomes of related members of the order Lagomorpha, including hares and pikas, do not contain RERV-H (Griffiths et al., 2002). This observation, together with the conservation of open reading frames (ORFs) for gag, pro and pol, indicates that this virus entered the rabbit genome relatively recently in evolution and raises the possibility that it is still active and infectious. Since its first discovery in human samples, we have been studying the function of several RERV-H proteins in vitro and here we describe our studies on the RERV-H viral protease (PR).
Retroviral PRs belong to the family of aspartic proteases, which has many members both in eukaryotes and prokaryotes (Davies, 1990; Hill & Phylip, 1997
). This family is characterized by sequence conservation around the active site, an acid pH optimum and similarity of three-dimensional structure (Wlodawer & Gustchina, 2000
). The cellular aspartic proteases are bilobal in structure, with two domains of similar amino acid sequence in a single polypeptide (Davies, 1990
). In contrast, retroviral PRs are dimeric enzymes composed of two separate but identical subunits (Bianchi et al., 1990
; Katoh et al., 1985
). The N- and C-terminal amino acids of each subunit are essential for dimer stability (Miller et al., 1989
; Wlodawer et al., 1989
).
Among retroviruses, several strategies are used to control expression of PR (Hatfield et al., 1992). In common with other betaretroviruses, the PR of RERV-H is encoded within a separate ORF (the pro gene), in fusion with an N-terminal deoxyuridine triphosphatase (dUTPase, DU) domain (Elder et al., 1992
) and its expression requires a ribosomal frameshifting event (Hatfield et al., 1992
). Thus, the PR domain is contained in both the GagPro and GagProPol precursors. Mature retroviral PRs are able to autocatalyse their own release from these polyproteins either during or shortly after assembly and budding. They also cleave the various Gag polyprotein precursors to yield the mature viral proteins as a late event in assembly. These tightly regulated proteolytic events result in morphological changes in the virus particle that are essential for virus infectivity (Kohl et al., 1988
; Sommerfelt et al., 1992
). Here, we show that RERV-H PR cloned from human DNA and expressed in a prokaryotic expression system is active on RERV-H proteins and is sensitive to known inhibitors of retroviral PRs.
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Methods |
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Molecular cloning.
NC-DU-PR PCR products were first blunt-end cloned into EcoRV-digested pBluescript KS(-) and then subcloned into BamHI/HindIII-digested pAlter-1 (Promega) using sites engineered into the PCR primers, to generate plasmid pNC-PR (Fig. 1a). The PR region (nt 7171283) was obtained by PCR and subcloned into the bacterial expression vector pET21b (Novagen) in-frame with the C-terminal hexahistidine tag to create plasmid pPR(wt). pNC-PR was mutated (nt 799800, Fig. 1a
) to substitute the active site aspartate with alanine using the oligonucleotide 5'-ATTATACTTACATCTGCACCTGTGGCTAGCAGGCCCTG-3' and the Altered sites II mutagenesis system (Promega, as recommended), to generate plasmid pNC-PR(D172A). This mutated PR fragment was subcloned into pET21b as above to create pPR(D172A).
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Plasmids pPR160, pPR160N and pPR160C were created by subcloning fragments of the original HRV-5/RERV-H PR clone (Griffiths et al., 1997; GenBank acc. no. U46939; corresponding to amino acids 173332, 173255 and 247332 of Fig. 1a
respectively) into EcoRI-digested pET21b in-frame with a C-terminal c-Myc tag (EQKLISEEDL) engineered into the reverse PCR primer. The RERV-H gag gene obtained from plasmid pHRV5.6 (Griffiths et al., 2002
) was cloned into pGex6p1 (Pharmacia) in fusion with the N-terminal glutathione S-transferase (GST) domain and with C-terminal c-Myc and hexahistidine tags introduced by PCR to create plasmid pGex-Gag.
Protein expression and analysis.
Escherichia coli strain BL21-CodonPlus(DE3)-RIL (Stratagene) was used as the host for all expression experiments. Transformed bacteria containing each expression plasmid were grown overnight at 37 °C in LuriaBertani broth (LB) containing 100 µg ampicillin ml-1 and 30 µg chloramphenicol ml-1. Bacteria were then diluted 1 in 10 in fresh LB with antibiotics and grown for 1 h at 37 °C before protein expression was induced with 1 mM IPTG for 3 h at 37 °C.
Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Amersham) using established procedures (Sambrook et al., 1989). Monoclonal antibodies (mAbs) to RERV-H PR (mAb-Pro1 and mAb-Pro9) were generated in rats as described (Dean et al., 1986
; Griffiths, 1996
). Polyclonal anti-RERV-H Gag antibodies raised in rabbits were a kind gift from John Hackett, Abbott Laboratories. RERV-H PR was detected by rat anti-PR mAbs or the mouse anti-c-Myc mAb 9E10 (Evan et al., 1985
; obtained from the Institute of Cancer Research, Sutton, UK). The secondary antibodies were goat anti-rat IgG, goat anti-mouse IgG or goat anti-rabbit Ig conjugated to horseradish peroxidase (Harlan). Bound antibodies were detected by enhanced chemiluminescence using the ECL Western blotting detection reagents (Amersham) as recommended.
Affinity purification of recombinant proteins.
Cultures (500 ml) of E. coli BL21-CodonPlus(DE3)-RIL transformed with either pGex-Gag or pPR(wt) were grown at 37 °C in LB with antibiotics to an optical density at 600 nm of 0·6. IPTG was added to 1 mM and cells were grown for a further 3 h before harvesting by centrifugation (4000 g, 10 min, 4 °C). The pellets were suspended in 5 ml of buffer L (100 mM NaH2PO4 pH 8·0, 10 mM Tris/HCl pH 8·0, 6 M guanidium . HCl) per gram of bacterial pellet (2·6 g for 500 ml culture) and shaken gently for 1 h at room temperature. The bacterial lysate was cleared by centrifugation (10 000 g, 30 min, room temperature) and the supernatant was added to 3 ml of 50 % NiNTA resin (Qiagen, pre-equilibrated in buffer L) and mixed gently for 1 h at room temperature. The resin was pelleted by centrifugation, resuspended in 10 ml of buffer D (100 mM NaH2PO4 pH 8·0, 10 mM Tris/HCl pH 8·0, 8 M urea, 20 mM imidazole) and mixed for 1 h at room temperature before loading into a column and washing with 10 ml of buffer D. Bound proteins were eluted with four 1 ml aliquots of buffer D containing either 250 mM imidazole [PR(wt)] or 175 mM imidazole (GSTGag). Purified PR(wt) proteins (800 µg in 500 µl of buffer D) were dialysed against 4 l of distilled water for 15 h at 4 °C and then for 6 h against 100 ml of activity buffer (5 mM DTT, 250 mM NaCl, 2 mM PMSF, 2 mM EDTA, 100 mM sodium phosphate pH 5·3) at 4 °C in a mini slide with a 3500 Da cut-off (Pierce).
Protease activity.
For analysis of autoprocessing, lysates were prepared from 3 ml of IPTG-induced bacterial culture. Cells were pelleted by centrifugation (4000 g, 4 °C, 10 min) and suspended in 400 µl of buffer T (20 mM Tris/HCl pH 8·0, 6 mM MgCl2, 1 mM EDTA, 1 % Triton X-100, 10 % glycerol). The resulting lysates were incubated for 10 min on ice with 0.1 units Benzonase (Merck) ml-1, loading buffer was added (5x times; is 250 mM Tris/HCl pH 8·0, 5 % -mercaptoethanol, 20 % glycerol, 5 % SDS, 0.5 % bromophenol blue) and the extracts analysed by SDS-PAGE and immunoblotting.
For assessing PR cleavage of RERV-H Gag polyproteins, 1·5 ml of E .coli culture expressing the PR clone under test (cultured and induced as above) was mixed with 0·5 ml of bacteria expressing GSTGag. This mixture was pelleted, suspended in 250 µl of activity buffer and sonicated three times for 30 s on ice. Bacterial lysates were incubated for 10 min on ice with Benzonase (Merck) and incubated for 3 h at 37 °C before analysing by SDS-PAGE and immunoblotting.
Protease assays on GSTGag proteins eluted from SDS-PAGE gels were performed with GSTGag that had been affinity purified on NiNTA and separated on a 10 % polyacrylamide gel. After SYPRO-Orange staining (Bio-Rad), a piece of gel containing the full-length 95 kDa GSTGag protein was excised, finely sliced and transferred to a tube containing 300 µl of activity buffer. The gel fragments were centrifuged (14 000 g, 4 °C, 10 min) and the supernatant containing the GSTGag eluted protein was retained. As a control, a gel piece containing no protein was treated in the same way. Bacterial cultures (3 ml) expressing either PR(wt) or PR(D172A) were centrifuged and the pellet suspended in 400 µl of activity buffer before sonicating and treating the resulting lysates with Benzonase. GSTGag proteins eluted from an SDS-PAGE gel (100 µl) were mixed with 100 µl of PR(wt) or PR(D172A) bacterial lysate and incubated for 3 h at 37 °C.
