Using Ion Mobility Spectrometry–Mass Spectrometry to Decipher the Conformational and Assembly Characteristics of the Hepatitis B Capsid Protein

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.     

Using Ion Mobility Spectrometry–Mass Spectrometry to Decipher the Conformational and Assembly Characteristics of the Hepatitis B Capsid Protein

The structural and functional analysis of the core protein of hepatitis B virus is important for a full understanding of the viral life cycle and the development of novel therapeutic agents. The majority of the core protein (CP149) comprises the capsid assembly domain, and the C-terminal region (residues 150–183) is responsible for nucleic acid binding. Protein monomers associate to form dimeric structural subunits, and helices 3 and 4 (residues 50–111 of the assembly domain) have been shown to be important for this as they constitute the interdimer interface. Here, using mass spectrometry coupled with ion mobility spectrometry, we demonstrate the conformational flexibility of the CP149 dimer. Limited proteolysis was used to locate involvement in this feature to the C-terminal region. A genetically fused CP dimer was found to show decreased disorder, consistent with a more restricted C-terminus at the fusion junction. Incubation of CP149 dimer with heteroaryldihydropyrimidine-1, a small molecule known to interfere with the assembly process, was shown to result in oligomers different in shape to the capsid assembly-competent oligomers of the fused CP dimer. We suggest that heteroaryldihydropyrimidine-1 affects the dynamics of CP149 dimer in solution, likely affecting the ratio between assembly active and inactive states. Therefore, assembly of the less dynamic fused dimer is less readily misdirected by heteroaryldihydropyrimidine-1. These studies of the flexibility and oligomerization properties of hepatitis B virus core protein illustrate both the importance of C-terminal dynamics in function and the utility of gas-phase techniques for structural and dynamical biomolecular analysis.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

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.     

Structural tolerance versus functional intolerance to mutation of hydrophobic core residues surrounding cavities in a parvovirus capsid

The structural and functional relevance of amino acid residues surrounding cavities within the hydrophobic core of the protein subunits that form the capsid of parvoviruses has been investigated. Several of the evolutionarily conserved, hydrophobic residues that delimit these cavities in the capsid of the minute virus of mice were replaced by other hydrophobic residues that would affect the size and/or shape of the cavity. When four or more methylene-sized groups were introduced, or six or more groups removed, capsid assembly was drastically impaired. In contrast, the introduction or removal of up to three groups had no significant effect on capsid assembly or thermostability. However, many of these mutations affected a capsid conformational transition needed for viral infectivity. Replacement of some polar residues around the largest cavity showed that capsid assembly requires a carboxylate buried within this cavity, but both aspartate and glutamate are structurally accepted. Again, only the aspartate allowed the production of infectious viruses, because of a specific role in encapsidation of the viral genome. These observations provide evidence of a remarkable structural tolerance to mutation of the hydrophobic core of the protein subunits in a viral capsid, and of an involvement of core residues and internal cavities in capsid functions needed for infectivity.     

Biophysical Analysis of the MHR Motif in Folding and Domain Swapping of the HIV Capsid Protein C-Terminal Domain

Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR), within the carboxyterminal domain (CTD) of the capsid protein. In a modified CTD dimer, MHR is swapped between monomers. While no evidence for MHR swapping has been provided by structural models of retroviral capsids, it is unknown whether it may occur transiently along the virus assembly pathway. Whatever the case, the MHR-swapped dimer does provide a novel target for the development of anti-HIV drugs based on the concept of trapping a nonnative capsid protein conformation. We have carried out a thermodynamic and kinetic characterization of the domain-swapped CTD dimer in solution. The analysis includes a dissection of the role of conserved MHR residues and other amino acids at the dimerization interface in CTD folding, stability, and dimerization by domain swapping. The results revealed some energetic hotspots at the domain-swapped interface. In addition, many MHR residues that are not in the protein hydrophobic core were nevertheless found to be critical for folding and stability of the CTD monomer, which may dramatically slow down the swapping reaction. Conservation of MHR residues in retroviruses did not correlate with their contribution to domain swapping, but it did correlate with their importance for stable CTD folding. Because folding is required for capsid protein function, this remarkable MHR-mediated conformational stabilization of CTD may help to explain the functional roles of MHR not only during immature capsid assembly but in other processes associated with retrovirus infection. This energetic dissection of the dimerization interface in MHR-swapped CTD may also facilitate the design of anti-HIV compounds that inhibit capsid assembly by conformational trapping of swapped CTD dimers.     

