California State University, Long Beach, Department of Biological Sciences, 1250 Bellflower Blvd, Long Beach, CA 90840, USA1
Author for correspondence: Editte Gharakhanian.Fax +1 562 985 8878. e-mail eghara{at}csulb.edu
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Abstract |
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Introduction |
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We have previously reported the development of a cell-free system to assay proteinprotein interactions among SV40 structural proteins (Gharakhanian et al., 1988 , 1995
). The cell-free lysate was used to map the Vp1-interactive domain of Vp3 and to show the formation of Vp1 pentamers and multi-pentamer complexes in vitro. Here, we have utilized the cell-free system to identify the specific Vp1 cysteines involved in inter-pentamer disulfide linkage. Systematic site-directed mutagenesis of individual Vp1 cysteine codons into serine codons was followed by transcription and translation of wild-type and mutant Vp1 genes in rabbit reticulocyte lysates. Translation products were assayed for multimerization by non-reducing SDSPAGE and by sucrose-gradient sedimentation. Using this methodology, we have previously reported formation of non-covalently linked 7S Vp1 pentamers followed by formation of disulfide-linked 12S, multi-pentameric complexes of Vp1 (Gharakhanian et al., 1995
). In this study, destruction of all seven cysteine codons resulted in no 12S complexes, directly implicating disulfide bonds in stabilization of inter-pentamer Vp1 interactions. Vp1 proteins that contained serine residues instead of cysteines at Cys49, Cys87, Cys254 and Cys267 continued to form disulfide-linked 12S complexes similar to wild-type Vp1, whereas Cys9, Cys104 and Cys207 triple mutant Vp1 proteins did not. Single and double mutants at the three cysteines affected continued to form 12S complexes, but to lesser degrees. Thus, our results show that Cys9, Cys104 and Cys207 are sufficient and essential for formation of Vp1 inter-pentamer disulfides in cell-free lysates. While single cysteines at positions 9, 104 or 207 are sufficient for formation of inter-pentamer disulfide bonds in vitro, all three cysteines participate in the process for maximum complex formation. These results are in agreement with SV40 structural studies which have implicated the same three Vp1 cysteines by mercury labelling (Liddington et al., 1991
), and which have actually detected Cys104-mediated disulfide bonds only at the inter-pentamer level of organization (Stehle et al., 1996
).
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Methods |
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Plasmids.
The pSp6Vp1 plasmid (Gharakhanian et al., 1995 ) is a transcription vector that contains the SP6 promoter, an ampicillin resistance gene and the SV40 Vp1 gene. This plasmid was used during oligo-directed mutagenesis to generate the mutant Vp1 genes. Another plasmid, pUC19M (Clontech), was used in the mutagenesis reaction as a control. This plasmid has an amber stop codon in the lacZ gene.
Restriction and modifying enzymes.
The restriction enzymes used were from New England Biolabs unless otherwise indicated. The T4 DNA polymerase and T4 DNA ligase were from Clontech.
Construction of mutant pSp6Vp1 plasmids by oligo-directed mutagenesis.
Mutagenesis was performed following the protocols in the Transformer Site-Directed Mutagenesis Kit from Clontech (1993) with some minor modifications. Two primers, the selection and the mutagenic primers, were annealed to the template at the same time. The mutagenic primer encoded a point mutation that changed a specific cysteine codon into a serine codon. The selection primer was used to change a unique restriction site on the vector to another unique restriction site. In this case, the AflIII restriction site on the plasmid was changed to a BglII restriction site.
After mutagenesis, DNA was extracted from mutant colonies and subjected to restriction digest analysis using AflIII and BglII enzymes. Mutant samples that were linearized by BglII but not by AflIII were further analysed for the desired G-to-C base change at a given cysteine codon by dideoxynucleotide sequencing using the Sequenase Quick-Denature Plasmid Sequencing Kit (Amersham). Polyacrylamide (6%)urea sequencing gels were fixed with a solution of 5% acetic acid15% ethanol for 3 min. Dried gels were exposed to Kodak BioMax MR1 film overnight at room temperature. The autoradiograms were developed in an automatic film processor (Kodak).
Using this method, four types of Cys-to-Ser mutants in pSp6Vp1 were generated. Three single Cys-to-Ser codon mutants at positions 9, 104 and 207 (pSp6Vp1.C9S, pSp6Vp1.C104S and pSp6Vp1.C207S, respectively) were generated. Additionally, one double Cys-to-Ser codon mutant at positions 9 and 207 (pSp6Vp1.C9S.C207S), one triple Cys-to-Ser codon mutant at positions 9, 104 and 207 (pSp6Vp1.C9S.C104S.C207S) and one quadruple Cys-to-Ser codon mutant at positions 49, 87, 254 and 267 (pSp6Vp14X) were produced. Please see Table 1.
