Poliovirus empty capsid morphogenesis: evidence for conformational differences between self- and extract-assembled empty capsids.

In this paper we describe the use of specific proteinases, surface-specific radioiodination, and antigenic reactivity in conjunction with isoelectric focusing for probing the conformations of different polioviral empty capsid species. Naturally occurring empty capsids (called procapsids) with an isoelectric point of 6.8 were resistant to proteolytic digestion by trypsin or chymotrypsin, as were empty capsids assembled in vitro in the presence of a cytoplasmic extract prepared from poliovirus-infected HeLa cells. In contrast, self-assembled empty capsids (isoelectric point, 5.0) were sensitive to both proteinases. Capsid proteins VP0 and VP1 were attacked predominantly, whereas VP3 was resistant to cleavage. Unpolymerized 14S particles possessed a trypsin sensitivity which was qualitatively similar to that of self-assembled empty shells. Surface-specific iodination of virions and procapsids labeled VP1 exclusively. In contrast, radioiodination of self-assembled empty capsids labeled predominantly VP0. After radioiodination the sedimentation coefficient corrected to water at 20 degrees C, the isoelectric point, and the trypsin resistance of the procapsids remained unchanged. Procapsids and extract-assembled empty capsids were N antigenic, whereas self-assembled empty capsids were H antigenic. Self-assembled empty capsids were not converted to pH 6.8 trypsin-resistant structures by incubation with a virus-infected cytoplasmic extract. However, 14S particles assembled in the presence of a mock-infected extract formed empty capsids, 20% of which resembled extract-assembled empty shells as determined by the above-described criteria. These and related findings are discussed in terms of empty capsid structure and morphogenesis.     

Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids.

Highly purified 14S subunit particles were obtained from alkali-dissociated poliovirus type 1 procapsids (naturally occurring empty capsids in poliovirus-infected cells) to compare their morphological and biophysical properties with those of naturally occurring 14S particles. Procapsid-derived 14S particles (PC-14S), like naturally occurring 14S particles, were capable of self-assembly into an empty shell in buffer or extracts from uninfected cells. These empty capsids always exhibited pIs more acidic than those of procapsids but were themselves distinguishable by their respective pIs. Nevertheless, if PC-14S or naturally occurring 14S particles were incubated with extracts made from poliovirus-infected cells, procapsidlike empty shells were formed. This clearly showed that the 14S particle, however obtained, possesses the information to form an empty shell of correct dimensions but of improper conformation, unless a factor present in poliovirus-infected cells is present. With the electron microscope, the PC-14S subunit frequently was seen as a pentagonal structure with a diameter of 20.4 +/- 1.4 nm, a size somewhat larger than expected for a subunit composing 1/12th of the poliovirus surface. Upon self-assembly in vitro, the empty shell formed exhibited a diameter of 29 +/- 1 nm and a wall thickness of ca. 6 to 7 nm. It was necessary to avoid CsCl banding of procapsids in their preparation as this treatment altered both their pI and their sensitivity to alkali dissociation into 14S subunits. The relevance of these findings to the nature and role of procapsids and the requirement for a morphopoietic factor in poliovirus morphogenesis is discussed.     

Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.

The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50.     

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Capsid intermediates assembled in a foot-and-mouth disease virus genome RNA-programmed cell-free translation system and in infected cells.

Structural protein complexes sedimenting at 140S, 70S (empty capsids), and 14S were isolated from foot-and-mouth disease virus-infected cells. The empty capsids were stable, while 14S complexes were relatively short-lived. Radioimmune binding assays involving the use of neutralizing monoclonal antibodies to six distinct epitopes on type A12 virus and polyclonal antisera to A12 structural proteins demonstrated that native empty capsids were indistinguishable from virus. Infected cell 14S particles possessed all the neutralizing epitopes and reacted with VP2 antiserum. Cell-free structural protein complexes sedimenting at 110S, 60S, and 14S containing capsid proteins VP0, VP3, and VP1 are assembled in a rabbit reticulocyte lysate programmed with foot-and-mouth viral RNA. These structures also contain the six epitopes, and cell-free 14S structures like their in vivo counterparts reacted with VP2 antiserum. Capsid structures from infected cells and the cell-free complexes adsorbed to susceptible cells, and this binding was inhibited, to various degrees, by saturating levels of unlabeled virus. These assays and other biochemical evidence indicate that capsid assembly in the cell-free system resembles viral morphogenesis in infected cells. In addition, epitopes on the virus surface possibly involved in interaction with cellular receptor sites are found early in virion morphogenesis.     

