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PubMed1998

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

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

Antibodies, Monoclonal↗

PubMed1987

The structure of the adenovirus capsid. III. Hexon packing determined from electron micrographs of capsid fragments.

The orientation and relative positions of all 240 hexons in the icosahedral outer capsid of adenovirus have been determined. Two types of capsid fragments, obtained after selective disruption of the virion, were analyzed using electron microscopy and image-processing techniques. Planar inverted groups-of-nine, arising from the central region of the capsid facet, were minimally stained to reveal the morphology of restricted regions of their component hexons. Images shown to be related by correspondence analysis were averaged and features of the individual hexon molecule, known from an X-ray crystallographic investigation, were used in their interpretation. The study confirms earlier observations that the hexons in the group-of-nine are distributed on a p3 net, shows that the hexons form a close-packed array using the pseudo-hexagonal shape of the hexon base, and provides their relative positions. Twenty interlocking groups-of-nine account for 180 of the 240 hexons present in the viral capsid. The orientation of the remaining 60 peripentonal hexons was obtained from a rotationally averaged image of a quarter-capsid, a novel viral fragment comprising five complete facets. Each peripentonal hexon forms planar asymmetric interactions with two neighbors in an adjacent group-of-nine so that it lies on an extension of the p3 net. The complete facet thus consists of 12 hexons arranged on a planar p3 net, with a shape that permits interlocking of hexons at the capsid edge. The relative positions of the hexons have been determined to within 5 A using the molecular model, and indicate that the pseudo-hexagonal basal regions are close-packed in a manner that maximizes the hexon-hexon contacts. The results confirm the model proposed earlier for the arrangement of hexons within the adenovirus capsid (Burnett, 1985), and show the power of the inter-disciplinary approach.

Adenoviridae↗

PubMed1998

The capsid protein of vesicular exanthema of swine virus serotype A48: relationship to the capsid protein of other animal caliciviruses.

Vesicular exanthema of swine virus (VESV), the prototype calicivirus, is the etiologic agent of the porcine disease vesicular exanthema of swine (VES). VES is characterized by vesicle formation on the extremities, mouth and snout and causes abortions and stillbirths if infection occurs during pregnancy. VESV is considered an exotic agent in the US, following its eradication in 1956. The single capsid protein gene of VESV serotype A48 was cloned and sequenced. The capsid amino acid sequence was 69% similar to the San Miguel sea lion virus serotype 1 (SMSV 1) and 89% similar to the SMSV serotype 4 (SMSV 4) capsid proteins. The six functional regions (A-F) previously identified in SMSV 1, SMSV 4, feline calicivirus and rabbit hemorrhagic disease virus capsid proteins were present in VESV A48. Two sets of PCR primers were designed which directed amplification of the 5' end (A region) and the hypervariable (E region) sequences of the capsid protein precursor gene of these viruses, as well as seven additional SMSV serotypes. Alignment and phylogenetic analysis of the N-terminal sequences demonstrated the close relationship of these viruses. Alignment of the hypervariable region amino acid sequences of the ten viruses confirmed that a great variety of sequence exists in this region; however, a consensus sequence (NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R)) was derived which is also present in the feline calicivirus capsid protein. Comparison of the E region sequences provides further evidence that this area of animal calicivirus capsid protein may contain the major antigenic determinants.

Amino Acid Sequence↗

PubMed1997

The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy.

Virus capsids constitute a diverse and versatile family of protein-bound containers and compartments ranging in diameter from approximately 200 A (mass approximately 1 MDa) to >1500 A (mass>250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 A, is disclosing novel structural motifs, assembly mechanisms, and the precise locations of major epitopes. Capsids are essentially symmetric structures, and icosahedral surface lattices have proved to be widespread. However, many capsid proteins exhibit a remarkable propensity for symmetry breaking, whereby chemically identical subunits in distinct lattice sites have markedly different structures and packing relationships. Temporal differences in the conformation of a given subunit are also manifested in the large-scale conformational changes that accompany capsid maturation. Larger and more complex capsids, such as DNA bacteriophages and herpes simplex virus, are formed not by simple self-assembly, but under the control of tightly regulated programs that may include the involvement of viral scaffolding proteins and cellular chaperonins, maturational proteolysis, and conformational changes on an epic scale. In addition to its significance for virology, capsid-related research has implications for biology in general, relating to the still largely obscure assembly processes of macromolecular complexes that perform many important cellular functions.

Antigens, Viral↗

PubMed2005

Herpes simplex virus type 1 capsids transit by the trans-Golgi network, where viral glycoproteins accumulate independently of capsid egress.

Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.

Animals↗

PubMed2006

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.

Animals↗

PubMed1997

Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus.

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.

Aphthovirus↗

PubMed2006

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus.

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.

Animals↗

PubMed2006

The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids.

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.

Animals↗

PubMed1993

Rescue of infectivity by sequential in vitro transcapsidation of rotavirus core particles with inner capsid and outer capsid proteins.