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Construction of mutant plasmids by directional subcloning.
The double Cys-to-Ser Vp1 mutant plasmids at positions 9 and 104, and at positions 104 and 207, (pSp6Vp1.C9S.C104S and pSp6Vp1.C104S.C207S, respectively) were generated by directional subcloning. The restriction endonucleases AflIII and ApaI (Boehringer Mannheim) cut at 1699 and 2258 bp, respectively, to produce a band of 559 bp. This 559 bp fragment contains the cysteine codons at positions 87, 104, and 207. The remaining 3·7 kb contains the cysteine codons at positions 9, 254 and 267. To generate pSp6Vp1.C9S.C104S, the 3·7 kb region from pSp6Vp1.C9S was used as the vector and the 559 bp region from pSp6Vp1.C104S was used as the insert. To generate pSp6Vp1.C104S.C207S, the 3·7 kb region of the wild-type pSp6Vp1 was used as the vector and the 559 bp region of pSp6Vp1.C9S.C104S.C207S was used as the insert.
Plasmid DNA was digested first with AflIII for 1 h at 37 °C then with ApaI at 30 °C for 2 h. After digestion, the samples were electrophoresed in 1% agarose. The appropriate bands were purified from the gel using the QiaExII DNA Extraction Kit (Qiagen). After DNA extraction, 1:1 molar ratios of insert to vector were prepared. Before ligation, the insertvector solution was placed in a beaker with boiling water until the water came to room temperature. T4 DNA ligase (Boehringer Mannheim) was added and the solution was kept at 12 °C overnight to allow the reaction to proceed. Plasmid DNA was then transformed into E. coli DH5 cells. To confirm successful subcloning, plasmid DNA was extracted from the resulting colonies and mutagenized region(s) were analysed by dideoxynucleotide sequencing. After the mutant plasmids were identified, large-scale plasmid isolations were done to prepare plasmid DNA for subsequent reactions using the Qiagen Plasmid Maxi Kit (Qiagen).
Multimerization assays of the wild-type and mutant proteins
In vitro transcriptiontranslation.
Production of wild-type and mutant Vp1 proteins was accomplished using the TnT Coupled Reticulocyte Lysate System from Promega. As directed by the manufacturer, transcription and translation were performed at 30 °C for 2 h in one reaction using 1 µg plasmid DNA and 60 µCi l-[35S]methionine (>1000 Ci/mmol; Amersham). To confirm that Vp1 protein is the major translation product of the Vp1 gene in the system, the translation product was immunoprecipitated as described by Gharakhanian et al. (1995) using polyclonal antibodies that were raised against a neutralizing Vp1 peptide (Fasching, 1998
; C. Fasching and others, unpublished). Samples were then incubated at 37 °C for up to 5 h for post-translational Vp1 interactions. Total incubation time at 30 and 37 °C was 7 h. Previous experiments (E. Gharakhanian, unpublished) showed that going beyond 7 h resulted in degraded products and non-specific aggregations.
During the 37 °C incubation, 10 µl samples were removed every hour. Half of the sample from each time-point was treated with 5 µl 20 mM DTT for 30 min at 37 °C as controls; the other half received 5 µl double-distilled water. Five µl 50 mM iodoacetamide was added to each sample to alkylate the free SH groups; the reaction was carried out in the dark at room temperature for 30 min. The reactions were stopped by addition of 15 µl 2x SDSPAGE sample buffer. The samples were either stored at -70 °C or used in SDSPAGE.
The samples were subjected to 10% non-reducing SDSPAGE. Thirty min after the tracking dye ran off, the gels were stopped. This was done to see 40 to 200 kDa bands on the same gel. The gels were fixed with 5% acetic acid5% isopropyl alcohol for 15 min and were subjected to fluorography using Autofluor (National Diagnostics). 14C-labelled molecular mass markers were also included on the gel.
Densitometric analyses were performed on selected autoradiograms using the Optimas Profile 1-D Gel Densitometry program. Using this program, relative amounts of the Vp1 monomeric bands in reduced and non-reduced samples at 5 h post-translation were tracked. Zero was given as the absorbance value whenever the program could not detect a peak. Vp1 monomer values were calculated by dividing the absorption total value of the non-reduced Vp1 monomeric band by the absorption total value of the reduced Vp1 monomeric band for each of the Vp1 samples. Values are expressed in percentage Vp1 monomers.
Separation of translation products by sucrose-gradient sedimentation.
