Disulfide bonds stabilize JC virus capsid-like structure by protecting calcium ions from chelation

To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus-like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast-expressed VP1 was self-assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non-reducing SDS–PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

Mutational analysis of the carboxyl-terminal region of the SV40 major capsid protein VP1

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, alpha-alpha'-alpha', beta-beta' and gamma-gamma. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301-312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt alpha-alpha'-alpha'-type and/or beta-beta'-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs.     

Assembly of the capsid protein of red-spotted grouper nervous necrosis virus during purification,and role of calcium ions in chromatography

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.     

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.     

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

Disulfide Linkage and Structure of Highly Stable Yeast-derived Virus-like Particles of Murine Polyomavirus

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys¹¹⁵–Cys²⁰, Cys¹²–Cys²⁰, Cys¹⁶–Cys¹⁶, Cys¹²/ Cys¹⁶–Cys¹¹⁵, and Cys²⁷⁴–Cys²⁷⁴), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.     

A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface.

Core particles of hepatitis B virus are assembled from dimers of a single 185-residue (subtype adw) viral capsid or core protein (p21.5) which possesses two distinct domains: residues 1 to 144 form a minimal capsid assembly domain, and the arginine-rich, carboxyl-terminal residues 150 to 185 form a protamine-like domain that mediates nucleic acid binding. Little is known about the topography of the p21.5 polypeptide within either the p21.5 capsids or dimers. Here, using site-specific proteases and monoclonal antibodies, we have defined the accessibility of p21.5 residues in dimers and capsids assembled from wild-type and mutant hepatitis B virus core proteins in Xenopus oocytes and in vitro. The data reveal the protamine region to be accessible to external reagents in p21.5 dimers but largely cryptic in wild-type capsids. Strikingly, in capsids the only protease target region was a 9-residue peptide covering p21.5 residues Glu-145 to Asp-153, which falls largely between the two core protein domains. By analogy with protease-sensitive interdomain regions in other proteins, we propose that this peptide constitutes a hinge between the assembly and nucleic acid binding domains of p21.5. We further found that deletion or replacement of the terminal Cys-185 residue greatly increased surface exposure of the protamine tails in capsids, suggesting that a known disulfide linkage involving this residue tethers the protamine region inside the core particles. We propose that disruption of this disulfide linkage allows the protamine region to appear transiently on the surface of the core particle.     

Identification of domains in canine parvovirus VP2 essential for the assembly of virus-like particles.

Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VPl. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.     

Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding

Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.     

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

Chimeric infectious bursal disease virus-like particles expressed in insect cells and purified by immobilized metal affinity chromatography.

Chimeric virus-like particles (VLPs) of infectious bursal disease virus (IBDV) were produced by coinfecting Spodoptera frugiperda (Sf-9) insect cells with two recombinant baculoviruses, vIBD-7 and vEDLH-22. vIBD-7 encodes VP2, VP3, and VP4 of the IBDV structural proteins. vEDLH-22 encodes VP2 with five histidine residues at the carboxy-terminus (VP2H). Coinfection produced hybrid VLPs composed of VP2, VP2H, and VP3. The additional histidine residues on VP2H enabled the efficient purification of VLPs based on immobilized metal affinity chromatography (IMAC). These results demonstrated that the VLPs formed are comprised of chimeric subunits with attached affinity ligands, and further, that sufficient His5 ligand was available for binding to the IMAC metal-chelating resin. Additionally, these novel particles were fully characterized for antigenicity by a series of monoclonal antibodies, and appeared identical to the two wild-type IBDV strains contributing subunits to the chimeric VLP. IMAC purification provides a promising low-cost and simple scheme to purify VLPs as vaccines.     

