- Email: [email protected]
Purification and secondary structure determination of simaian immunodeficiency virus p27
J. Mol. Biol. (1990) 216, 207-211
Purification and Secondary Structure Determination of Simian Immunodeficiency Virus p27 Nigel R. Burns ~, Stewart Craig 1, Sheena R. Lee ~, S. Mark H. Richardson 1 Nigel Stenner ~, Sally E. A d a m s ~, Susan M. K i n g s m a n 2 and Alan J. K i n g s m a n l'z ~British Bio-technology Ltd Watlington Road, Oxford OX4 5L Y, U.K. Virus Molecular Biology Group Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, U.K. (Received 2 August 1990; accepted 3 August 1990) We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of a-helix, fl-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.
show considerable anti-picornavirai activity. The proposed similarity in folding topology led to the suggestion that compounds with similar activities could be developed for SIV/HIV. In order to test the fl-barrel model directly, we have determined the relative amounts of the secondary structure elements of purified SIV p27 by performing circular dichroism measurements. SIV p27 was purified following enzymic cleavage of a fusion protein expressed in yeast via the plasmid pOGS249. The fusion protein comprises amino acid residues 1 to 381 of the protein pl, encoded by the T YA gene of the yeast transposon Ty 1-15 (Mellor et al., 1985), and SIV GAG corresponding to residues 116 to 363 (Fig. 1). The SIV GAG moiety of the fusion protein therefore contains the 20 C-terminal residues of p17 contiguous with full-length p27. The junction between p17 and p27 is one of the cleavage sites of the virally encoded proteinase. A stop codon has been introduced immediately following the coding sequence of p27, thereby ensuring the authentic C-terminal sequence. The protein pl is capable of assembling into virus-like particles (VLPs) in the absence of any other Ty-encoded proteins (Adams et al., 1987). pl "fusion proteins, including the p l : p l 7 :p27 fusion described here, retain the ability to assemble into particles (Adams et al., 1987; Malim et al., 1987), known as hybrid Ty-VLPs (Fig. 1). The formation of such particulate structures provides a very simple means by which they can be purified (Burns et al., 1990).
As with other retroviruses, the genomic RNA of the mature simian immunodeficiency virus (SIVt) virion is packaged within a core particle that also contains reverse transcriptase and endonuclease/ integrase (for a review, see Dickson et al., 1984). The key structural protein responsible for the formation of these cores is the gag-derived protein, p27. The core particle is enveloped within a plasma membrane, derived from the host cell from which the core buds, and in which are embedded viral envelope proteins. The formation of the mature particle involves the cleavage of p27 from a larger precursor polyprotein (Prp55) by means of a virally encoded aspartyl prot6inase. A similar morphogenic pathway is observed in the closely related human immunodeficiency virus (HIV), for which SIV is currently the best available animal model (for a review, see Desrosiers, 1988). On the basis of sequence homologies, Argos (1989) has proposed that SIV p27, and its HIV homologue, p24, are folded into a tertiary structural motif commonly seen in viral capsid proteins. This motif is described as an eight-stranded antiparallel //-barrel. The same model for HIV p24 had been proposed by Rossman (1988) on the basis of sequence "fingerprints". Both of the above reports drew attention to compounds that bind into the hydrophobic pocket formed in such fl-barrels, and ~"Abbreviations used: SIV, simian immunodeficiency virus; HIV, human immunodeficiency virus; VLP, virus-like particle; c.d., circular dichroism. 0022-2836/90/220207-05 $03.00/0
207
© 1990AcademicPress Limited
208
N., R: Burns et al.
(o
(
)
z, ~:i:!i~i :i~i ~
~"
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f
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I (d)~ (b)
!
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TYA(d )
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0
0 Q O 0
Free p27
Q
Figure 1. The expression of hybrid Ty VLPs and production of SIV p27. (a) SIV p27 is expressed as a pl : p17 : p27 fusion protein from plasmid pOGS249. Plasmid pOGS249 is a 2/~/pBR322 yeast]E, coli shuttle vector with a hybrid promoter, from the yeast PGK and GAL-IO genes driving expression. Transcription and translation are terminated in the PGK terminator region (PGKt). This plasmid is maintained in yeast at a copy number of about 200. (b) The fusion protein contains the C-terminal 20 amino acid residues of p17 contiguous with p27 and therefore contains a retroviral aspartyl proteinase cleavage site; the seissile bond is marked with an asterisk (*). (c) Schematic representation of hybrid pl : pl7 : p27 VLPs. (d) Incubation of the pl : p17 : p27 VLPs with recombinant HIV-1 proteinase effects cleavage at the pI7-p27 junction to produce p27 whilst the pl : pl7 component remains particulate.
