Crystals of Rous Sarcoma Virus Capsid Protein Show a Helical Arrangement of Protein Subunits

Kovari, Ladislau C.; Momany, Cory; Miyagi, Faith; Lee, Sukyeong; Campbell, Stephen J.; Vuong, Bao Q.; Vogt, Volker M.; Rossmann, Michael G.

doi:10.1006/viro.1997.8807

Cited by 10 publications

(10 citation statements)

References 26 publications

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“…Monomeric CA-SP and CA-S. Recombinant CA-SP, 249-residue processing intermediate, and 240-residue mature CA-S proteins were expressed in E. coli and purified for comparison with the mature CA protein (237 residue) that has been extensively characterized in vitro (46)(47)(48)52). Purified CA-S and CA-SP, like CA, were almost exclusively monomeric ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

England

Purdy

Ropson

et al. 2014

J Virol

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Section: Resultsmentioning

confidence: 99%

“…RSV Prague C CA-S (240 residues) and CA-SP (249 residues) were cloned into pET-24(ϩ) and expressed in Escherichia coli BL21(DE3), as described previously (46)(47)(48)(49). Proteins were purified by ammonium sulfate precipitation, DEAE cation exchange, and size exclusion chromatography (46,47).…”

Section: Methodsmentioning

confidence: 99%

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

England

Purdy

Ropson

et al. 2014

J Virol

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“…The purification protocol was adapted from a previously described protocol (40). The bacterial pellet was resuspended in buffer containing 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 10% glycerol, 1 mM EDTA, 10 mM dithiothreitol, Complete protease inhibitor cocktail (Roche), and lysozyme (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Critical Role of Conserved Hydrophobic Residues within the Major Homology Region in Mature Retroviral Capsid Assembly

Purdy¹,

Flanagan

Ropson

et al. 2008

J Virol

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During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.The retroviral CA protein plays critical structural roles in each of the two distinct stages of virion assembly. When an immature particle is formed by the polymerization of the Gag polyprotein, the N-terminal CA domain (NTD) and C-terminal CA domain (CTD) embedded within Gag control packing and assembly (1,3,12,14,17,41,54,77,80). Subsequently, the processing of Gag by the viral protease initiates a maturation process in which the structural proteins MA (matrix), CA (capsid), and NC (nucleocapsid) are released from Gag. The free CA polymerizes as a capsid shell around the genomic RNA and NC protein, creating the core of the mature virion. The maturation events are complex and include disruption of the CA-CA interfaces that held the Gag proteins together, conformational changes within each domain of CA, and formation of new CA-CA interfaces of the mature capsid shell (6,11,12,24,31,44,52,53,58,74,(79)(80)(81).In spite of limited sequence similarity, the three-dimensional structure of mature CA is highly conserved among retroviruses and consists of the two mostly ␣-helical domains, NTD and CTD, connected by an interdomain linker (8,18,19,24,29,36,37). After maturation is completed, the final capsid shell consists of a lattice of CA hexamers, established by NTD-NTD interactions and linked by CTD-CTD dimerization (24,25,27,43,48,51,53,79). The dimer interface is formed by the dimerization helix, the second helix of the CTD. A third interface, an NTD-CTD interdomain interaction that forms during maturation, was originally predicted by a genetic study of the Alpharetrovirus Rous sarcoma virus (RSV) and subsequently confirmed and mapped by biochemical and st...

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“…As expected, both the calculated self-rotation of the helicalbundle and the coiled-coils show an arc of dyads, which are perpendicular to another dyad as well as to peaks in the = 30 to 180 sections. Moreover, when the helical-bundle is arrayed in a crystal lattice of the monoclinic crystal system, as taken from the crystallization report from Kavori et al [18], the self-rotation contains a similar low resolutionlooking arc to the self-rotation of Sec2pN. That is, the selfrotation contains seven apparent but evenly spaced dyads that at the time suggested a sevenfold screw rotation axis of the molecule.…”

Section: What Does the Self-rotation Of Other Helical Proteins Look Lmentioning

confidence: 96%

“…The helical-bundle is taken from the C-terminus of the Rous Sarcoma Virus capsid protein[18], which was solved by NMR methods. (See PDBid: 1EOQ) 2 Modeled from tropomyosin (PDBid:1C1G) 256 Protein & Peptide Letters, 2007, Vol.…”

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confidence: 99%

Structural Insights into the Exchange Domain of Sec2p: Expression, Purification,Crystallization, and Preliminary X-Ray Diffraction Data Analysis

Gill

2007

PPL

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Sec2p is an essential yeast gene and is part of the cell polarization process that leads to budding. The N-terminal domain of sec2p (Sec2pN)--the guanine-nucleotide exchange factor for sec4p--has been expressed in Escherichia coli, purified, and crystallized. Crystals belong to the space group P2(1) with unit cell dimensions 178.1 x 98.4 x 180.0 A, beta = 91.7( degrees ), and diffract synchrotron-generated X-rays to better than 3.6 A resolution. Pseudo-precession plots reveal a Laue symmetry of 2/m, corresponding to the aforementioned space group, and unusual weak diffraction in the approximately 5-7 A resolution range. The Matthews number calculations for a typical crystal density suggest a range of 28 to 64 molecules per asymmetric unit. Self-rotation and native Patterson calculations demonstrate a pure helical array of protein subunits. Based on the X-ray diffraction data analysis and amino-acid sequence alignments, the paper presents a hypothetical model of the exchange domain of sec2p as a pair of coiled-coil helices that binds to sec4p and facilitates nucleotide disassociation.

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Crystals of Rous Sarcoma Virus Capsid Protein Show a Helical Arrangement of Protein Subunits

Cited by 10 publications

References 26 publications

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

Potential Role for CA-SP in Nucleating Retroviral Capsid Maturation

Critical Role of Conserved Hydrophobic Residues within the Major Homology Region in Mature Retroviral Capsid Assembly

Structural Insights into the Exchange Domain of Sec2p: Expression, Purification,Crystallization, and Preliminary X-Ray Diffraction Data Analysis

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