Wild-type and mutant Vp1 proteins were produced by in vitro transcriptiontranslation as described above and were subjected to post-translational manipulations as described (Gharakhanian et al., 1995 ) with the following modifications. After translation, one-third of each sample was frozen at -70 °C, one-third was further incubated at 37 °C for 1 h and one-third was incubated for 3 h, to allow for Vp1Vp1 interactions. At the end of each incubation time, samples were stored at -70 °C. Thirty µl of each thawed sample was loaded onto a 520% sucrose gradient with a 50% sucrose cushion. The samples were centrifuged at 35000 r.p.m. at 4 °C for 23 h in an SW41 rotor. Fifteen 0·75 ml fractions were collected from the bottom of each tube and 100 µl of each fraction was precipitated with trichloroacetic acid (TCA). An equal volume of cold 50% TCA was added to each sample in wells lined with filter paper. This was incubated for 15 min at 4 °C. The supernatant was aspirated and the filter paper was gently rinsed with more cold 50% TCA, air dried, and precipitable radioactivity was determined by liquid-scintillation counting. In repeated fractionations, bovine serum albumin (4·5S) peaked at fraction 12, native immunoglobulin G (7S) peaked at fraction 10 and catalase B (11·3S) peaked at fraction 7.
To reconfirm the molecular masses of the 7S and 12S species, 500 µl each of the wild-type Vp1 fraction 10 (7S Vp1) and fraction 6 (12S Vp1) were subjected to gel filtration using Sephacryl S-300 (Sigma) in standard TE buffer. Fractions (1 ml) were collected and 100 µl of each fraction was TCA-precipitated and Vp1 radioactivity was determined as described above. Yeast alcohol dehydrogenase (150 kDa), sweet potato ß-amylase (200 kDa), horse spleen apoferritin (443 kDa) and bovine thyroglobulin (670 kDa) were used as markers for calibration (Sigma marker kit).
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Results |
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Next, a Cys-to-Ser triple mutant Vp1 (at Cys9, Cys104 and Cys207) was assayed for multimerization as described above. In contrast to wild-type and quadruple mutant Vp1 and similar to septuple mutant Vp1, the triple mutant did not form disulfide-linked post-pentameric complexes in both non-reducing SDSPAGE (Fig. 2d) and in sucrose gradients (Fig. 3d
), yet continued to form non-disulfide linked 7S pentameric complexes by 1 h post-translation as assessed by sucrose-gradient sedimentation (Fig. 3d
).
The results suggest that one or two or all three cysteine residues at positions 9, 104 and 207 play a role in inter-pentamer Vp1 disulfide interactions in vitro. To distinguish between these possibilities, Cys-to-Ser single and double mutants at Cys9, Cys104 and Cys207 were assayed for multimerization as described above. The single mutant proteins at Cys9, Cys104 and Cys207 continued to form SS bonds in non-reducing SDSPAGE (Fig. 2eg
) and sedimented as 12S species by 3 h post-translation (Fig. 3e
g
). In all three single mutants, a small peak of 7S pentamers remained after 3 h as assayed by sedimentation (Fig. 3e
g
), and densitometric analysis of the non-reducing SDSPAGE showed slightly higher persistence of non-disulfide linked Vp1 at 5 h post-translation (data not shown).
Similar to wild-type Vp1, all three double mutants at Cys9, Cys104 and Cys207 formed disulfide-linked 12S complexes by 3 h post-translation (Fig. 2hj
and 3h
j
). However, both non-reducing SDSPAGE and sucrose-gradient analysis of time-dependent multimerization revealed a decrease in disulfide-linked multimerization relative to wild-type.
These results show that cysteine residues at positions 9, 104 and 207 are essential and sufficient for disulfide-linked stabilization of inter-pentamer complexes in vitro, and that while all three are necessary for maximum complex formation, involvement of any one of the three is sufficient for the process.
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Discussion |
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A septuple mutant at all seven cysteine residues of Vp1 formed no disulfide-linked multi-pentamer complexes as assayed by non-reducing SDSPAGE and by sucrose-gradient fractionations, directly implicating cysteine-mediated disulfide linkage in Vp1 inter-pentamer stabilization. In the same assays, a quadruple mutant at Cys49, Cys87, Cys254 and Cys267 continued to form 12S complexes. The triple mutant protein Vp1.C9S.C104S.C207S, however, was severely defective in formation of inter-pentamer disulfides and sedimented as pentameric 7S species at both 1 and 3 h post-translation. Collectively, the results suggest that inter-pentamer disulfides at Cys9, Cys104 and Cys207 are essential and sufficient for inter-pentamer disulfide-linked stabilization of Vp1 molecules in cell-free lysates. This result is in complete agreement with the structural mercury-labelling experiments that led to the labelling of the four unaffected cysteines, suggesting that only Cys9, Cys104 and Cys207 can be occupied in disulfide linkage (Liddington et al., 1991 ). The size and S value of disulfide-linked complexes seen in this study show that disulfide bonds play a role in the stability of Vp1 complexes at the inter-pentamer level. This is consistent with the Liddington et al. study where disulfide bonds were not detected at Vp1 monomer level nor at pentamer level by X-ray crystallography; inter-pentamer complexes were not studied in that report. More recently, Stehle et al. (1996)
actually detected inter-pentamer disulfides between B and B' Vp1 molecules at Cys104. Cys-to-Ser single and double mutant Vp1 proteins at Cys9, Cys104 and Cys207 were only partially affected and continued to form disulfide-linked 12S complexes. The fact that mutant proteins containing only one of the three critical cysteines continue to form disulfide-linked 12S complexes strongly suggests that the disulfide bridges are between identical cysteines (e.g. Cys104Cys104). In fact, the only structurally visualized SV40 Vp1 disulfides are between Cys104 residues (Stehle et al., 1996
). Lack of drastic changes from wild-type in the assembly of double mutants precluded meaningful results from mixed-mutant experiments.