To obtain authentic, full-length p27 we isolated pl : pl7 : p27 VLPs and incubated them in the presence of a crude preparation of recombinant HIV-1 proteinase, derived from Escherichia coli. This proteinase, which catalyses the homologous reaction in HIV-1, is clearly active upon the corresponding SIV sequence, since quantitative cleavage of the fusion protein is observed (Fig. 2). As stated above, the formation of the hybrid Ty-VLPs is determined by the pl moiety, which retains its particulate nature after proteinase digestion. In contrast, the SIV p27 does not form multimerie structures under these conditions, and therefore the sedimentation rate of the Ty component far exceeds that of the p27. The bulk of the Ty component could therefore
be separated from p27 by ultracentrifugation, with the pl being pelleted, whilst p27 remained in the supernatant. The very small amount of residual pl : p l 7 and other contaminating proteins were removed from the supernatant by ion-exchange chromatography to yield p27 that is homogeneous, as judged by Coomassie-stained SDS/ polyacrylamide electrophoresis gels (Fig. 2). N-terminal sequencing confirmed the fidelity of the cleavage site (S. Craig, data not shown). SIV p27 purified as described above was used to determine the circular dichroism (c.d.) spectrum. The near-ultraviolet c.d. spectrum of p27 shows an intense negative elliptieity with distinct minima centred around 276nm, 287 nm and 294 nm
Communications o
b
c
209
d
97-----~ '~['----- pl:pl 7:p27
se----I~ :
~s--~
,41~
L
6
~
'
pJ:pl7
.4F---- p2r
s g ~
Figure 2. Purification of SIV p27 as analysed by SDS/polyacrylamide electrophoresis. Lane a, purified, uncleared pl:pl7:p27 VLPs. Lane b, p l : p l 7 : p 2 7 VLPs cleaved with recombinant HIV-1 proteinase. Lane c, supernatant following ultracentrifugation of cleaved VLPs. Lane d, crude HIV-I proteinase preparation. Lane e, purified p27 followed ion-exchange chromatography. Lanes a and b were loaded with 12 pg of protein and lane e was loaded with 20 pg of protein. Methods. pl : pl7 : p27 VLPs were purified according to previously published procedures (Burns et al., 1990). HIV-I proteinase was used as a crude preparation from transformed E. coli according to unpublished procedures, VLPs at l mg/ml were incubated with 5% (v/v) crude HIV-1 proteinase solution in 20 mM-Tris'HCl (pH 7"0) for 2 h at 37°C. Following cleavage the residual VLPs were removed by centrifugation at 40,000 revs/min for 60 rain in an SW41 rotor (Beckman). The resulting supernatant was applied to a 1 cm x 10 cm Q Sepharose column (Pharmaeia LKB) equilibrated in 20 mM-Tris"HCI (pH 8"0). SIV p27 was eluted by developing the column with a 0 to 500 mM-NaC1 gradient in 20 mM-Tris"HCI (pH 8"0). (Fig. 3). The band centred at 276 nm is characteristic of tyrosine in an asymmetric environment with the fine structure at higher wavelengths arising from tryptophan, p27 contains eight tyrosine and five tryptophan residues, making assignments of the contributions difficult. The intensity of the spectrum indicates a well-folded tertiary structure, with specific aromatic environments. The c.d. spectrum in the range of 190 to 250 nm shows a moderately strong intensity with minima centred at 208 and 220 nm, with a crossover point at 200 nm and a maximum at about 195 nm (Fig. 3). These features are typical of a protein with appreciable a-helical content. Indeed, analysis of the spectrum by the program CONTIN (Provencher, 1982) gives values of 44% a-helix and 56% fl-sheet. The structure of a number of icosahedral RNA-containing viruses has been solved by X-ray diffraction of crystals (for a review, see Rossman & Rueckert, 1987). With the exception of the phage MS2 (Valega~d et al., 1990), the major capsid proteins of all these viruses share a common tertiary structure. This is in the form of eight strands of antiparallel fl-sheet, joined by connecting loops that are predominantly in an unordered conformation. Variations between the structure of different viral capsid proteins tends to be confined to the loops, with the fl-sheet component being relatively invariant. Occasionally, short a-helices are seen in the strand-connecting loops. Thus typically, such