Dimeric rous sarcoma virus capsid protein structure relevant to immature Gag assembly

The structure of the N-terminal domain (NTD) of Rous sarcoma virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroviruses can assemble in vitro into virus-like particles closely resembling in vivo-assembled immature virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a “shape determinant” promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no β-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, immature Gag particles, the dimer interface may represent a structural feature that is essential in retrovirus assembly.     

The isolated major homology region of the HIV capsid protein is mainly unfolded in solution and binds to the intact protein

Assembly of the mature human immunodeficiency virus type 1 (HIV-1) capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, the CA protein undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. The most conserved region of CA, the major homology region (MHR), is located in the C-terminal domain of CA (CTD). The MHR is involved in both immature and mature virus assembly; however, its exact function during both assembly stages is unknown. To test its conformational preferences and to provide clues on its role during CA assembly, we have used a minimalist approach by designing a peptide comprising the whole MHR (MHRpep, residues Asp152 to Ala174). Isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CTD with an affinity of ~30μM (as shown by fluorescence and ITC); the CTD binding region comprises residues belonging to α-helices 10 and 11. In the immature virus capsid, the MHR and α-helix 11 regions of two CTD dimers also interact [Briggs JAG, Riches JD, Glass B, Baratonova V, Zanetti G and Kr?usslich H-G (2009) Proc. Natl. Acad. Sci. USA 106, 11090-11095]. These results can be considered a proof-of-concept that the conformational preferences and binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.     

Structure-based design and biochemical evaluation of sulfanilamide derivatives as hepatitis B virus capsid assembly inhibitors

Virus capsid structure is essential in virion maturation and durability, so disrupting capsid assembly could be an effective way to reduce virion count and cure viral diseases. However, currently there is no known antiviral which affects capsid inhibition, and only a small number of assembly inhibitors were experimentally successful. In this present study, we aimed to find hepatitis B virus (HBV) capsid assembly inhibitor which binds to the HBV core protein and changes protein conformation. Several candidate molecules were found to bind to certain structure in core protein with high specificity. Furthermore, these molecules significantly changed the protein conformation and reduced assembly affinity of core protein, leading to decrease of the number of assembled capsid or virion, both in vitro and in vivo. In addition, prediction also suggests that improvements in inhibition efficiency could be possible by changing functional groups and ring structures.     

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.     

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser(106). PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser(106) mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

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.     

Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli

Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coli were examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.     

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.     

Mapping of Homologous Interaction Sites in the Hepatitis B Virus Core Protein

Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.     

Structural features of the scaffold interaction domain at the N terminus of the major capsid protein (VP5) of herpes simplex virus type 1

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 capsid. A key interaction occurs between the C terminus of the scaffold protein and the N terminus of the major capsid protein (VP5). Results from alanine-scanning mutagenesis of hydrophobic residues in the N terminus of VP5 revealed seven residues (I27, L35, F39, L58, L65, L67, and L71) that reside in two predicted alpha helices (helix 1(22-42) and helix 2(58-72)) that are important for this bimolecular interaction. The goal of the present study was to further characterize the VP5 scaffold interaction domain (SID). Amino acids at the seven positions were replaced with L, M, V or P (I27); I, M, V, or P (L35, L58, L65, L67, and L71); and H, W, Y, or L (F39). Replacement with a hydrophobic side chain did not affect the interaction with scaffold protein in yeast cells or the ability of a virus specifying the mutation from replicating in cells. The mutation to the proline side chain abolished the interaction in all cases and was lethal for virus replication. Mutant viruses with proline substitutions in helix 1(22-42) at positions 27 and 35 assembled large open capsid shells that did not attain closure. Proline substitutions in helix 2(58-72) at either position 59, 65, or 67 abolished the accumulation of VP5 protein, and, at 58 and 71, although VP5 did accumulate, capsid shells were not assembled. Thus, the second SID, SID2, is highly structured, and this alpha helix (helix 2(58-72)) is likely involved in capsomere-capsomere interactions during shell accretion. Conserved glycine G59 in helix 2(58-72) was also mutated. G59 may act as a flexible "hinge" in helix 2(58-72) because decreasing the movement of this side chain by replacement with valine impaired capsid assembly. Thus, the N terminus of VP5 and the alpha helices embedded in this domain, as in the capsid shell proteins of some double-stranded DNA phages, are a key regulator of shell accretion and stabilization.