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Journal of Virology, May 2002, p. 4341-4349, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4341-4349.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Characterization of the Protease of a Fish Retrovirus, Walleye Dermal Sarcoma Virus

Sharon K. Fodor^, and Volker M. Vogt^*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

Received 10 July 2001/ Accepted 4 February 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4°C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from Escherichia coli as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus associated with skin tumors in walleyes (2). It is the prototype of the epsilonretrovirus genus, which includes the closely related viruses walleye epidermal hyperplasia virus type 1 (WEHV-1) and WEHV-2 (11, 18, 31). In comparisons of reverse transcriptase (RT) sequences across retrovirus genera, the walleye viruses show the highest similarity with the mammalian C-type viruses, now called gammaretroviruses, typified by the murine leukemia viruses (MuLVs) (31).

Two other characteristics of the walleye viruses are also shared with the mammalian C-type viruses, suggesting a close evolutionary relationship. First, termination suppression apparently is the mechanism for production of the Gag-Pro-Pol polypeptide, based on the presence of an amber stop codon located between gag and pro. Second, the nucleocapsid (NC) domain of the Gag protein has a single Cys-His motif, unlike retroviruses of other genera, which have two such motifs. However, unlike their mammalian relatives, the walleye viruses contain three additional open reading frames, one of which encodes a cyclin D homologue (11, 17). Furthermore, these fish viruses are unique in that replication in natural circumstances occurs in the cold, near 4°C.

The mature structural proteins of WDSV, derived from the Gag and Env polyproteins, have been placed onto the genomic sequence based on N-terminal sequencing (11). However, little is known about the function of the enzymes encoded by the virus. The goal of the present study was to purify and characterize a recombinant WDSV protease and to use the recombinant protein to identify the protease cleavage sites in the Gag-Pro-Pol polyprotein, which gives rise to PR, RT, and integrase (IN).

Retroviral structural and enzymatic proteins are initially expressed as Gag and Gag-Pro-Pol (and in some cases Gag-Pro) polyprotein precursors, respectively. Polyprotein cleavage to yield the mature viral proteins is a late event in assembly. The viral PR, as a domain of Gag-Pro-Pol, cleaves itself out of the precursor either during or shortly after assembly and budding. The released PR then acts in trans on Gag and Gag-Pro-Pol to produce mature proteins. The structures and enzymatic properties of several retroviral PRs are well known (reviewed in reference 34). For all retroviruses, PR is a dimeric aspartyl protease made up of two identical monomers, each contributing an aspartic acid residue to the active site. A key feature of the dimer that is essential for its stability is a short, four-stranded antiparallel ß-sheet formed by the N- and C-terminal residues (23, 35). Substrate peptides bind to the enzyme in an extended ß-strand-like conformation within the active site, which is located at the interface of the two subunits.

PR at a minimum interacts with seven amino acid residues of the substrate, P4-P3-P2-P1/P1'-P2'-P3', the scissile bond being located between residues P1 and P1'. No tight consensus sequence defining a cleavage site has been found, even for a PR of one virus species. However, cleavage sites do share some general features. Best known among these features is the universal lack of a beta-branched amino acid residue in the P1 position (5, 26).

Based on the similarities between mammalian C-type viruses and WDSV, we expected MuLV PR to be the most suitable model for WDSV PR. The N terminus of the mature MuLV PR begins with the last four amino acids of the NC domain of Gag, followed by the glutamine residue that is inserted during termination suppression at the stop codon separating Gag and Pro (36). Hence, we constructed an Escherichia coli plasmid that expresses a WDSV PR fusion protein containing additional amino acid residues from NC and RT flanking the predicted PR domain. The purified PR precursor was found to undergo autoprocessing in vitro, producing mature PR. The enzymological properties of this PR were assessed, and its cleavage sites within the Pro-Pol protein were determined. Sequence comparisons between WDSV and WEHV-1 and WEHV-2 indicate that the walleye viruses contain a conserved glutamine residue at position P2 in all cleavage sites, a characteristic unique to this genus of retroviruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning, expression, and purification of recombinant PR. The WDSV PR coding sequence (from nucleotides 2513 to 3034, containing a CAG glutamine codon inserted at the TAG stop codon between gag and pro) was PCR amplified and inserted into the His-tagged vector pET28A (Novagen). The resulting plasmid, pPR22, expresses a PR fusion protein with 11 NC residues fused to the N terminus, 23 RT residues fused to the C terminus, and 6-His tags at both termini. The D39S active-site mutant, in which the aspartic acid codon (GAT) at nucleotide 2666 was mutated to a serine codon (TCA), was generated by PCR and also cloned into pET28A.

