Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.     

A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses

The outer surface protein C (OspC) of Borrelia burgdorferi, the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10(4) B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.     

Internal core protein cleavage leaves the hepatitis B virus capsid intact and enhances its capacity for surface display of heterologous whole chain proteins

Virus capsids find increasing use as nanoparticulate platforms for the surface display of heterologous ligands, including as multivalent vaccine carriers. Presentation on the icosahedral hepatitis B virus capsid (HBcAg) is known to strongly enhance immunogenicity of foreign sequences, most efficiently if they are inserted into the dominant c/e1 B cell epitope, a surface-exposed loop in the center of the constituent core protein primary sequence. Even some complete proteins were successfully inserted but others, e.g. the outer surface protein A (OspA) of the Lyme disease agent Borrelia burgdorferi, impaired formation of capsid-like particles (CLPs). This difference can be rationalized by the requirement for the termini of the insert to fit into the predetermined geometry of the two acceptor sites in the carrier. We reasoned that cleavage of one of the two bonds connecting insert and carrier should relieve these constraints, provided the cleaved protein fragments remain competent to support the particle structure. Indeed, HBcAg CLPs containing a recognition site for tobacco etch virus (TEV) protease in the c/e1 loop remained intact after cleavage, as did CLPs carrying a 65-residue peptide insertion. Most importantly, in situ cleavage of a core-OspA fusion protein by coexpressed TEV protease strongly enhanced CLP formation compared with the uncleaved protein. These data attest to the high structural stability of the HBcAg CLP and they significantly widen its applicability as a carrier for heterologous proteins. This approach should be adaptable to any protein-based particle with surface-exposed yet sequence-internal loops.     

In vitro assembly of mosaic hepatitis B virus capsid-like particles (CLPs): rescue into CLPs of assembly-deficient core protein fusions and FRET-suited CLPs

Hepatitis B virus core protein self-assembles into icosahedral, highly immunogenic capsid-like particles (CLPs) that can serve as molecular platforms for heterologous proteins. Insertion into the centrally located c/e1 epitope leads to surface display, fusion to the C terminus to internal disposition of the foreign domains. However, symmetry-defined space restrictions on the surface and particularly inside the CLPs limit the size of usable heterologous fusion partners. Further, CLPs carrying differing foreign domains are desirable for applications such as multivalent vaccines, and for structure probing by distance sensitive interactions like fluorescence resonance energy transfer (FRET). Here, we report an in vitro co-assembly system for such mosaic-CLPs allowing successful CLP formation with a per se assembly-deficient fusion protein, and of CLPs from two different fluoroprotein-carrying fusions that exert FRET in an assembly-status dependent way.     

Surface localization determinants of Borrelia OspC/Vsp family lipoproteins

The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.     

Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 A. OspC, a largely alpha-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long alpha-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.     

Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF), and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC). Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc) derived capsid-like particles (CLPs) to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.     

Alphavirus capsid protein helix I controls a checkpoint in nucleocapsid core assembly

The assembly of the alphavirus nucleocapsid core has been investigated using an in vitro assembly system. The C-terminal two-thirds of capsid protein (CP), residues 81 to 264 in Sindbis virus (SINV), have been previously shown to have all the RNA-CP and CP-CP contacts required for core assembly in vitro. Helix I, which is located in the N-terminal dispensable region of the CP, has been proposed to stabilize the core by forming a coiled coil in the CP dimer formed by the interaction of residues 81 to 264. We examined the ability of heterologous alphavirus CPs to dimerize and form phenotypically mixed core-like particles (CLPs) using an in vitro assembly system. The CPs of SINV and Ross River virus (RRV) do not form phenotypically mixed CLPs, but SINV and Western equine encephalitis virus CPs do form mixed cores. In addition, CP dimers do not form between SINV and RRV in these assembly reactions. In contrast, an N-terminal truncated SINV CP (residues 81 to 264) forms phenotypically mixed CLPs when it is assembled with full-length heterologous CPs, suggesting that the region that controls the mixing is present in the N-terminal 80 residues. Furthermore, this result suggests that the dimeric interaction, which was absent between SINV and RRV CPs, can be restored by the removal of the N-terminal 80 residues of the SINV CP. We mapped the determinant that is responsible for phenotypic mixing onto helix I by using domain swapping experiments. Thus, discrimination of the CP partner in alphavirus core assembly appears to be dependent on helix I sequence compatibility. These results suggest that helix I provides one of the important interactions during nucleocapsid core formation and may play a regulatory role during the early steps of the assembly process.     

Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking.

The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus. Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle. To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine. A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region. Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization. Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface. Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues. Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds. The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities. The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.     

A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8–23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection

Multiple tandem copies of an immunogenic epitope comprising amino acids 8–23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C-terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains. Expression of the longer fusions was best in strains harbouring a lesion in htrA , a stress protein gene. SL3261, an aroA strain, did not effectively express the longer fusions. Mice immunised with an S. typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC. A non-immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion. In contrast, the S. typhimurium SL3261 aroA vaccine expressing the TetC-tetrameric fusion did not elicit antibody to the peptide. Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro , and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna. We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge. These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV.     

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.     

Characterization of a unique borreliacidal epitope on the outer surface protein C of Borrelia burgdorferi

The outer surface protein C (OspC) of the Lyme disease agent, Borrelia burgdorferi, is an immunoprotective antigen in laboratory models of infection. However, to understand its protective effects, it is important to identify the key epitopes of this protein. We produced a borreliacidal anti-OspC monoclonal antibody specific to the B31 strain and identified its binding site. The specificity of MAb 16.22 was determined by Western blot reactivity using OspC derived from different Borrelia isolates which had varying amino acid sequences. Comparison of the OspC sequences and binding data suggested that MAb 16.22 binds to amino acids 133-147 of the OspC protein. To test this hypothesis, we synthesized a 15-amino acid peptide containing the target sequence and, using competition enzyme-linked immunosorbent assay (ELISA), we found that this peptide included the epitope of MAb 16.22. In addition, we determined that MAb 16.22 is able to kill of B. burgdorferi in a complement-independent fashion.     

Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi

Abstract The expression of outer surface protein C (OspC) was determined for North American Borrelia burgdorferi isolates HB19, DN127c19-2, 25015 and both low and high culture passage B31. A monoclonal antibody detected the presence of OspC protein in only two isolates, while polyclonal antiserum identified this protein in all five isolates. The ospC gene was cloned and sequenced for isolates HB19, DN127c19-2 and 25015, and compared with the published ospC sequences of other Lyme disease spirochetes. Bothe the nucleotide and amino acid sequences were found to vary as much among isolates from the same geographic area as between isolates of different species.     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores.

Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.