The activity of purified and dialysed RERV-H PR was assayed on GSTGag. Cells pelleted from 3 ml of culture expressing GSTGag were suspended in 400 µl of activity buffer and sonicated. The lysates were treated with Benzonase and a 100 µl aliquot was mixed with 100 µl of purified and dialysed RERV-H PR(wt) or PR(D172A), and incubated for 3 h at 37 °C.
N-terminal microsequencing.
Purified and dialysed PR(wt) proteins were separated by SDS-PAGE and transferred to PVDF membrane (Immobilon-P, Millipore) in transfer buffer (10 mM Tris/HCl, pH 8·3, 100 mM glycine, 15 % methanol) for 90 min at 230 mA. Following transfer, filters were stained (0·1 % Coomassie Brilliant blue R250 in 40 % methanol), destained in 50 % methanol and rinsed several times in water. Bands were excised and sequenced using an Applied Biosystems 477 pulse-liquid protein sequencer with on-line phenylthiohydantoin analysis.
Inhibitor assays.
Inhibition assays were performed as protease assays, with addition of increasing amounts of pepstatin A (Roche) or PYVPheStaAMT (kindly provided by Jan Konvalinka) solubilized in DMSO. Whatever the inhibitor concentration, each assay contained the same concentration of DMSO (9·4 %). Products were detected after immunoblotting with an anti-c-Myc mAb.
Sequence analysis.
DNA sequencing was performed using a BeckmanCoulter CEQ2000 automated DNA sequencer. Computer-aided analysis of protein and nucleotide sequences was performed with the Sequencher program (Gene Code). The program CORA (Orengo, 1999) was used to construct the structural alignment of retroviral PRs and this alignment was then used to generate the theoretical structural model of the RERV-H PR with Modeller 4.0 (Sali & Blundell, 1993
) using the human immunodeficiency virus type-1 (HIV-1) and Rous sarcoma virus (RSV) PR monomers as templates.
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Results |
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Autoprocessing activity of the RERV-H protease
The activity of the RERV-H PR was studied in E. coli using a variety of expression constructs (Fig. 1b). To determine whether the recombinant RERV-H PR is active, we first tested its ability to cleave itself out of a larger precursor molecule since this is a required function for retroviral PRs (Wan et al., 1996
). Because we were not certain of the termini of the mature RERV-H PR, we expressed a fragment, denoted PR(wt), that begins 27 residues upstream of the DTG active site (estimated from a structural analysis of retroviral PRs, see below) and ends at the natural stop codon of the pro gene (Fig. 1a, b
). To facilitate affinity purification on nickelagarose beads, the PR(wt) protein was expressed in fusion with a C-terminal hexahistidine tag. The tagged PR(wt) is a 222 amino acid protein with a calculated molecular mass of 24 665 Da.
Expression of the PR(wt) polypeptide was induced in E. coli and examined by immunoblotting using two mAbs raised against a fragment of RERV-H PR. These are mAb-Pro9, which recognizes a central epitope on RERV-H PR, and mAb-Pro1, which binds a C-terminal epitope (Fig. 1c). As shown in Fig. 2
, mAb-Pro9 detected five prominent bands at approximately 24, 22, 20, 18 and 16 kDa while bands of 24, 22 and 12 kDa and doublets of 17 and 15 kDa were detected with mAb-Pro1.
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Processing of RERV-H Gag polyprotein
We next wished to establish whether RERV-H PR expressed in bacteria is capable of processing its cognate Gag protein in trans. We successfully expressed a 95 kDa recombinant RERV-H Gag polyprotein fused with GST at the N terminus and C-terminal hexahistidine and c-Myc tags (GSTGag; Fig. 1b). Note that the GST moiety was introduced to increase the level of expression of the full-length Gag and was not used to purify the protein on glutathioneagarose.