His-tagged proteins were expressed in E. coli BL21(DE3) cells. Protein expression was induced for 3 h with the addition of 100 µg of IPTG (isopropylthiogalactopyranoside) per ml. Cells were collected by centrifugation and resuspended in TN500 buffer (50 mM Tris [pH 8.0], 500 mM NaCl) containing 0.2% sodium deoxycholate. Cells were lysed by sonication, and inclusion bodies were collected by centrifugation in an SA600 rotor at 12,000 rpm for 15 min. Inclusion bodies were dissolved in TN500 containing 8 M urea. His-tagged proteins were purified by binding to nickel resin in TN500-8 M urea buffer. Bound proteins were washed with TN500-8 M urea containing 20 mM imidazole and eluted with TN500-8 M urea containing 1 M imidazole. The eluate was dialyzed into TN500-8 M urea containing 1 mM dithiothreitol (DTT). Inclusion of DTT at this step was essential for recovery of PR activity. The PR22 protein was stored in TN500-8 M urea-1 mM DTT at -20°C or subjected to autocleavage as described below.

Cleavage reactions. To achieve autocleavage, purified PR22 in 8 M urea was either dialyzed at room temperature into cleavage buffer (50 mM Tris [pH 7.0], 1 M NaCl, 1 mM DTT) and incubated for 2 to 15 h or diluted 10-fold with the same buffer. The residual 0.8 M urea was found to enhance the solubility of PR22 but not to affect the processing reaction. After incubation, the solution was centrifuged in a microcentrifuge at 14,000 rpm for 5 min to remove precipitated protein. The supernatant could be stored in the same buffer at 4°C for up to 1 week. PR concentration was determined by absorbance at 280 nm.

Salt and pH requirements for autocleavage were evaluated by dilution of PR22 followed by overnight incubation at room temperature. Reactions contained 15 µM PR22, 1 mM DTT, 100 or 1,000 mM NaCl, and 100 mM buffer (potassium phosphate, pH 5.0 or 5.5; MES [morpholineethanesulfonic acid], pH 6.0 or 6.5; and Tris-HCl, pH 7.0, 7.5, 8.0, or 8.5). The resulting products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Sypro Orange (Molecular Probes), and quantitated by phosphorimager.

WDSV Gag was synthesized in the presence of [³⁵S]methionine in a rabbit reticulocyte lysate (T7 TNT; Promega) from plasmid pcR/Gag (WDSV coding sequences from nucleotides 1 to 2554 cloned into pcDNAI/amp [Invitrogen]). In analyses of Gag processing by PR, 2 µl of reticulocyte lysate and 2 µl of PR (1 µg) were incubated in a final volume of 20 µl. Reactions evaluating pH and salt requirements contained 200 mM potassium phosphate buffer, pH 6.0, and NaCl from 0.1 M to 2.0 M NaCl or 200 mM potassium phosphate buffer at pH 5.3 to 8.0 and 1.0 M NaCl. Samples were incubated for 1 h at room temperature and analyzed by SDS-PAGE and autoradiography.

The following peptides were used to test PR activity: WDSV p20/CA (PQYQHPIRNR), WDSV RT/IN (FLQRVLKKG), and Rous sarcoma virus (RSV) RT/IN (PFQAYPLREA). Reactions (100 µl) contained 40 nmol of peptide and 80 pmol of PR. Samples were incubated at room temperature for 2 h, and reactions were stopped by the addition of 10 µl of trifluoroacetic acid, centrifuged for 10 min at 14,000 rpm at room temperature, and stored on ice until analyzed by high-pressure liquid chromatography (HPLC). Samples of 50 µl were injected on a Vydac C18 column and eluted with a 30-min, 2 to 30% acetonitrile gradient containing 0.1% trifluoroacetic acid with a flow rate of 1 ml per min. Fractions containing products were collected and submitted to the Cornell Biotechnology Facility for amino acid analysis.