fl-barrel proteins comprise 30 to 40 °/o ]?-sheet, 45 to 60~/o unordered and from 0 to 16% a-helix. It would appear therefore that SIV p27 contains considerably more a-helix than any of the known fl-barrel proteins. If SIV p27 does form a fl-barrel, then this a-helix must be incorporated within the connecting loops, and/or extend from either end of the molecule. Secondary structure predictions using the algorithm of Chou & Fasman (1978), predicts that a-helix comprises about 40~/o of the protein and that these helices are dispersed at intervals throughout the sequence (N. Burns, data not shown). However, either of these configurations would be unlike any of the fl-barrel proteins for which the crystal structure is available. Although there is sufficient fl-sheet to form the strands of a fl-barrel, according to the analysis of the c.d. spectrum by CONTIN, there is insufficient unordered polypeptide to accommodate the strandconnecting loops that are typical of such structures. A globular protein can be expected to have a minimum in the region of 15~/o of its polypeptide backbone in conformations other than a-helix and fl-sheet. If SIV p27 contains such a minimum of non-a/non-fl structure, then our inability to detect it may have occurred due to experimental error in the determination of the spectrum. Larger errors are likely only if there is a systematic error in the analysis used. Since the shape of the spectrum is highly typical of a protein with considerable helical content, we assume that any large-scale under-
210
N . R . Burns et al. 10 40,000
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30,000
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20,000 A 'T
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E
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u
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-20,000 260
270
280 290 300 W0vlenoth (nm)
310
(a)
' 200
' ' ~ 210 220 230 Wavlength (nm)
240
250
(b)
Figure 3. (a) Near-ultraviolet circular dichroism of SlY p27. Spectrum is an average of 4 scans with the baseline subtracted. Spectra were recorded with a 1 cm pathlength celt and l nm bandwidth. (b) Far-ultraviolet circular dichroism of SIV p27. The spectrum is an average of 8 scans with the baseline subtracted. Spectra were recorded with a 0"01 cm pathlength cell and 2 nm bandwidth. Methods: Circular dichroism spectra were measured on a Jobin-Yvon Dichrographe IV, which was calibrated using (+)-10-camphol~utphonic acid. Spectra were recorded at 20°C and at a protein concentration of 0"381 mg/ml. Protein concentrations were measured using a value, A = 14"2 at 280 nm. calculated from the amino acid composition. The mean molar residue molecular weight was calculated as ! 10.
estimate of unordered structure has been made by overestimating the amount of fl-sheet. Interestingly, analysis of the c.d. spectrum of t u m o u r necrosis factor, a classical fl-barrel protein, using the C O N T I N program (Wingfield et al., 1987), gave values for fl-sheet and unordered structure t h a t agreed a l m o s t exactly with the s t r u c t u r e derived from X - r a y diffraction (Jones et al., ]989). Given the a b o v e considerations, we conclude t h a t c o n t r a r y to previously published models and sequence alignments, the analysis of the c.d. spect r u m of S I V p27 does not s u p p o r t the theory of this protein forming the fl-barrel m o t i f so c o m m o n l y seen in viral capsid proteins. Therefore, a search for compounds t h a t are able to bind inside the pocket of such barrels, and thereby inhibit S I V / H I V , is likely to be futile. However, disrupting the activity of the structural c o m p o n e n t s of these retroviruses remains an a t t r a c t i v e route to identify effective antiviral agents. The identification and rational design of such c o m p o u n d s will be greatly facilitated b y a detailed structural analysis of the relevant proteins. We thank Professor g. Pain, University of Newcastle, for helpful discussions, and Nicola Graft and Gary Stabler for excellent technical assistance. The supply of reagents from the MRC AIDS Directed Programme resource
project, in particular pNIBSCI ti'om P. Kitchin. is gratefully acknowledged.
