Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.     

A micromolar pool of antigenically distinct precursors is required to initiate cooperative assembly of hepatitis B virus capsids in Xenopus oocytes.

Assembly of hepatitis B virus capsid-like (core) particles occurs efficiently in a variety of heterologous systems via aggregation of approximately 180 molecules of a single 21.5-kDa core protein (p21.5), resulting in an icosahedral capsid structure with T = 3 symmetry. Recent studies on the assembly of hepatitis B virus core particles in Xenopus oocytes suggested that dimers of p21.5 represent the major building block from which capsids are generated. Here we determined the concentration dependence of this assembly process. By injecting serially diluted synthetic p21.5 mRNA into Xenopus oocytes, we expressed different levels of intracellular p21.5 and monitored the production of p21.5 dimers and capsids by radiolabeling and immunoprecipitation, by radioimmunoassay, or by quantitative enzyme-linked immunosorbent assay analysis. The data revealed that (i) p21.5 dimers and capsids are antigenically distinct, (ii) capsid assembly is a highly cooperative and concentration-dependent process, and (iii) p21.5 must accumulate to a signature concentration of approximately 0.7 to 0.8 microM before capsid assembly initiates. This assembly process is strikingly similar to the assembly of RNA bacteriophage R17 as defined by in vitro studies.     

The morphogenic linker peptide of HBV capsid protein forms a mobile array on the interior surface

Many capsid proteins have peptides that influence their assembly. In hepatitis B virus capsid protein, the peptide STLPETTVV, linking the shell-forming 'core' domain and the nucleic acid-binding 'protamine' domain, has such a role. We have studied its morphogenic properties by permuting its sequence, substituting it with an extraneous peptide, deleting it to directly fuse the core and protamine domains and assembling core domain dimers with added linker peptides. The peptide was found to be necessary for the assembly of protamine domain-containing capsids, although its size-determining effect tolerates some modifications. Although largely invisible in a capsid crystal structure, we could visualize linker peptides by cryo-EM difference imaging: they emerge on the inner surface and extend from the capsid protein dimer interface towards the adjacent symmetry axis. A closely sequence-similar peptide in cellobiose dehydrogenase, which has an extended conformation, offers a plausible prototype. We propose that linker peptides are attached to the capsid inner surface as hinged struts, forming a mobile array, an arrangement with implications for morphogenesis and the management of encapsidated nucleic acid.     

Hepatitis B virus capsid assembly is enhanced by naturally occurring mutation F97L

In chronic hepatitis B virus (HBV) infections, one of the most common mutations to the virus occurs at amino acid 97 of the core protein, where leucine replaces either phenylalanine or isoleucine, depending on strain. This mutation correlates with changes in viral nucleic acid metabolism and/or secretion. We hypothesize that this phenotype is due in part to altered core assembly, a process required for DNA synthesis. We examined in vitro assembly of empty HBV capsids from wild-type and F97L core protein assembly domains. The mutation enhanced both the rate and extent of assembly relative to those for the wild-type protein. The difference between the two proteins was most obvious in the temperature dependence of assembly, which was dramatically stronger for the mutant protein, indicating a much more positive enthalpy. Since the structures of the mutant and wild-type capsids are essentially the same and the mutation is not involved in the contact between dimers, we suggest that the F97L mutation affects the dynamic behavior of dimer and capsid.     

Identification of residues in the hepatitis C virus core protein that are critical for capsid assembly in a cell-free system

Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation.     

Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes.

Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60% (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.     

Role of the propeptide in controlling conformation and assembly state of hepatitis B virus e-antigen