N-terminal sequencing and mass spectrometry. Mature PR was separated from contaminating proteins by SDS-PAGE, and the PR band was transferred electrophoretically to a polyvinylidene difluoride membrane. The membrane was stained with Ponceau S for 5 min, and the background was cleared by rinsing with water. The protein band was excised and submitted to the Cornell Biotechnology Facility for N-terminal amino acid sequencing. For analysis by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-mass spectrometry, purified mature PR was dialyzed five times against 1 liter of distilled H₂O at 4°C. The dialysate was dried in a Speed Vac and submitted to the Cornell Biotechnology Facility for analysis.

Nucleotide sequence accession numbers and sequence alignments. Nucleic acid and amino acid sequences were analyzed using the DNAStar suite of programs. Sequence alignments were performed using the Clustal method with the PAM250 residue weight table. Accession numbers for the virus sequences described herein are as follows: feline leukemia virus (FeLV), M18247; Moloney MuLV, AF033811; WDSV, AF033822; WEHV-1, AF133051; and WEHV-2, AF133052.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression and purification of WDSV PR. In order to study WDSV PR in the absence of knowledge of the N and C termini of the mature protein, we designed a recombinant precursor (PR22) that encompasses the entire PR domain as well as neighboring residues of NC and RT (Fig. 1). The amber stop codon separating NC and PR was replaced by a glutamine codon, mimicking termination suppression at the gag-pro junction in MuLV (36). PR22 has the last 11 residues of NC at its N terminus and the first 23 residues of RT at its C terminus, plus a 6-His tag at each end. The protein was expressed in E. coli from an inducible vector, pET28A.

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FIG. 1. Cloning and expression of recombinant protease. (A) Genomic organization of WDSV gag, pro, and pol genes. The Gag-Pro-Pol polyprotein is translated via termination suppression, where the TAG stop codon separating gag and pol is read as a glutamine codon (CAG). (B) The stop codon was mutated to a glutamine codon (CAG) using a mutagenic PCR primer, and the product was cloned into the His-tagged vector pET28A. Upon protein induction in E. coli, a His-tagged protease precursor is expressed and subsequently affinity purified with nickel resin. The purified precursor self-cleaves to produce the mature protease. Figure not drawn to scale.

Lysates of induced cells (Fig. 2A, lane 1) showed three overexpressed bands on SDS-PAGE, which we identified as the full-length protein (PR22, 22 kDa), a partially processed product (PR18, 18 kDa), and the mature PR (15 kDa). Like PRs and PR-containing proteins of other retroviruses, PR22 proved insoluble in the crude extract. Hence, the insoluble protein fraction was dissolved in 8 M urea, and then the 6-His-tagged proteins were purified by means of a nickel-chelating resin (Fig. 2A, lane 2). Removal of the urea by dialysis into cleavage buffer led to precipitation of approximately 50% of the protein, the majority of which consisted of PR22 and PR18 (Fig. 2A, lane 4). PR itself, which accumulated during dialysis, remained soluble (Fig. 2A, lane 3).

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FIG. 2. Purification of recombinant protease. (A) Purification of His-tagged PR22. Lanes: M, size markers; 1, induced cell lysate postsonication; 2, uncleaved, purified PR22; 3, supernatant after PR cleavage; 4, pellet after PR cleavage. (B) Purified PR active-site mutant. Lanes: M, size markers; 1, PR22; 2, D39S mutant PR.

Even as early as 30 min after induction, PR18 was evident in crude extracts (data not shown). To verify that this product was due to authentic PR-mediated processing in E. coli rather than processing by a bacterial protease, we constructed a mutant version of PR22 with an active-site mutation (D39S). Upon expression of this mutant (Fig. 2B, lane 2), the only induced polypeptide species migrated at 22 kDa. We interpret this result to mean that both the 18-kDa and 15-kDa products result from enzymatic activity of the viral PR. Preparations of both the purified PR22 and the equivalent D39S mutant protein contained a minor contaminant similar in size to PR (Fig. 2B, lanes 1 and 2), but this contaminant could be differentiated from PR with higher-resolution gels (data not shown).