References
Adams, S. E., Mellor, J , Gull, K., Sim, 1-¢.B., Tuite, M. F., Kingsman, S. M. & Kingsman, A. J. (1987). The Functions and Relationships of Ty-VLP Proteins in Yeast Reflect Those of Mammalian Retroviral Proteins. Cell, 49, 111-198. Argos, P. (1989) A Possible Homology Between Immunodeficieney Virus p24 Core Protein and Picornaviral VP2 Coat Protein: Prediction of HIV p24 Antigen Sites. EMBO J. 8, 779-785. Burns, N. R., Gilmore, J. E. M., Kingsman, S. M., Kingsman, A. J. & Adams, S. E. (1990). Production and Purification of Hybrid Ty-VLPs. Methods in Molecular Biology, Humana Press, in the press. Chou. P. Y. & Fasman, G. D. (1978). Prediction of Secondary Structure of Proteins from their Amino Acid Sequence. Advan. Enzymol. 47, 45-147. Desrosiers, R. C. (1988). Simian Immunodeficiency Virus. Annu. Rev. Microbiol. 42, 607-625. Dickson, C., Eisenman, R., Fan, H., Hunter, E. & Teich, N. (1984). Protein Biosynthesis and Assembly. In R N A Tumor Viruses (Wiess, R,., Teich, N., Varmus, H. & Coffin, J., eds), vol. l, pp. 513-648, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Jones, E. Y., Stuart, D. I. & Walker, N. P. C. (1989). Structure of Tumor Necrosis Factor. Nature (London), 388, 225-228.
Communications Malim, M. H., Adams, S. E., Gull, K., Kingsman, A. J. & Kingsman, S. M. (1987). The Production of Hybrid T y : I F N Virus-like Particles. Nucl. Acids Res. 15, 7571-7580. Mellor, J., Fulton, A. M., Dobson, M. J., Wilson, W., Kingsman, A. J. & Kingsman, S. M. (1985). The Ty Transposon of Saecharomyces cerevisiae Determines the Synthesis of at Least Three Proteins. Nucl. Acids Res. 13, 6249-6263. Provencher, S. W. (1982). CONTIN. A General Purpose Constrained Regularization Program for Inverting Noisy Linear Algebraic and Integral Equations. Comput. Phys. Commun. 27, 229-249.
211
Rossman, M. G. (1988). Antiviral Agents Targeted to Interact with Viral Capsid Proteins and a Possible Application to Human Immunodeficieney Virus. Proc. Nat. Acad. Sci., U.S.A. 85, 4625-4627. Rossman, M. G. & Ruekert, It. R. (1987). What Does the Molecular Structure of Viruses Tell Us About Viral Functions? Microbiol. Sci. 4, 206-215. Valeg£rd, K., Liljas, L., Fridborg, K. & Unge, T. (1990). The Three-dimensional Structure of the Bacterial Virus MS2. Nature (London), 345, 36-41. Wingfield, P., Pain, R. H. & Craig. S. (1987). Tumor Necrosis Factor is a Compact Trimer. FEBS Letters, 211, 179-184.
Edited by A. Klug
Purification and Secondary Structure Determination of Simian Immunodeficiency Virus p27 Nigel R. Burns ~, Stewart Craig 1, Sheena R. Lee ~, S. Mark H. Richardson 1 Nigel Stenner ~, Sally E. A d a m s ~, Susan M. K i n g s m a n 2 and Alan J. K i n g s m a n l'z ~British Bio-technology Ltd Watlington Road, Oxford OX4 5L Y, U.K. Virus Molecular Biology Group Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, U.K. (Received 2 August 1990; accepted 3 August 1990) We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of a-helix, fl-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.
show considerable anti-picornavirai activity. The proposed similarity in folding topology led to the suggestion that compounds with similar activities could be developed for SIV/HIV. In order to test the fl-barrel model directly, we have determined the relative amounts of the secondary structure elements of purified SIV p27 by performing circular dichroism measurements. SIV p27 was purified following enzymic cleavage of a fusion protein expressed in yeast via the plasmid pOGS249. The fusion protein comprises amino acid residues 1 to 381 of the protein pl, encoded by the T YA gene of the yeast transposon Ty 1-15 (Mellor et al., 1985), and SIV GAG corresponding to residues 116 to 363 (Fig. 1). The SIV GAG moiety of the fusion protein therefore contains the 20 C-terminal residues of p17 contiguous with full-length p27. The junction between p17 and p27 is one of the cleavage sites of the virally encoded proteinase. A stop codon has been introduced immediately following the coding sequence of p27, thereby ensuring the authentic C-terminal sequence. The protein pl is capable of assembling into virus-like particles (VLPs) in the absence of any other Ty-encoded proteins (Adams et al., 1987). pl "fusion proteins, including the p l : p l 7 :p27 fusion described here, retain the ability to assemble into particles (Adams et al., 1987; Malim et al., 1987), known as hybrid Ty-VLPs (Fig. 1). The formation of such particulate structures provides a very simple means by which they can be purified (Burns et al., 1990).