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 A

Human rhinovirus 14 has a pseudo T = 3 icosahedral structure in which 60 copies of the three larger capsid proteins VP1, VP2 and VP3 are arranged in an icosahedral surface lattice, reminiscent of T = 3 viruses such as tomato bushy stunt virus and southern bean mosaic virus. The overall secondary and tertiary structures of VP1, VP2 and VP3 are very similar. The structure of human rhinovirus 14, which was refined at a resolution of 3.0 A [R = 0.16 for reflections with F greater than 3 sigma(F)], is here analyzed in detail. Quantitative analysis of the surface areas of contact (proportional to hydrophobic free energy of association) supports the previously assigned arrangement within the promoter, in which interactions between VP1 and VP3 predominate. Major contacts among VP1, VP2 and VP3 are between the beta-barrel moieties. VP4 is associated with the capsid interior by a distributed network of contacts with VP1, VP2 and VP3 within a promoter. As the virion assembly proceeds, the solvent-accessible surface area becomes increasingly hydrophilic in character. A mixed parallel and antiparallel seven-stranded sheet is composed of the beta C, beta H, beta E and beta F strands of VP3 in one pentamer and beta A1 and beta A2 of VP2 and the VP1 amino terminus in another pentamer. This association plays an essential role in holding pentamers together in the mature virion as this contact region includes more than half of the total short non-bonded contacts between pentamers. Contacts between protomers within pentamers are more extensive than the contacts between pentamers, accounting in part for the stability of pentamers. The previously identified immunogenic regions are correlated with high solvent accessibility, accessibility to large probes and also high thermal parameters. Surface residues in the canyon, the putative cellular receptor recognition site, have lower thermal parameters than other portions of the human rhinovirus 14 surface. Many of the water molecules in the ordered solvent model are located at subunit interfaces. A number of unusual crevices exist in the protein shell of human rhinovirus 14, including the hydrophobic pocket in VP1 which is the locus of binding for the WIN antiviral agents. These may be required for conformational flexibility during assembly and disassembly. The structures of the beta-barrels of human rhinovirus 14 VP1, VP2 and VP3 are compared with each other and with the southern bean mosaic virus coat protein.     

Mechanism of assembly of recombinant murine polyomavirus-like particles

VP1 is the major viral coat protein of murine polyomavirus and can be used for the generation of virus-like particles in vitro. Here, we demonstrate that capsid assembly is an equilibrium reaction followed by oxidation of intracapsomere disulfide bonds, which are not essential for the formation of virus-like particles but enable complete particle assembly and prevent capsid disassembly.     

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.     

人多瘤病毒BK株VP1的C端阳电荷残基对病毒样颗粒形成的影响

VP1是人多瘤病毒BK株的主要结构蛋白,使用重组杆状病毒表达系统在体外表达 VP1 可以形成病毒样颗粒(VLP)。为了探讨VP1的C末端阳电荷残基R 281, R 285, K 288, R 290, R 292, K 293, R 294,和 K297 对VLP形成和其结合DNA的影响,我们分别改变将阳电荷残基变成丙氨酸,然后表达 VP1 蛋白。结果发现用丙氨酸替代K 288,R 290,R 292,K 293,R 294后仍能形成VLP, 但与野毒株相比,在 VLP分泌以及衣壳蛋白与细胞DNA的结合方面有差异。有趣的是,R 281被丙氨酸取代后仅在细胞中形成少量的 VLP,而 R 285 被丙氨酸取代后不能形成VLP。该研究证实阳电荷氨基酸残基 R 281 和 R 285 是形成 VLP所必须的,K 288、R 290、R 292、K 293、R 294和K 297则影响VLP和DNA的结合。     

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus

Human interleukin 4 is a 129 amino acid lymphokine secreted by activated T cells that exerts pleiotropic biological effects on B and T lymphocytes and other hematopoietic cells. Structure-function relations were studied by employing selective proteolytic cleavage of purified recombinant human interleukin 4 (rhuIL-4). Limited proteolysis with endoprotease Glu-C from Staphylococcus aureus (V8) produced two digestion products that were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weight values of 19K (I) and 15K (II), respectively. These species were isolated by reversed-phase HPLC. Amino acid sequencing indicated that species II was an 84 amino acid core fragment extending from Gln-20 to Glu-103 and containing a hydrolyzed peptide bond at Glu-26. On the basis of known disulfide bond assignments, it was concluded that species II was stabilized by two disulfide bonds (Cys-24/Cys-65 and Cys-46/Cys-99). Analysis of its secondary structure by circular dichroism revealed a high content of alpha helix. Species I was the full-length rhuIL-4 with selective cleavage at Glu-26 and Glu-103. Both species I and II were inactive in an in vitro assay based on proliferation of peripheral blood lymphocyte blasts and lacked the ability to bind to teh rhuIL-4 receptor on Daudi cells. In order to elucidate further the role of the residues removed by S. aureus V8 protease, rabbit antisera were raised to synthetic peptides corresponding to residues 1-26 at the N-terminus and 104-129 at the C-terminus. Only antisera directed to the C-terminal peptide inhibited binding of 125I-rhuIL-4 to Daudi cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection.

VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.