In order to identify the N terminus of mature PR and to further characterize PR18, N-terminal amino acid sequencing was performed on these polypeptides. The N terminus of the PR18 was found to be the 6-His tag engineered into the protein, implying that the first processing event removes the C terminus, presumably at the PR-RT junction, as shown schematically in Fig. 1. The N terminus of the mature PR was found to be the sequence PIDCP, corresponding to the second residue after the glutamine insertion at the Gag-Pro junction (Fig. 1B). In order to locate the C terminus of PR, we used the indirect approach of measuring the molecular mass of the mature PR by mass spectrometry (data not shown). A mass of 15,052 Da was found, in excellent agreement with a protein of 138 residues beginning with the sequence PIDCP and ending with the sequence YHKQL, which has a predicted mass of 15,050 Da. Cleavage at any other position would produce a protein differing in mass by at least 100 Da.

Autocleavage of the PR precursor. Maturation of an immature virus particle requires that the PR domain cleave itself out of the Gag-Pro-Pol polyprotein, an event experimentally represented by the processing observed for PR22. We tested the conditions affecting this reaction in vitro (Fig. 3). Retroviral proteases are most active at low pH and high ionic strength, and thus autoprocessing was evaluated from pH 5.0 to 8.5 in 100 or 1,000 mM NaCl. The reaction products were separated by SDS-PAGE and quantitated by phosphorimaging. Invariably some precipitation occurred in these incubations (see Fig. 2). Therefore, 0.8 M urea was included in autocleavage reactions to maximize the solubility of PR22 and PR18 and minimize precipitation. However, as we have not specifically evaluated precipitation as a function of pH, we presume that the products formed reflect the efficiency with which processing occurs before precipitation of the substrates terminates the reaction and may not necessarily reflect the pH optimum of the enzyme.

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FIG. 3. Effects of pH on protease autocleavage. Cleavage of PR22 precursor was tested at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 at low and high salt concentrations. Cleavage products were analyzed by SDS-PAGE (A), stained with Sypro Orange, and quantitated on a phosphorimager (B and C). (A) Lanes: 1 to 10, 100 mM NaCl; 11 to 20, 1,000 mM NaCl; 1, 10, 11, and 20, uncleaved precursor; 2 and 12, pH 5.0; 3 and 13, pH 5.5; 4 and 14, pH 6.0; 5 and 15, pH 6.5; 6 and 16, pH 7.0; 7 and 17, pH 7.5; 8 and 18, pH 8.0; 9 and 19, pH 8.5. (B) Cleavage products at 100 mM NaCl. (C) Cleavage products at 1,000 mM NaCl. Circles, PR22; squares, PR18; triangles, mature PR. The lane marked urea represents the input protein before adjustment to cleavage conditions.

Another complication inherent in this system is the requirement for two cleavage events to liberate mature PR. Processing was evaluated both as accumulation of PR and as disappearance of PR22. When the former was used, the most efficient reaction conditions were neutral pH and high salt (70% of input protein ended up as PR; Fig. 3C, pH 7.0). When disappearance of PR22 was used to assess PR activity, the most efficient conditions were also neutral pH and high salt, although the differences over the range of pH values were less pronounced (Fig. 3B and 3C). That processing by WDSV PR is most effective at pH 7.0 is surprising because other retroviral proteases have been found to be most active at lower pHs (15, 21, 22, 30).

Accumulation of PR18 revealed that the requirements for processing at the N-terminal versus C-terminal cleavage sites are different. At high pH, PR18 accumulated in the absence of mature PR, implying that processing under these conditions occurred exclusively at the C-terminal site (Fig. 3B and 3C). At the lowest pH, 5.0, PR22 did not undergo autocatalytic cleavage at all, although PR18 did process itself under the same conditions (Fig. 3B and 3C). These two observations suggest that cleavage at the C terminus of the PR domain is a prerequisite for cleavage at the N terminus. Sequential cleavage has also been observed for human immunodeficiency virus type 1 (HIV-1) PR, except in that case the order of cleavage is reversed (19, 20).