As with other retroviruses, the genomic RNA of the mature simian immunodeficiency virus (SIVt) virion is packaged within a core particle that also contains reverse transcriptase and endonuclease/ integrase (for a review, see Dickson et al., 1984). The key structural protein responsible for the formation of these cores is the gag-derived protein, p27. The core particle is enveloped within a plasma membrane, derived from the host cell from which the core buds, and in which are embedded viral envelope proteins. The formation of the mature particle involves the cleavage of p27 from a larger precursor polyprotein (Prp55) by means of a virally encoded aspartyl prot6inase. A similar morphogenic pathway is observed in the closely related human immunodeficiency virus (HIV), for which SIV is currently the best available animal model (for a review, see Desrosiers, 1988). On the basis of sequence homologies, Argos (1989) has proposed that SIV p27, and its HIV homologue, p24, are folded into a tertiary structural motif commonly seen in viral capsid proteins. This motif is described as an eight-stranded antiparallel //-barrel. The same model for HIV p24 had been proposed by Rossman (1988) on the basis of sequence "fingerprints". Both of the above reports drew attention to compounds that bind into the hydrophobic pocket formed in such fl-barrels, and ~"Abbreviations used: SIV, simian immunodeficiency virus; HIV, human immunodeficiency virus; VLP, virus-like particle; c.d., circular dichroism. 0022-2836/90/220207-05 $03.00/0
207
© 1990AcademicPress Limited
208
N., R: Burns et al.
(o
(
)
z, ~:i:!i~i :i~i ~
~"
I:p17:p27 VLP
f
HIV-I Proteose
I (d)~ (b)
!
phpl7 VLP
TYA(d )
@ 0
GGNY~+PVQQ
0
0 Q O 0
Free p27
Q
Figure 1. The expression of hybrid Ty VLPs and production of SIV p27. (a) SIV p27 is expressed as a pl : p17 : p27 fusion protein from plasmid pOGS249. Plasmid pOGS249 is a 2/~/pBR322 yeast]E, coli shuttle vector with a hybrid promoter, from the yeast PGK and GAL-IO genes driving expression. Transcription and translation are terminated in the PGK terminator region (PGKt). This plasmid is maintained in yeast at a copy number of about 200. (b) The fusion protein contains the C-terminal 20 amino acid residues of p17 contiguous with p27 and therefore contains a retroviral aspartyl proteinase cleavage site; the seissile bond is marked with an asterisk (*). (c) Schematic representation of hybrid pl : pl7 : p27 VLPs. (d) Incubation of the pl : p17 : p27 VLPs with recombinant HIV-1 proteinase effects cleavage at the pI7-p27 junction to produce p27 whilst the pl : pl7 component remains particulate.