Hepatitis B virus “e-antigen” (HBeAg) is thought to be a soluble dimeric protein that is associated with chronic infection. It shares 149 residues with the viral capsid protein “core-antigen” (HBcAg), but has an additional 10-residue, hydrophobic, cysteine-containing amino-terminal propeptide whose presence correlates with physical, serological, and immunological differences between the two proteins. In HBcAg dimers, the subunits pair by forming a four-helix bundle stabilized by an intermolecular disulfide bond. The structure of HBeAg is probably similar but, instead, has two intramolecular disulfide bonds involving the propeptide. To compare the proteins directly and thereby clarify the role of the propeptide, we identified mutations and solution conditions that render both proteins as either soluble dimers or assembled capsids. Thermally induced unfolding monitored by circular dichroism, and electrophoresis of oxidized and reduced dimers, showed that the propeptide has a destabilizing effect and that the intramolecular disulfide bond forms preferentially and blocks the formation of the intermolecular disulfide bond that otherwise stabilizes the dimer. The HBeAg capsids are less regular than the HBcAg capsids; nevertheless, cryo-electron microscopy reconstructions confirm that they are constructed of dimers resembling those of HBcAg capsids. In them, a portion of the propeptide is visible near the dimer interface, suggesting that it intercalates there, consistent with the known formation of a disulfide bond between C(− 7) in the propeptide and C61 in the dimer interface. However, this intercalation distorts the dimer into an assembly-reluctant conformation.     

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.     

A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids.

We have cloned in Escherichia coli both the complete core gene of hepatitis B virus and a truncated version of it, leading to the synthesis of high levels of a core-antigen-equivalent polypeptide (r-p22) and of an e-antigen-equivalent polypeptide (r-p16), respectively. We then compared the structural and antigenic properties of the two polypeptides, as well as their ability to bind viral nucleic acids. r-p16 was found to self-assemble into capsid-like particles that appeared similar, when observed under the electron microscope, to those formed by r-p22. In r-p16 particles, disulfide bonds linked the truncated polypeptides in dimers, assembled in the particle by noncovalent interactions. In r-p22 capsids, further disulfide bonds, conceivably involving the carboxy-terminal cysteines of r-p22 polypeptides, joined the dimers together, converting the structure into a covalently closed lattice. The protamine-like domain was at least partly exposed on the surface of r-p22 particles, since it was accessible to selective proteolysis. Finally, r-p22, but not r-p16, was shown to bind native and denatured DNA as well as RNA. Taken together, these results suggest that the protamine-like domain in core polypeptides is a nucleic acid-binding domain and is dispensable for the correct folding and assembly of amino-terminal and central regions.     

A hydrophobic heptad repeat of the core protein of woodchuck hepatitis virus is required for capsid assembly.

The capsid particle of hepadnaviruses is assembled from its dimer precursors. However, the mechanism of the protein-protein interaction is still poorly understood. A small region in the capsid protein of woodchuck hepatitis virus (WHV) contains four hydrophobic residues, including leucine 101, leucine 108, valine 115, and phenylalanine 122, that are conserved and spaced every seventh residue in the primary sequence to form a hydrophobic heptad repeat (hhr). A hydrophobic force often plays an important role in the interaction of proteins. Therefore, to investigate the role of this region in capsid assembly, we individually changed the codons specifying these four hydrophobic amino acids to codons specifying alanine or proline. In addition, we examined the in vivo infectivity of a WHV genome bearing a naturally occurring single amino acid change (histidine 104-->proline) in the hhr region. The phenotype of each altered genome was determined in both eukaryotic and prokaryotic systems by a capsid protein assay and electron microscopic examination. We show that replacement of any one of the four hydrophobic residues with alanine did not prevent capsid assembly. However, assembled capsid particles were not detected if combinations of any two of the four residues were substituted with alanines or if the spacing of these four hydrophobic residues was changed. An individual introduction of a proline (which dramatically changes the secondary structure of proteins) into different positions of this small region also abolished capsid assembly in vitro or viral replication in vivo. These results suggested that the hhr region of the core protein of WHV was critical for capsid assembly.     

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.     

Assembly Pathway of Hepatitis B Core Virus-like Particles from Genetically Fused Dimers

Macromolecular complexes are responsible for many key biological processes. However, in most cases details of the assembly/disassembly of such complexes are unknown at the molecular level, as the low abundance and transient nature of assembly intermediates make analysis challenging. The assembly of virus capsids is an example of such a process. The hepatitis B virus capsid (core) can be composed of either 90 or 120 dimers of coat protein. Previous studies have proposed a trimer of dimers as an important intermediate species in assembly, acting to nucleate further assembly by dimer addition. Using novel genetically-fused coat protein dimers, we have been able to trap higher-order assembly intermediates and to demonstrate for the first time that both dimeric and trimeric complexes are on pathway to virus-like particle (capsid) formation.     

Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry

Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.     

Residue conservation in viral capsid assembly

To evaluate the evolutionary constraints placed on viral proteins by the structure and assembly of the capsid, we calculate Shannon entropies in the aligned sequences of 45 polypeptide chains in 32 icosahedral viruses, and relate these entropies to the residue location in the three-dimensional structure of the capsids. Three categories of residues have entropies lower than the chain average implying that they are better conserved than average: residues that are buried within a subunit (the protein core), residues that contain atoms buried at an interface between subunits (the interface core), and residues that contribute to several such interfaces. The interface core is also conserved in homomeric proteins and in transient protein-protein complexes, which have only one interface whereas capsids have many. In capsids, the subunit interfaces implicate most of the polypeptide chain: on average, 66% of the capsid residues are at an interface, 34% at more than one, and 47% at the interface core. Nevertheless, we observe that the degree of residue conservation can vary widely between interfaces within a capsid and between regions within an interface. The interfaces and regions of interfaces that show a low sequence variability are likely to play major roles in the self-assembly of the capsid, with implications on its mechanism that we discuss taking adeno-associated virus as an example.     

Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein.

As a step toward understanding the assembly of the hepatitis B virus (HBV) nucleocapsid at a molecular level, we sought to define the primary sequence requirements for assembly of the HBV core protein. This protein can self assemble upon expression in Escherichia coli. Applying this system to a series of C-terminally truncated core protein variants, we mapped the C-terminal limit for assembly to the region between amino acid residues 139 and 144. The size of this domain agrees well with the minimum length of RNA virus capsid proteins that fold into an eight-stranded beta-barrel structure. The entire Arg-rich C-terminal domain of the HBV core protein is not necessary for assembly. However, the nucleic acid content of particles formed by assembly-competent core protein variants correlates with the presence or absence of this region, as does particle stability. The nucleic acid found in the particles is RNA, between about 100 to some 3,000 nucleotides in length. In particles formed by the full-length protein, the core protein mRNA appears to be enriched over other, cellular RNAs. These data indicate that protein-protein interactions provided by the core protein domain from the N terminus to the region around amino acid 144 are the major factor in HBV capsid assembly, which proceeds without the need for substantial amounts of nucleic acid. The presence of the basic C terminus, however, greatly enhances encapsidation of nucleic acid and appears to make an important contribution to capsid stability via protein-nucleic acid interactions. The observation of low but detectable levels of nucleic acid in particles formed by core protein variants lacking the Arg-rich C terminus suggests the presence of a second nucleic acid-binding motif in the first 144 amino acids of the core protein. Based on these findings, the potential importance of the C-terminal core protein region during assembly in vivo into authentic, replication-competent nucleocapsids is discussed.     

Differentiation-dependent interpentameric disulfide bond stabilizes native human papillomavirus type 16

Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.     

Conformation and mobility of the RNA-binding N-terminal part of the intact coat protein of cowpea chlorotic mottle virus. A two-dimensional proton nuclear magnetic resonance study

Two-dimensional proton nuclear magnetic resonance (n.m.r.) experiments were performed on the coat protein of cowpea chlorotic mottle virus (molecular mass: 20.2 kDa) present as dimer (pH 7.5) or as capsid consisting of 180 protein monomers (pH 5.0). The spectra of both dimers and capsids showed resonances originating from the flexible N-terminal region of the protein. The complete resonance assignment of a synthetic pentacosapeptide representing this N terminus made it possible to interpret the spectra in detail. The capsid spectrum showed backbone amide proton resonances arising from the first eight residues having a flexible random coil conformation, and side-chain resonances arising from the first 25 N-terminal amino acids. The dimer spectrum showed also side-chain resonances of residues 26 to 33, which are flexible in the dimer but immobilized in the capsid. The n.m.r. experiments indicated that the conformation of the first 25 amino acids of the protein in dimers and capsids is comparable to the conformation of the synthetic peptide, which alternates among extended and helical conformations on the n.m.r. time-scale. It is suggested that the alpha-helical region, situated in the region between residues 10 and 20, binds to the RNA during assembly of the virus particle.