Gag cleavage by WDSV PR. To determine if the mature PR derived in vitro from PR22 is functionally active against its natural Gag substrate, we synthesized WDSV Gag in the presence of [³⁵S]methionine in a rabbit reticulocyte lysate and subjected this substrate to cleavage by mature PR and analysis by SDS-PAGE and autoradiography. Complete cleavage of Gag was observed, and the first product to be released was NC, based on its size (data not shown).

The requirements for pH and ionic strength in Gag processing were evaluated in this system (Fig. 4). As found for autocleavage, processing of Gag occurred best at neutral pH (Fig. 4, lanes 2 to 12). High ionic strength inhibited processing (Fig. 4, lanes 14 to 19). High ionic strength and low pH, optimal conditions for Gag processing in vitro by most retroviral proteases, nearly abolished Gag cleavage by WDSV PR (Fig. 4, lane 19). Inhibition of PR-mediated Gag cleavage at high ionic strength has also been observed for HIV-1 PR (14, 16). However, HIV-1 PR prefers high ionic strength when peptides are used as substrates. A convincing mechanistic explanation for the difference in salt dependence for the two types of substrates is not apparent.

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FIG. 4. Gag cleavage by recombinant WDSV protease. Processing of reticulocyte lysate-synthesized WDSV Gag by WDSV PR was evaluated from pH 5.0 to 8.0 and 0.1 to 2.0 M NaCl. Lanes 1 and 13, uncleaved Gag; lanes 2 to 12, 1.0 M NaCl at pH 5.0, 5.3, 5.6, 5.9, 6.2, 6.5, 6.8, 7.1, 7.4, 7.7, or 8.0, respectively; lanes 14 to 19, pH 6.0 at 0.1, 0.25, 0.5, 1.0, 1.5, or 2.0 M NaCl, respectively.

Peptide cleavage by WDSV PR. Quantitative analysis of processing of reticulocyte-synthesized Gag is difficult because of the several cleavage sites. Therefore, we designed a peptide substrate for PR using the sequence at the junction of the p20 and CA domains of Gag (PQYQHPIRNR). Enzymatic activity was measured as a function of temperature, pH, and ionic strength, using the appearance of the product peptide on HPLC to quantitate activity. In these experiments the reaction conditions yielding maximal product were defined as 100% activity.

The activity was approximately linear as a function of temperature (Fig. 5A). The optimal pH was 6.5 to 7.0 (Fig. 5B), with a sharp decrease in activity at more acid pHs. In the peptide assay, PR was most active at very high salt concentrations, from 1.5 to 3.0 M NaCl (Fig. 5C), similar to other retroviral proteases. For example, the optimal conditions for peptide cleavage reported for MuLV PR, HIV-1 PR, and bovine leukemia virus (BLV) PR are 3 M, 2.5 to 3.5 M, and 1 to 2 M, respectively (1, 21, 22). In our hands, at pH 6.5 and 2.0 M NaCl, the turnover number for WDSV PR for this peptide was calculated to be 6.4 min^-1, and the specific activity was calculated to be 200 pmol/min/µg of PR. However, the enzyme preparation may contain some inactive enzyme due to improper refolding, and the calculated specific activity would therefore be an underestimation.

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FIG. 5. Temperature, pH, and salt requirements for PR activity. (A) The p20/CA peptide was cleaved for 2 h at pH 7.0 and 1.0 M NaCl at 0, 4, 16, 26, and 37°C. (B) PR activity was determined at 1.0 M NaCl for pH 6.0, 6.5, 7.0, 7.5, and 8.0. The p20/CA peptide was cleaved with WDSV PR at room temperature. (C) PR activity was determined at pH 6.5 for 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M NaCl. All values are reported as percent activity, where 100% was set as the condition yielding maximum activity.

Identification of cleavage sites in Pro-Pol. In examining the cleavage sites within WDSV Gag (11), we noted a conserved glutamine residue at the P2 position in these sites (Table 1). Alignment of the WDSV, WEHV-1, and WEHV-2 Gag sequences predicted that the P2 glutamine in Gag proteins is also conserved in WEHV-1 and WEHV-2 (data not shown). We then aligned the Pro-Pol polyproteins of WDSV, WEHV-1, WEHV-2, MuLV, and FeLV to identify potential cleavage sites (data not shown). Some gaps located between enzyme domains were apparent in the alignment, reflecting insertion or deletion of sequences in evolution (Fig. 6A). When the gaps were removed for purposes of comparison, conserved glutamine residues became evident in the sequences of the walleye viruses near the known Pro-Pol cleavage sites of MuLV and FeLV (Fig. 6B and 6C). Thus, all PR cleavage sites in Gag and Gag-Pro-Pol in the three walleye viruses are predicted to contain a conserved glutamine residue at the P2 position (Table 1).