When GSTGag was expressed in E. coli, the largest band detected on immunoblots with the anti-c-Myc mAb was 95 kDa, which is the expected size of the full-length GSTGag precursor (Fig. 3a). Bands at approximately 65, 45, 42, 30 and 20 kDa were also observed and these may represent partial degradation products of the Gag precursor, or may be the result of internal translation initiation. The instability of retroviral Gag polyproteins expressed in E. coli has been described previously (Debouck et al., 1987
; Graves et al., 1988
; Hansen et al., 1988
; Mueller-Lantzsch et al., 1993
).
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While we were able to show that the RERV-H PR is able to cleave the RERV-H Gag polyprotein, the presence of bacterial protease cleavage fragments made this somewhat difficult to interpret. In an attempt to establish a PR assay free of such degradation, the 95 kDa full-length GSTGag polyprotein precursor was recovered from a gel slice following SDS-PAGE. The eluted GSTGag polyprotein was cleaved only once by PR(wt) (Fig. 3b), releasing a 42 kDa fragment detected using anti-c-Myc antibodies and which probably corresponds to a CA-NC polypeptide. The other c-Myc tagged fragments observed following cleavage of the native Gag were not detected and no cleavage was observed when the eluted GSTGag was incubated with PR(D172A).
Incubation of GSTGag and PR(wt) bacterial lysates in buffers containing SDS or with ground SDS-polyacrylamide gel pieces also gave rise to the 42 kDa cleavage product only (data not shown), indicating that the SDS-PAGE eluted GSTGag was a poor substrate for PR activity. This is perhaps not surprising although previous data has shown that for avian sarcoma leukaemia virus, complete cleavage of Gag can be achieved following SDS-gel purification (Vogt et al., 1979) whereas HIV-1 Gag isolated from SDS-PAGE is completely resistant to protease treatment in vitro (Hansen et al., 1988
). Differences in the particular gel systems used may account for the variable results.
Plasmid pPR(wt) was designed to express a form of PR fused to a C-terminal hexahistidine tag to allow affinity purification of the recombinant protein over a nickel column (Leuthardt & Roesel, 1993). However, following autocleavage of the PR(wt) polypeptide, the mature PR should no longer have the hexahistidine tag. Nevertheless, the 24 kDa full-length PR(wt) and the cleavage products detected by the C-terminal-specific mAb-Pro1 still possess this tag (Fig. 2
). We therefore purified the full-length PR(wt) and C-terminal cleavage fragments from the crude bacterial lysates. We found that these proteins were insoluble and had to be purified under denaturing conditions (data not shown). Typically, 2·4 mg of the RERV-H PR(wt) 24 kDa precursor, 0·3 mg of RERV-H 22 kDa, 1 mg of RERV-H 17 kDa and 3 mg of RERV-H 15 kDa polypeptides could be purified from a 500 ml culture. The purified proteins were dialysed and PR activity was assayed using a bacterial lysate containing GSTGag as a substrate. We observed that the purified protein mixture retained its activity following dialysis and produced an identical cleavage pattern on the Gag substrate as before purification (Fig. 3c
). Since PR(wt) was purified under strongly denaturing conditions, it most likely refolded during dialysis to regain its activity.
Characterization of the RERV-H PR autoprocessing sites
In order to identify RERV-H PR autoprocessing sites and define the limits of the mature PR, N-terminal amino acid sequencing was performed on the 24, 17 and 15 kDa purified His-tagged PR(wt) polypeptides. The N terminus of the 24 kDa polypeptide was found to be AALS (band 1, Fig. 4a). Thus, the 24 kDa protein observed is not the complete polypeptide encoded by plasmid pPR(wt) but an N-terminal autoprocessed intermediate. This most likely explains the size difference observed on SDS-PAGE between PR(wt) and PR(D172A) since PR(D172A) is not autoprocessed.
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To determine whether the PR predicted from microsequencing experiments was active, we expressed a 103 amino acid form of the protein (Short-PR, amino acids 145247, see Fig. 1a, b) in E. coli and analysed its processing activity on the GSTGag substrate. As shown in Fig. 4(b)
, the shortPR(wt) is active, but not as much as PR(wt) since the only cleavage product obtained was 42 kDa.
The reduced activity of the 103 residue shortPR(wt) could be due either to a truncated N or C terminus. For human T-lymphotropic virus type I and HIV-1, the C-terminal amino acids are required for enzymatic activity because their involvement in the beta-sheet stabilizes the structure of the dimer (Hayakawa et al., 1992; Zhang et al., 1991
). If this were the case for RERV-H PR, the truncated C terminus (compared to HIV) would be expected to result in reduced dimer stability and less activity. Similarly, it has been shown that additional or missing amino acids at the N terminus of PR are deleterious to the activity of PRs of avian retroviruses (Grinde et al., 1992
; Pichova et al., 1992
; Sellos-Moura & Vogt, 1996
).
