Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein

Science

Volumn 278, Issue 5339, 1997, Pages 849-853

  Gamble, Theresa R ^a   Yoo, Sanghee ^a   Vajdos, Felix F ^a   Von Schwedler, Uta K ^a   Worthylake, David K ^a   Wang, Hui ^a   McCutcheon, John P ^a   Sundquist, Wesley I ^a   Hill, Christopher P ^a  

^a UNIVERSITY OF UTAH SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CAPSID PROTEIN;

AMINO TERMINAL SEQUENCE; ARTICLE; CARBOXY TERMINAL SEQUENCE; CRYSTAL STRUCTURE; DIMERIZATION; HUMAN IMMUNODEFICIENCY VIRUS 1; HYDROGEN BOND; NONHUMAN; PRIORITY JOURNAL; PROTEIN SECONDARY STRUCTURE; PROTEIN STRUCTURE; VIRUS ASSEMBLY; VIRUS CAPSID;

AMINO ACID SEQUENCE; BINDING SITES; CAPSID; CELL LINE; CLONING, MOLECULAR; CLONING, ORGANISM; CRYSTALLOGRAPHY, X-RAY; DIMERIZATION; HIV-1; HUMANS; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; MUTAGENESIS, SITE-DIRECTED; PEPTIDYLPROLYL ISOMERASE; PROTEIN CONFORMATION; VIRUS REPLICATION;

HUMAN IMMUNODEFICIENCY VIRUS; HUMAN IMMUNODEFICIENCY VIRUS 1; MIRIDAE;

EID: 0030693391     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5339.849     Document Type: Article