We recently developed an in vitro transcapsidation system in which infectivity of single-shelled (ss) rotavirus particles was successfully rescued (Chen and Ramig, Virology [1993]). Here, we report the rescue of infectivity of rotavirus core particles using virus strain B223 (G serotype 10) as the core donor and strain SA11-4F (G3) as the capsid donor. Core particles of B223 were obtained by CaCl2 treatment of B223 ss-particles followed by isopycnic CsCl gradient centrifugation. Inner capsid protein VP6 of SA11-4F was prepared by CaCl2 treatment of SA11-4F ss-particles, followed by removal of core particles by two rounds of centrifugation. Outer capsid proteins VP4 and VP7 of SA11-4F were prepared by EDTA treatment of ds-particles, followed by three rounds of centrifugation to remove ss-particles and minimize residual infectivity. No infectivity (< 3 PFU/ml) was detectable in any of the donor preparations. Transcapsidated ss-particles were obtained by mixing B223 core particles and a 5-fold excess of SA11-4F VP6 at neutral pH. The formation of transcapsidated ss-particles was confirmed by electron microscopy, protein composition analysis, and density determination. Along with the formation of ss-particles by in vitro transcapsidation, some infectivity was also detected and transcriptase activity was reconstituted. Semi-purified transcapsidated ss-particles were then mixed with SA11-4F outer capsid proteins VP4 and VP7 at acidic pH to obtain transcapsidated ds-particles, as described previously. The formation of ds-like particles was also confirmed by electron microscopy, protein composition, and density determination. As the result of formation of transcapsidated ds-like particles, viral infectivity increased significantly (80-fold) relative to that of transcapsidated ss-particles. The infectivity of transcapsidated ds-particles was neutralized by polyclonal anti-SA11 serum, but not by polyclonal anti-B223 serum. The transcapsidated particles formed small plaques like B223 (core donor), and all the progeny plaques contained B223 genomes. These results demonstrate that the infectivity of rotavirus core particles can be rescued by sequential addition of inner and outer capsid proteins in vitro.

Antigens, Viral↗

PubMed1999

Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1.

VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.

Capsid↗

PubMed2005

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: a strategy for efficient display of large full-length proteins.

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc(-) phage particles. Binding was specific, stable, and of high affinity. This defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.

Animals↗

PubMed2005

Structure of a mutant T=1 capsid of Sesbania mosaic virus: role of water molecules in capsid architecture and integrity.

Deletion of the N-terminal 31 amino acids from the coat protein (CP) of Sesbania mosaic virus (SeMV) results in the formation of T = 1 capsids. The X-ray crystal structure of CP-NDelta31 mutant capsids reveals that the CP adopts a conformation similar to those of other T = 1 mutants. The 40 N-terminal residues are disordered in CP-NDelta31. The intersubunit hydrogen bonds closely resemble those of the native capsid. The role of water molecules in the SeMV structure has been analyzed for the first time using the present structure. As many as 139 of the 173 waters per subunit make direct contacts with the protein atoms. The water molecules form a robust scaffold around the capsid, stabilize the loops and provide integrity to the subunit. These waters constitute a network connecting diametrically opposite ends of the subunit. Such waters might act as nodes for conveying signals for assembly or disassembly across a large conformational space. Many water-mediated interactions are observed at various interfaces. The twofold interface, which has the smallest number of protein-protein contacts, is primarily held by water-mediated interactions. The present structure illuminates the role of water molecules in the structure and stability of the capsid and points out their possible significance in assembly.

Binding Sites↗

PubMed1995

Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus.

Amino Acid Sequence↗

PubMed1997

Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly.

The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.

Amino Acid Sequence↗

PubMed2001

A minor capsid protein P30 is essential for bacteriophage PRD1 capsid assembly.

Bacteriophage PRD1 is a double-stranded DNA virus infecting Gram-negative hosts. It has a membrane component located in the interior of the isometric capsid. In addition to the major capsid protein P3, the capsid contains a 9 kDa protein P30. Protein P30 is proposed to be located between the adjacent facets of the icosahedral capsid and is required for stable capsid assembly. In its absence, an empty phage-specific membrane vesicle is formed. The major protein component of this vesicle is a phage-encoded assembly factor, protein P10, that is not present in the final structure.

Bacteriophage PRD1↗

PubMed1994

Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site.

In previous studies of encapsidation of Sindbis virus RNA, we identified a 570nt fragment (nt 684-1253) from the 12 kb genome that binds to the viral capsid protein with specificity and is required for packaging of Sindbis virus defective interfering RNAs. We now show that the capsid binding activity resides in a highly structured 132nt fragment (nt 945-1076). We had also demonstrated that a 68 amino acid peptide derived from the capsid protein retained most of the binding activity of the original protein and have now developed an RNA mobility shift assay with this peptide fused to glutathione-S-transferase. We have used this assay in conjunction with the original assay in which the intact capsid protein was immobilized on nitrocellulose to analyze more extensive deletions in the 132-mer. All of the deletions led to a reduction in binding, but the binding of a 5' 67-mer was enhanced by the addition of nonspecific flanking sequences. This result suggests that the stability of a particular structure within the 132nt sequence may be important for capsid recognition.

Base Sequence↗

PubMed1994

Conserved primary structures in core capsid proteins and reassembly of core particles and outer capsids between rice gall dwarf and rice dwarf phytoreoviruses.

The nucleotide sequence of the S3 genome segment of rice gall dwarf virus, a phytoreovirus, consisted of 3224 bp and sequence analysis showed that the segment potentially encoded a 116K major core capsid protein of the virus. This 116K protein and the 114K major core capsid protein of rice dwarf virus are similar in size and were found to have amino acid sequence homology as high as that between the major outer capsid proteins of the two viruses. Core particles and outer capsids of these viruses were interchangeable, suggesting conformational similarity in the three-dimensional architecture of their core and outer capsid proteins.

Amino Acid Sequence↗