Inhibition of RERV-H PR activity
The effect of two aspartic protease inhibitors on RERV-H PR activity was assessed. We first used pepstatin A, a broad spectrum inhibitor of both retroviral and cellular aspartic proteases (Nam & Hatanaka, 1986; Ratner et al., 1985
). Processing of the GSTGag precursor by RERV-H PR(wt) was partially inhibited by 0·1 mM pepstatin A and was completely inhibited at 1·5 mM (Fig. 5
a). The concentration of pepstatin A required to give inhibition of activity was several orders of magnitude higher than the concentration required for inhibition of cellular aspartic proteases such as pepsin and renin (Umezawa et al., 1970
). However, this requirement for a high pepstatin A concentration agrees with results previously reported for retroviral PRs (Dreyer et al., 1989
; Katoh et al., 1987
).
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Modelling of RERV-H PR structure
The amino acid sequence of the RERV-H PR itself provides strong evidence that it encodes an aspartic protease, since it contains the canonical active site sequence motif (LXDTGAD) between positions 23 and 29 (Fig. 6a, HIV-1 numbering) and a second conserved motif, GRD/N, at position 8688 (Fig. 6a
). Moreover, the spacing between these two motifs is consistent with other retroviral PRs. To complement the biochemical analysis, we sought to derive a structural model for the RERV-H PR from the structures of those retroviral PRs that have been crystallized.
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Retroviral PRs range in length from 99 (HIV-1) to 125 (RSV) residues, with structural conservation of the N- and C-terminal regions that form the intersubunit beta-sheet (Rao et al., 1991). On the basis of the structural alignment analysis (Fig. 6a
) a three-dimensional atomic model of a 107 residue RERV-H PR was constructed using the program Modeller 4 (Fig. 6b
), and this model of RERV-H PR illustrates how the sequence may fold. Regions of sequence conservation from the structural alignment can be spatially arranged such that they form an active site that is consistent with all other retroviral PRs. Existing data on retroviral PRs shows that they exhibit little structural variation around the substrate-binding pocket but have most variation within three external loop structures and this is reflected in the model of RERV-H PR. This model of the RERV-H PR shows that two of the external loops are smaller than those of RSV PR and are similar in size to those of HIV-1 PR. The alignment and modelling of RERV-H PR both show that structural and sequence motifs are conserved with other retroviral PRs and are consistent with the biochemical analysis.
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Discussion |
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Zoonotic transfer of RERV-H from rabbits to humans is regarded as unlikely but has not been formally excluded and currently no data exist on the expression or activity of RERV-H proteins. Although some proviral loci are defective, full-length ORFs for gag, pro and pol have been cloned from rabbit and human samples so the possibility remains that some intact loci exist that could encode replication-competent viruses. Since such proviruses have not yet been identified and in the absence of a culture system for this virus, we have sought to demonstrate functional activity for the viral enzymes. In this report we have characterized the RERV-H PR. Although the clones used were amplified by PCR from human DNA samples, they do not differ significantly from sequences previously reported in rabbits (Griffiths et al., 2002).
Using a prokaryotic expression system to prepare recombinant RERV-H PR enzyme and Gag substrate, we have shown that the RERV-H PR can catalyse the cleavage of both PR-containing substrates (Fig. 2) and RERV-H Gag precursors (Figs 3 and 4
). As expected, RERV-H PR is an aspartic protease as shown by structural analysis and inhibition assays (Figs 5 and 6
). Due to cleavage by bacterial proteases, we had difficulty in identifying the termini of the mature PR, although a 103 amino acid protein with PR activity was produced (Fig. 4b
). From this we conclude that RERV-H does encode a functional protease.
Future work will focus on other aspects of RERV-H function such as Gag assembly and the activity of the other RERV-H enzymes. Preliminary data indicate that the RERV-H Gag polyprotein can assemble into cores when expressed in mammalian cells. The bacterial expression system described here will permit the production of large amounts of normal and altered RERV-H PR proteins, which will be useful for studying the biochemical properties of PR and permit mutational analysis of the proteolytic function. This will be important should RERV-H prove to represent a genuine zoonotic human infection.
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ACKNOWLEDGEMENTS |
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Received 18 June 2002;
accepted 25 August 2002.