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21. 3 data as derivatives [V. Ramakrishnan and V. Biou, Methods Enzymol. 276, 538 (1997)]. All four selenium sites were located in difference Patterson and Fourier maps. Refinement of selenium atom parameters with MLPHARE [Collaborative Computing Project 4, Acta Crystallogr. D50, 760 (1994)] gave a mean figure of merit of 0.627. Phase refinement by solvent flattening and histogram shifting with DM (ibid.) gave a very clear map. A model consisting of the first 70 residues of CA(151-231) was built using O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)] and was refined with X-PLOR (25) against 1.7 Å data that were collected on a MAR Imaging Plate detector at beamline 1-5 of the Stanford Synchrotron Radiation Laboratory (SSRL).
22. 3 data as derivatives [V. Ramakrishnan and V. Biou, Methods Enzymol. 276, 538 (1997)]. All four selenium sites were located in difference Patterson and Fourier maps. Refinement of selenium atom parameters with MLPHARE [Collaborative Computing Project 4, Acta Crystallogr. D50, 760 (1994)] gave a mean figure of merit of 0.627. Phase refinement by solvent flattening and histogram shifting with DM (ibid.) gave a very clear map. A model consisting of the first 70 residues of CA(151-231) was built using O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)] and was refined with X-PLOR (25) against 1.7 Å data that were collected on a MAR Imaging Plate detector at beamline 1-5 of the Stanford Synchrotron Radiation Laboratory (SSRL).
23. 3 data as derivatives [V. Ramakrishnan and V. Biou, Methods Enzymol. 276, 538 (1997)]. All four selenium sites were located in difference Patterson and Fourier maps. Refinement of selenium atom parameters with MLPHARE [Collaborative Computing Project 4, Acta Crystallogr. D50, 760 (1994)] gave a mean figure of merit of 0.627. Phase refinement by solvent flattening and histogram shifting with DM (ibid.) gave a very clear map. A model consisting of the first 70 residues of CA(151-231) was built using O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)] and was refined with X-PLOR (25) against 1.7 Å data that were collected on a MAR Imaging Plate detector at beamline 1-5 of the Stanford Synchrotron Radiation Laboratory (SSRL).
24. 3 data as derivatives [V. Ramakrishnan and V. Biou, Methods Enzymol. 276, 538 (1997)]. All four selenium sites were located in difference Patterson and Fourier maps. Refinement of selenium atom parameters with MLPHARE [Collaborative Computing Project 4, Acta Crystallogr. D50, 760 (1994)] gave a mean figure of merit of 0.627. Phase refinement by solvent flattening and histogram shifting with DM (ibid.) gave a very clear map. A model consisting of the first 70 residues of CA(151-231) was built using O [T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)] and was refined with X-PLOR (25) against 1.7 Å data that were collected on a MAR Imaging Plate detector at beamline 1-5 of the Stanford Synchrotron Radiation Laboratory (SSRL).
25. 184, which clearly differ in detail between the CA(151-231) and CA(146-231) structures. The CA(146-231) structure is currently being refined at 2.5 Å resolution.
26. A database search [L. Holmand and C. Sander, J. Mol. Biol. 233, 123 (1993)] revealed that CA(151-231) most closely resembles the putative actin-binding subdomain of myosin [root-mean-square (rms) difference of 2.6 Å for 52 Cα atom pairs], with best overlap (rms difference, 1.0 Å) shown for helices 1 and 2 of CA(151-231) against myosin residues 524 to 536 and 545 to 551 [I. Rayment et al., Science 261, 58 (1993)]. Although the biological relevance of this similarity is not yet clear, the result is intriguing because Gag associates with the actin cytoskeleton [O. Rey, J. Canon, P. Krogstad, Virology 220, 530 (1996)], and actin has been detected within HIV-1 at 200 molecules per virion [D. E. Ott et al., J. Virol. 70, 7734 (1996)].
27. A database search [L. Holmand and C. Sander, J. Mol. Biol. 233, 123 (1993)] revealed that CA(151-231) most closely resembles the putative actin-binding subdomain of myosin [root-mean-square (rms) difference of 2.6 Å for 52 Cα atom pairs], with best overlap (rms difference, 1.0 Å) shown for helices 1 and 2 of CA(151-231) against myosin residues 524 to 536 and 545 to 551 [I. Rayment et al., Science 261, 58 (1993)]. Although the biological relevance of this similarity is not yet clear, the result is intriguing because Gag associates with the actin cytoskeleton [O. Rey, J. Canon, P. Krogstad, Virology 220, 530 (1996)], and actin has been detected within HIV-1 at 200 molecules per virion [D. E. Ott et al., J. Virol. 70, 7734 (1996)].
28. A database search [L. Holmand and C. Sander, J. Mol. Biol. 233, 123 (1993)] revealed that CA(151-231) most closely resembles the putative actin-binding subdomain of myosin [root-mean-square (rms) difference of 2.6 Å for 52 Cα atom pairs], with best overlap (rms difference, 1.0 Å) shown for helices 1 and 2 of CA(151-231) against myosin residues 524 to 536 and 545 to 551 [I. Rayment et al., Science 261, 58 (1993)]. Although the biological relevance of this similarity is not yet clear, the result is intriguing because Gag associates with the actin cytoskeleton [O. Rey, J. Canon, P. Krogstad, Virology 220, 530 (1996)], and actin has been detected within HIV-1 at 200 molecules per virion [D. E. Ott et al., J. Virol. 70, 7734 (1996)].
