Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.     

Crystal structure of an RNA bacteriophage coat protein-operator complex

The RNA bacteriophage MS2 is a convenient model system for the study of protein-RNA interactions. The MS2 coat protein achieves control of two distinct processes--sequence-specific RNA encapsidation and repression of replicase translation--by binding to an RNA stem-loop structure of 19 nucleotides containing the initiation codon of the replicase gene. The binding of a coat protein dimer to this hairpin shuts off synthesis of the viral replicase, switching the viral replication cycle to virion assembly rather than continued replication. The operator fragment alone can trigger self-assembly of the phage capsid at low protein concentrations and a complex of about 90 RNA operator fragments per protein capsid has been described. We report here the crystal structure at 3.0 A resolution of a complex between recombinant MS2 capsids and the 19-nucleotide RNA fragment. It is the first example of a structure at this resolution for a sequence-specific protein-RNA complex apart from the transfer RNA synthetase complexes. The structure shows sequence-specific interactions between conserved residues on the protein and RNA bases essential for binding.     

Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy

Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease. The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg). Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined. Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical. Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts. By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure. In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits.     

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.     

Cryoelectron microscopic examination of human immunodeficiency virus type 1 virions with mutations in the cyclophilin A binding loop

The human immunodeficiency virus type 1 capsid protein contains a conserved P217X4PX2PX5P231 motif. Mutation at Pro-222 decreases virion incorporation of cyclophilin A, while mutation at Pro-231 abolishes infectivity. Although viral RNA incorporation and protease cleavage of the Gag precursor were not affected by these mutations, cryoelectron microscopy revealed a loss of virion maturation in P231A particles.     

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.     

Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.     

The crystal structure of bacteriophage Q beta at 3.5 A resolution

BACKGROUND: The capsid protein subunits of small RNA bacteriophages form a T = 3 particle upon assembly and RNA encapsidation. Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation. We have determined the crystal structure of phage Q beta with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages. RESULTS: The crystal structure of bacteriophage Q beta determined at 3.5 A resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes. As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers. A contiguous ten-stranded antiparallel beta sheet facing the RNA is formed in the dimer. The disulfide bonds lock the constituent dimers of the capsid covalently in the T = 3 lattice. CONCLUSIONS: The unusual stability of the Q beta particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid. A comparison with the structure of the related phage MS2 shows that although the fold of the Q beta coat protein is very similar, the details of the protein-protein interactions are completely different. The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding.     

RNA binding properties of core protein of the flavivirus Kunjin

Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.     

Formation of an RNA heterodimer upon heating of nodavirus particles

Flock House virus is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, which are believed to be encapsidated into a single viral particle. However, evidence to support this claim is circumstantial. Here we demonstrate that exposure of nodavirus particles to heat causes the two strands of viral RNA to form a stable complex, directly establishing that both RNAs are copackaged into one virion. The physical properties of the RNA complex, the effect of heat on the particles per se, and the possible relevance of these findings to the nodavirus life cycle are presented.     

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

Hepatitis B virus capsid: localization of the putative immunodominant loop (residues 78 to 83) on the capsid surface, and implications for the distinction between c and e-antigens

Hepatitis B virus capsid protein comprises a 149 residue "assembly" domain that polymerizes into icosahedral particles, and a 34 residue RNA-binding "protamine" domain. Recently, the capsid structure has been studied to resolutions below 10 A by cryo-electron microscopy, revealing much of its alpha-helical substructure and that it appears to have a novel fold for a capsid protein; however, the resolution is still too low for chain-tracing by conventional criteria. Aiming to establish a fiducial marker to aid in the process of chain-tracing, we have used cryo-microscopy to pinpoint the binding site of a monoclonal antibody that recognizes the peptide from residues 78 to 83. This epitope resides on the outer rim of the 30 A long spikes that protrude from the capsid shell. These spikes are four-helix bundles formed by the pairing of helix-turn-helix motifs from two subunits; by means of a tilting experiment, we have determined that this bundle is right-handed. Variants of the same protein present two clinically important and non-crossreactive antigens: core antigen (HBcAg), which appears early in infection as assembled capsids; and the sentinel e-antigen (HBeAg), a non-particulate form. Knowledge of the binding site of our anti-HBcAg antibody bears on the molecular basis of the distinction between the two antigens, which appears to reflect conformational differences between the assembled and unassembled states of the capsid protein dimer, in addition to epitope masking in capsids.     

Cross-functional analysis of the Microviridae internal scaffolding protein

The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.     

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.     

The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.     

Biophysical characterization of wild-type and mutant bacteriophage IKe major coat protein in the virion and in detergent micelles

Interactions between the filamentous bacteriophage major coat protein and its environment differ markedly between the membrane-bound assembly intermediate which spans the lipid bilayer and the phage coat protein which makes up the capsid of the virion. Nonetheless, both reflect successful strategies to sequester the hydrophobic regions of the coat protein away from the aqueous milieu. To characterize the roles of individual residues in the conformation, stability, and oligomerization of the coat protein in both the virion and in detergent micelles, wild-type IKe and M13 coat proteins, together with a library of over 40 IKe coat protein mutants, were studied using circular dichroism (CD), fluorescence, and solution nuclear magnetic resonance (NMR) spectroscopies. The largely helical conformations of coat protein in IKe wild-type and mutant virions were found to be very similar by CD, demonstrating that the overall organization of the phage can accommodate a diverse range of amino acid substitutions in the major coat protein. Intrinsic Trp fluorescence showed that the polarity of the Trp 29 environment in the virion was modulated by residues within one helical turn of this locus. Characterization of IKe phage growth and plaquing properties highlighted the importance of Pro 30 in maintaining viability. As well, the Pro 30 mutants were the only substitutions which rendered the detergent-solubilized coat protein less thermostable and additionally altered the polarity of the Trp 29 environment. The Pro 30 Gly mutant exhibited numerous 1H and 15N chemical shift changes between residues Ile 25 and Ala 38 in the 2D 1H-15N HSQC spectrum in myristoyllysophosphatidylglycerol (MPG) micelles, demonstrating that the effect of the substitution is propagated beyond adjacent residues. The overall results highlight the stabilizing effect of Pro in the first turn of a transmembrane helix and the importance of hydrophobicity in modulating the oligomerization and stability of coat protein both in the phage and in detergent micelles.     

Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.