Of the three cysteine residues discussed, Cys104 may indeed be the preferred residue for disulfide bond formation since this residue is highly conserved between SV40 and polyomavirus (Liddington et al., 1991 ; Gharakhanian, 1988
), and is located in a highly conserved hydrophobic region among related Vp1 proteins (Gharakhanian, 1988
). The SV40 Vp1 Cys104 is located in a loop on the Vp1 pentamer that is in contact with other Cys104-containing loops on neighbouring pentamers (Liddington et al., 1991
), making it an ideal cysteine residue for disulfide bonding in the post-pentameric Vp1 complex as detected by Stehle et al. (1996)
. Cys9 is also highly conserved between SV40 and polyomavirus (Gharakhanian, 1988
; Liddington et al., 1991
); it is located on the amino-terminal arm of Vp1 and is exposed on a Vp1 pentamer. The position of Cys9 in the capsid has not been visualized by crystallography, so this free amino-terminal arm on the pentamer may be an alternative site for disulfide bonding with other pentamers. Cys207 is located on an exposed loop on the pentamer (Liddington et al., 1991
), making it another possible alternative site for disulfide bond formation with other pentamers. While a single disulfide bond may be sufficient, all three may be involved to enhance stabilization of the complex. The fact that out of three possible disulfide bonds, any one is sufficient for Vp1 multimerization may also be an evolutionary advantage for the virus; if mutations occur in any two of the three cysteines, the potential for formation of one stabilizing disulfide bond is maintained. Alternatively, potential involvement of each of the three cysteines in inter-pentamer disulfides may be limited in the capsid where minor viral structural proteins, Vp2 and Vp3, and the SV40 minichromosome are present and higher-order configurations may be more constrictive. Additionally, within the capsid, the three cysteines may find themselves in differential redox states, and different cysteines may be favoured in the three differentially interactive clusters of Vp1 (Yan et al., 1996
).
At what point the disulfide bonds form during the assembly of SV40 virus remains speculative. Whether the disulfide bonds stabilizing the SV40 virus form during its assembly in the cell or sometime after completion of assembly and after cell lysis remains unclear. Our results show SV40 Vp1 inter-pentamer disulfide bond formation in the cytoplasmic environment of cell-free lysates where 0·51 mM DTT is present, suggesting that Vp1 disulfides may form in the reducing intracellular environment. Disulfide bonds are most commonly seen in the proteins of the secretory pathway due to the non-reducing environment found there. Recently, however, disulfide bonds have also been reported outside of the secretory pathway, especially among viral proteins (Monastryskaya et al., 1994 ; Volpers et al., 1994
; Kosukegawa et al., 1996
; Chang et al., 1997
; Sandalon & Oppenheim, 1997
). These observations are supported by recent reports which have established the ability of cytoplasmic and nuclear compartments to direct redox regulation of various proteins at cysteine residues (Schreck et al., 1991
; Pognonec et al., 1992
; Xanthoudakis et al., 1992
; Meyer et al., 1993
; Benerza, 1994
; Chinenov et al., 1998
). Our results, together with the cited reports, suggest that disulfides and other redox regulation of cysteines can occur in cytoplasmic or nuclear environments that are reducing overall. Non-reducing micro-environments may be found at the interface of interacting complexes due to inaccessible hydrophobic pockets, or to localized oxidative activities. To further study the spatial and temporal involvement of specific cysteines in disulfide-linked stabilization of the SV40 capsid, in vivo studies are in progress. The combination of in vitro and in vivo studies can increase our understanding of specific SV40 Vp1 cysteine-mediated interactions and their significance.
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Acknowledgments |
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Footnotes |
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c Present address: Dept of Microbiology & Immunology, University of California, Los Angeles, CA 90095, USA.
d Present address: Dept of Biology, University of California, San Diego, CA 92093, USA.
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References |
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Received 30 March 1999;
accepted 19 May 1999.