29. A database search [L. Holmand and C. Sander, J. Mol. Biol. 233, 123 (1993)] revealed that CA(151-231) most closely resembles the putative actin-binding subdomain of myosin [root-mean-square (rms) difference of 2.6 Å for 52 Cα atom pairs], with best overlap (rms difference, 1.0 Å) shown for helices 1 and 2 of CA(151-231) against myosin residues 524 to 536 and 545 to 551 [I. Rayment et al., Science 261, 58 (1993)]. Although the biological relevance of this similarity is not yet clear, the result is intriguing because Gag associates with the actin cytoskeleton [O. Rey, J. Canon, P. Krogstad, Virology 220, 530 (1996)], and actin has been detected within HIV-1 at 200 molecules per virion [D. E. Ott et al., J. Virol. 70, 7734 (1996)].
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33. d of 10 ± 3 μM. Only the 24.9 μM data and associated residuals are shown in Fig. 3. Equilibrium distributions of CA(151-231) (from initial protein concentrations of 22.4 and 79.8 μM) were globally fit to simple monomer models, yielding an estimated protein mass of 9.6 ± 0.1 kD, in reasonable agreement with the mass of the CA(151-231) monomer (9.1 kD). Equilibrium distributions of CA(W184A) (initial protein concentrations of 7.5, 15.5, and 23 μM) and CA(M185A) (initial protein concentrations of 5.7, 11.8, and 16.9 μM) were globally fit to simple monomer models, yielding estimated protein masses of 25.5 ± 0.2 kD [CA(W184A)] and 25.5 ± 0.1 kD [CA(M185A)], in excellent agreement with their monomeric masses (25.5 kD). Only the 16.9 μM CA(M185A) data and associated residuals are shown in Fig. 3C.
34. NL4-3 expression plasmid R9 [S. Swingler et al., J. Virol. 71, 4372 (1997)], which contains an SV-40 origin of replication. Viral particles were produced by transient calcium phosphate transfection of this plasmid into 293T human embryonic kidney cells. After 48 hours, particle production was reduced by 50 to 80% relative to a wild-type control R9 vector, as quantitated by both p24 enzyme-linked immunosorbent assay (DuPont) and a reverse transcriptase assay [S. P. Goff, P. Traktman, D. Baltimore, J. Virol. 38, 238 (1981)]. Particle infectivity was assayed by normalizing mutant and wild-type virus for reverse transcriptase activity and by then performing viral growth curves in both CEM and SupT1 human T cell lines. No replication of the mutant virus could be detected in reverse transcriptase assays performed throughout a 2-week period; replication was therefore reduced by at least three orders of magnitude relative to wild-type virus. For a detailed mutational analysis of the capsid dimer interface, see U. K. von Schwedler et al., in preparation.
35. NL4-3 expression plasmid R9 [S. Swingler et al., J. Virol. 71, 4372 (1997)], which contains an SV-40 origin of replication. Viral particles were produced by transient calcium phosphate transfection of this plasmid into 293T human embryonic kidney cells. After 48 hours, particle production was reduced by 50 to 80% relative to a wild-type control R9 vector, as quantitated by both p24 enzyme-linked immunosorbent assay (DuPont) and a reverse transcriptase assay [S. P. Goff, P. Traktman, D. Baltimore, J. Virol. 38, 238 (1981)]. Particle infectivity was assayed by normalizing mutant and wild-type virus for reverse transcriptase activity and by then performing viral growth curves in both CEM and SupT1 human T cell lines. No replication of the mutant virus could be detected in reverse transcriptase assays performed throughout a 2-week period; replication was therefore reduced by at least three orders of magnitude relative to wild-type virus. For a detailed mutational analysis of the capsid dimer interface, see U. K. von Schwedler et al., in preparation.
36. NL4-3 expression plasmid R9 [S. Swingler et al., J. Virol. 71, 4372 (1997)], which contains an SV-40 origin of replication. Viral particles were produced by transient calcium phosphate transfection of this plasmid into 293T human embryonic kidney cells. After 48 hours, particle production was reduced by 50 to 80% relative to a wild-type control R9 vector, as quantitated by both p24 enzyme-linked immunosorbent assay (DuPont) and a reverse transcriptase assay [S. P. Goff, P. Traktman, D. Baltimore, J. Virol. 38, 238 (1981)]. Particle infectivity was assayed by normalizing mutant and wild-type virus for reverse transcriptase activity and by then performing viral growth curves in both CEM and SupT1 human T cell lines. No replication of the mutant virus could be detected in reverse transcriptase assays performed throughout a 2-week period; replication was therefore reduced by at least three orders of magnitude relative to wild-type virus. For a detailed mutational analysis of the capsid dimer interface, see U. K. von Schwedler et al., in preparation.
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40. A. T. Brünger, X-PLOR version 3.843, a System for X-ray Crystallography and NMR (Yale University, New Haven, CT, 1996).
41. We thank M. Capel, C. Phillips, J. Phillips, R. Sweet, and F. Whitby for assistance with data collection; V. Ramakrishnan and members of the Sundquist and Hill laboratories for critical comments on the manuscript; M. Martin for plasmid pNL4-3; D. Trono for plasmid R9; and J. Cassatt for support and encouragement. Supported by NIH grants RO1 AI40333 and RO1 AI43036 (W.I.S. and C.P.H.), the Lucille P. Markey Charitable Trust, and a postdoctoral fellowship from the Cancer Research Institute (T.R.G.). Coordinates (1am3) and diffraction data (r1am3sf) for CA(151-231) have been deposited in the Protein Data Bank (Brookhaven National Laboratory).