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TABLE 1. Protease cleavage sites of WDSV, WEHV-1, and WEHV-2a

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FIG. 6. Predicted cleavage sites in Pol based on sequence alignment. Gapped regions between PR, RT, and IN domains in Pol alignments were used to predict PR cleavage sites in WDSV Pol. (A) PR-RT junction from Pol alignment. (B) Sequence surrounding the predicted PR-RT cleavage site is shown with gaps removed. (C) Sequence surrounding the predicted RT-IN cleavage site. Arrows, known cleavage sites of MuLV and FeLV; triangles, predicted cleavage sites of WDSV, WEHV-1, and WEHV-2.

As discussed above, the assignment of the WDSV PR-RT cleavage site is supported by mass spectrometry. To verify the predicted RT-IN cleavage site, a peptide representing this sequence (FLVQRVLKKG) was synthesized and used as a substrate for PR. The reaction products were separated by HPLC, and amino acid analyses were carried out to identify them. The resulting data verified that processing occurred as expected, between the R and V residues of the peptide (data not shown). Cleavage at these PR-RT and RT-IN sites in WDSV would yield an RT molecule 650 residues in length with a mass of 72.5 kDa and an IN molecule 380 residues in length with a mass of 42.8 kDa.

The predicted mass of RT is consistent with size estimates of enzymatically active WDSV RT purified from virions, about 70 kDa (6). In some retroviruses Pro-Pol is processed at other sites in addition to those at the PR-RT and RT-IN junctions. For example, in HIV-1, the RNase H domain of one of the two subunits of RT is removed by proteolytic processing (10, 12, 24, 25, 27, 32), while in RSV a C-terminal extension of IN is removed (7, 8). By contrast, in MuLV and FeLV, no other cleavage sites have been identified (3, 36). Hence, based on the absence of gaps in the sequence alignments between Pro and Pol of the walleye viruses and MuLV and FeLV, other than those at the PR-RT and RT-IN junctions, we predict that these are the only cleavage sites in the walleye viruses.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have purified a recombinant WDSV PR and shown that it can perform two functions required in vivo: self-cleavage out of the immature polypeptide and subsequent processing of the Gag precursor. Analysis of in vitro cleavage products and sequence alignments allowed us to identify the sites of scission in the Pro-Pol polypeptide that lead to the mature PR, RT, and IN proteins of this virus. Biochemical characterization identified two unique features of WDSV PR in comparison with other retroviral PRs: a pH optimum near neutrality and a conserved amino acid residue, glutamine, at the P2 position in all cleavage sites.

The conservation of the P2 glutamine apparently is shared with the other two walleye retroviruses, WEHV-1 and WEHV-2, based on sequence alignments of Gag-Pro-Pol. The assignment of the site at the N terminus of PR for those viruses is subject to some uncertainty, however, for the following reason. In WDSV there is a single glutamine residue near the Gag-Pro junction, and it is in the P2 position of the experimentally determined cleavage site. In WEHV-1 and WEHV-2 there is in addition a second glutamine five residues upstream of the Gag-Pro junction. If the latter residue represents the P2 position and cleavage takes place at that site, then the N terminus of PR in these viruses would be similar to that in MuLV, which is four residues upstream of the termination codon separating Gag and Pro (36). We suspect that the N termini of WEHV-1 and WEHV-2 PR indeed are generated at this site, since the P4, P2, and P1' residues then would be the same as in the slightly downstream site used in WDSV.