To obtain authentic, full-length p27 we isolated pl : pl7 : p27 VLPs and incubated them in the presence of a crude preparation of recombinant HIV-1 proteinase, derived from Escherichia coli. This proteinase, which catalyses the homologous reaction in HIV-1, is clearly active upon the corresponding SIV sequence, since quantitative cleavage of the fusion protein is observed (Fig. 2). As stated above, the formation of the hybrid Ty-VLPs is determined by the pl moiety, which retains its particulate nature after proteinase digestion. In contrast, the SIV p27 does not form multimerie structures under these conditions, and therefore the sedimentation rate of the Ty component far exceeds that of the p27. The bulk of the Ty component could therefore
be separated from p27 by ultracentrifugation, with the pl being pelleted, whilst p27 remained in the supernatant. The very small amount of residual pl : p l 7 and other contaminating proteins were removed from the supernatant by ion-exchange chromatography to yield p27 that is homogeneous, as judged by Coomassie-stained SDS/ polyacrylamide electrophoresis gels (Fig. 2). N-terminal sequencing confirmed the fidelity of the cleavage site (S. Craig, data not shown). SIV p27 purified as described above was used to determine the circular dichroism (c.d.) spectrum. The near-ultraviolet c.d. spectrum of p27 shows an intense negative elliptieity with distinct minima centred around 276nm, 287 nm and 294 nm
Communications o
b
c
209
d
97-----~ '~['----- pl:pl 7:p27
se----I~ :
~s--~
,41~
L
6
~
'
pJ:pl7
.4F---- p2r
s g ~
Figure 2. Purification of SIV p27 as analysed by SDS/polyacrylamide electrophoresis. Lane a, purified, uncleared pl:pl7:p27 VLPs. Lane b, p l : p l 7 : p 2 7 VLPs cleaved with recombinant HIV-1 proteinase. Lane c, supernatant following ultracentrifugation of cleaved VLPs. Lane d, crude HIV-I proteinase preparation. Lane e, purified p27 followed ion-exchange chromatography. Lanes a and b were loaded with 12 pg of protein and lane e was loaded with 20 pg of protein. Methods. pl : pl7 : p27 VLPs were purified according to previously published procedures (Burns et al., 1990). HIV-I proteinase was used as a crude preparation from transformed E. coli according to unpublished procedures, VLPs at l mg/ml were incubated with 5% (v/v) crude HIV-1 proteinase solution in 20 mM-Tris'HCl (pH 7"0) for 2 h at 37°C. Following cleavage the residual VLPs were removed by centrifugation at 40,000 revs/min for 60 rain in an SW41 rotor (Beckman). The resulting supernatant was applied to a 1 cm x 10 cm Q Sepharose column (Pharmaeia LKB) equilibrated in 20 mM-Tris"HCI (pH 8"0). SIV p27 was eluted by developing the column with a 0 to 500 mM-NaC1 gradient in 20 mM-Tris"HCI (pH 8"0). (Fig. 3). The band centred at 276 nm is characteristic of tyrosine in an asymmetric environment with the fine structure at higher wavelengths arising from tryptophan, p27 contains eight tyrosine and five tryptophan residues, making assignments of the contributions difficult. The intensity of the spectrum indicates a well-folded tertiary structure, with specific aromatic environments. The c.d. spectrum in the range of 190 to 250 nm shows a moderately strong intensity with minima centred at 208 and 220 nm, with a crossover point at 200 nm and a maximum at about 195 nm (Fig. 3). These features are typical of a protein with appreciable a-helical content. Indeed, analysis of the spectrum by the program CONTIN (Provencher, 1982) gives values of 44% a-helix and 56% fl-sheet. The structure of a number of icosahedral RNA-containing viruses has been solved by X-ray diffraction of crystals (for a review, see Rossman & Rueckert, 1987). With the exception of the phage MS2 (Valega~d et al., 1990), the major capsid proteins of all these viruses share a common tertiary structure. This is in the form of eight strands of antiparallel fl-sheet, joined by connecting loops that are predominantly in an unordered conformation. Variations between the structure of different viral capsid proteins tends to be confined to the loops, with the fl-sheet component being relatively invariant. Occasionally, short a-helices are seen in the strand-connecting loops. Thus typically, such
fl-barrel proteins comprise 30 to 40 °/o ]?-sheet, 45 to 60~/o unordered and from 0 to 16% a-helix. It would appear therefore that SIV p27 contains considerably more a-helix than any of the known fl-barrel proteins. If SIV p27 does form a fl-barrel, then this a-helix must be incorporated within the connecting loops, and/or extend from either end of the molecule. Secondary structure predictions using the algorithm of Chou & Fasman (1978), predicts that a-helix comprises about 40~/o of the protein and that these helices are dispersed at intervals throughout the sequence (N. Burns, data not shown). However, either of these configurations would be unlike any of the fl-barrel proteins for which the crystal structure is available. Although there is sufficient fl-sheet to form the strands of a fl-barrel, according to the analysis of the c.d. spectrum by CONTIN, there is insufficient unordered polypeptide to accommodate the strandconnecting loops that are typical of such structures. A globular protein can be expected to have a minimum in the region of 15~/o of its polypeptide backbone in conformations other than a-helix and fl-sheet. If SIV p27 contains such a minimum of non-a/non-fl structure, then our inability to detect it may have occurred due to experimental error in the determination of the spectrum. Larger errors are likely only if there is a systematic error in the analysis used. Since the shape of the spectrum is highly typical of a protein with considerable helical content, we assume that any large-scale under-
210
N . R . Burns et al. 10 40,000
-I0
30,000
-3O
20,000 A 'T
I 0
0
E
E
"0
=......