The conservation of a single amino acid residue at the same position in all cleavage sites for the Gag, Pro, and Pol proteins is surprising. No such conservation has been observed for any other retrovirus (26, 33). The importance of the amino acid at the P2 position has been noted previously in the avian retrovirus system. For example, placement of a proline residue at this position abolished the ability of proteins or peptides to be substrates for PR (9, 13, 29). To determine if the P2 glutamine is strictly required in WDSV, we tested a glutamine-containing peptide representing the RT-IN cleavage site of RSV (PFQAYPLREA) as a substrate for the PRs of WDSV and in parallel with avian myeloblastosis virus (AMV, a close relative of RSV) (data not shown). At 25°C AMV PR cut the peptide at the correct site, as expected, between the Y and P residues. Though it did so less efficiently, WDSV PR also cut the peptide, and analysis of the products showed them to be the same as for AMV PR, thereby placing the glutamine residue in P3. Thus, at least under these conditions, a P2 glutamine is not essential for functioning of WDSV PR. We speculate that at the naturally cold temperature of replication of this virus, the requirements for a P2 glutamine may be more stringent.

WDSV PR is unusual among retroviral PRs in that the pH optima for autoprocessing as well as for processing of peptides and of Gag protein are near neutrality. Other retroviral PRs prefer acid conditions, typically in the pH range of 5 to 6. For example, RSV is most active at pH 5.5 to 6.0 (15). BLV PR is active over the pH range 3.5 to 6.5. Both of these PRs show sharply reduced activity at pH 7.0 (15, 21). MuLV PR activity is optimal at pH 6.0 (22), although it is still capable of efficient cleavage at pH 7.0. HIV-1 PR shows somewhat less preference for acid pH (4). It has been inferred previously for HIV-1 PR acting on peptides that the primary effect of pH is on substrate binding (K_m), not catalytic efficiency (k_cat) (28).

WDSV is unusual among retroviruses in that the cleavage sites have a high number of potentially charged residues: four of the six known sites each have two such basic residues (R, K, or H) and one has two acidic residues (D). By comparison, of the 11 cleavage sites in RSV polyproteins, only 2 have a single charged residue, and in the 11 sites in HIV-1 polyproteins, 4 have a single charged residue and 2 have two charged residues. The interaction of the charged residues with the PR binding-site residues and with each other would be expected to lead to a major pH dependence. A likely example of this effect is the difference in autocleavage rates at the N and C termini in PR22. At low salt and pH >7.0, the N-terminal site is refractory to cleavage, but at high salt this inhibition is greatly lessened. This cleavage site is unique in WDSV in having two acidic residues. Charge interactions leading to poor binding could be overcome by either a reduction in pH or an increase in ionic strength. The biological significance of different pH optima among diverse retroviruses is unknown, since all proteolytic processing is presumed to occur near neutral pH, either in the cell in the final stages of budding or in the extracellular environment after liberation of an immature virion.

The relative differences in WDSV PR activity at different pHs and salt concentrations, as measured in the several assays described in this study, are likely due to multiple factors. Since the short peptides used as substrates are not expected to have significant secondary structure, the processing rate should be dependent only on binding (K_m) and the catalytic efficiency (k_cat) of PR. In other retrovirus systems, binding of peptide substrates is mediated primarily by hydrophobic interaction, which accounts in part for the high-salt preference. The presence of oppositely charged groups in the enzyme-binding pocket and in the peptide substrate would counteract the positive effect on binding due to the hydrophobic interactions, leading to a complicated dependence on ionic strength, as discussed above.

In protein substrates, pH and salt concentration in addition may affect local folding and hence the accessibility of the cleavage site. For the autoprocessing reaction, the parameters affecting cleavage may be still more complicated. Two PR domains must dimerize to form an active site. At least in mature retroviral PRs, the N and C termini are part of a four-stranded, antiparallel ß-sheet that itself is essential for dimer stability and hence enzyme activity. If the PR domain folds as does the mature PR, the cleavage sites used to generate the mature protein would be occluded by secondary structure. Thus, processing of the PR precursor may require destabilization of the dimer to render the cleavage sites accessible to the enzyme's active site. The implied dynamic equilibrium between different conformations remains to be explored in any retrovirus system.

 
This work was supported by USPHS grant CA 20081.

 
* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Biotechnology Bldg., Cornell University, Ithaca, NY 14853. Phone: (607) 255-2443. Fax: (607) 255-2428. E-mail: vmv1{at}cornell.edu.

Present address: Dept. of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
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Journal of Virology, May 2002, p. 4341-4349, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4341-4349.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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