t
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u
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~
•
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0
--70
--I0,000
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-20,000 260
270
280 290 300 W0vlenoth (nm)
310
(a)
' 200
' ' ~ 210 220 230 Wavlength (nm)
240
250
(b)
Figure 3. (a) Near-ultraviolet circular dichroism of SlY p27. Spectrum is an average of 4 scans with the baseline subtracted. Spectra were recorded with a 1 cm pathlength celt and l nm bandwidth. (b) Far-ultraviolet circular dichroism of SIV p27. The spectrum is an average of 8 scans with the baseline subtracted. Spectra were recorded with a 0"01 cm pathlength cell and 2 nm bandwidth. Methods: Circular dichroism spectra were measured on a Jobin-Yvon Dichrographe IV, which was calibrated using (+)-10-camphol~utphonic acid. Spectra were recorded at 20°C and at a protein concentration of 0"381 mg/ml. Protein concentrations were measured using a value, A = 14"2 at 280 nm. calculated from the amino acid composition. The mean molar residue molecular weight was calculated as ! 10.
estimate of unordered structure has been made by overestimating the amount of fl-sheet. Interestingly, analysis of the c.d. spectrum of t u m o u r necrosis factor, a classical fl-barrel protein, using the C O N T I N program (Wingfield et al., 1987), gave values for fl-sheet and unordered structure t h a t agreed a l m o s t exactly with the s t r u c t u r e derived from X - r a y diffraction (Jones et al., ]989). Given the a b o v e considerations, we conclude t h a t c o n t r a r y to previously published models and sequence alignments, the analysis of the c.d. spect r u m of S I V p27 does not s u p p o r t the theory of this protein forming the fl-barrel m o t i f so c o m m o n l y seen in viral capsid proteins. Therefore, a search for compounds t h a t are able to bind inside the pocket of such barrels, and thereby inhibit S I V / H I V , is likely to be futile. However, disrupting the activity of the structural c o m p o n e n t s of these retroviruses remains an a t t r a c t i v e route to identify effective antiviral agents. The identification and rational design of such c o m p o u n d s will be greatly facilitated b y a detailed structural analysis of the relevant proteins. We thank Professor g. Pain, University of Newcastle, for helpful discussions, and Nicola Graft and Gary Stabler for excellent technical assistance. The supply of reagents from the MRC AIDS Directed Programme resource
project, in particular pNIBSCI ti'om P. Kitchin. is gratefully acknowledged.
References
Adams, S. E., Mellor, J , Gull, K., Sim, 1-¢.B., Tuite, M. F., Kingsman, S. M. & Kingsman, A. J. (1987). The Functions and Relationships of Ty-VLP Proteins in Yeast Reflect Those of Mammalian Retroviral Proteins. Cell, 49, 111-198. Argos, P. (1989) A Possible Homology Between Immunodeficieney Virus p24 Core Protein and Picornaviral VP2 Coat Protein: Prediction of HIV p24 Antigen Sites. EMBO J. 8, 779-785. Burns, N. R., Gilmore, J. E. M., Kingsman, S. M., Kingsman, A. J. & Adams, S. E. (1990). Production and Purification of Hybrid Ty-VLPs. Methods in Molecular Biology, Humana Press, in the press. Chou. P. Y. & Fasman, G. D. (1978). Prediction of Secondary Structure of Proteins from their Amino Acid Sequence. Advan. Enzymol. 47, 45-147. Desrosiers, R. C. (1988). Simian Immunodeficiency Virus. Annu. Rev. Microbiol. 42, 607-625. Dickson, C., Eisenman, R., Fan, H., Hunter, E. & Teich, N. (1984). Protein Biosynthesis and Assembly. In R N A Tumor Viruses (Wiess, R,., Teich, N., Varmus, H. & Coffin, J., eds), vol. l, pp. 513-648, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Jones, E. Y., Stuart, D. I. & Walker, N. P. C. (1989). Structure of Tumor Necrosis Factor. Nature (London), 388, 225-228.
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Edited by A. Klug