Characterization of post-translational products of herpes simplex virus gene 35 proteins binding to the surfaces of full capsids but not empty capsids.

We report on the properties of a genetically and immunologically related family of structural (gamma) polypeptides of herpes simplex virus 1 designated as infected cell polypeptides (ICP) 35. The members of this family were identified and studied with the aid of a panel of monoclonal antibodies exemplified by H745. This monoclonal antibody reacted with six bands (ICP35a to 35f) formed by ICPs contained in either HEp-2 or Vero cell lysates electrophoretically separated in denaturing gels and transferred to nitrocellulose sheets. The six bands had apparent molecular weights in the range 39,000 to 50,000. Traces of ICP35 with apparent molecular weights of 37,000 were also observed in some preparations. On two-dimensional separation ICP35 family members formed at least 20 spots reactive with H745. These differed in both isoelectric properties and electrophoretic mobility in denaturing gels. Pulse-chase experiments, together with results published earlier, indicate that ICP35a to 35d are cytoplasmic precursors to nuclear products. One of these corresponds to virion protein 22a, a component of capsids containing DNA accumulating in the nuclei of infected cells. ICP35 was labeled by 32Pi added to the medium, but the extent of phosphorylation varied and may be a determinant of isoelectric properties. Iodination studies indicate that ICP35e and 35f are the predominant forms of ICP35 present on the surface of full, nuclear capsids containing DNA. None of the members of the ICP35 family were detected in empty capsids. Surface iodination labeled the major capsid protein (ICP5) of empty capsids, but not of full capsids, indicating that ICP35e and 35f coat the surface of the viral capsid and block access to sites for iodination of ICP5, the major capsid protein.     

C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.     

Antigenic hepatitis A virus structures may be produced in Escherichia coli

The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in Escherichia coli. HAV pentamers (14S) self-assembled into capsids (70S) in vitro. The antibodies induced by these structures recognized and neutralized HAV.     

Antigenic Hepatitis A Virus Structures May Be Produced in Escherichia coli

The synthesis of 14S pentamers and 70S empty capsids of hepatitis A virus (HAV) has been accomplished by expressing the viral genome for periods of time longer than 4 h in Escherichia coli. HAV pentamers (14S) self-assembled into capsids (70S) in vitro. The antibodies induced by these structures recognized and neutralized HAV.     

Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.     

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.     

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids.

The capsid proteins of papillomavirus self-assemble to form empty capsids or virus-like particles that appear quite similar to naturally occurring virions by conventional electron microscopy. To characterize such virus-like particles more fully, cryoelectron microscopy and image analysis techniques were used to generate three-dimensional reconstructions of capsids produced by vaccinia virus recombinants (V capsids) that expressed human papillomavirus type 1 L1 protein only or both L1 and L2 proteins. All V capsids had 72 pentameric capsomers arranged on a T = 7 icosahedral lattice. Each particle (approximately 60 nm in diameter) consisted of an approximately 2-nm-thick shell of protein with a radius of 22 nm with capsomers that extend approximately 6 nm from the shell. At a resolution of 3.5 nm, both V capsid structures appear identical to the capsid structure of native human papillomavirus type 1 (T. S. Baker, W. W. Newcomb, N. H. Olson, L. M. Cowsert, C. Olson, and J. C. Brown, Biophys. J. 60:1445-1456, 1991), thus implying that expressed and native capsids are structurally equivalent.     

Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened.     

ATP-Dependent localization of the herpes simplex virus capsid protein VP26 to sites of procapsid maturation

The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutant tsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.     

Capsid size determination by Staphylococcus aureus pathogenicity island SaPI1 involves specific incorporation of SaPI1 proteins into procapsids

The Staphylococcus aureus pathogenicity island SaPI1 carries the gene for the toxic shock syndrome toxin (TSST-1) and can be mobilized by infection with S. aureus helper phage 80α. SaPI1 depends on the helper phage for excision, replication and genome packaging. The SaPI1-transducing particles comprise proteins encoded by the helper phage, but have a smaller capsid commensurate with the smaller size of the SaPI1 genome. Previous studies identified only 80α-encoded proteins in mature SaPI1 virions, implying that the presumptive SaPI1 capsid size determination function(s) must act transiently during capsid assembly or maturation. In this study, 80α and SaPI1 procapsids were produced by induction of phage mutants lacking functional 80α or SaPI1 small terminase subunits. By cryo-electron microscopy, these procapsids were found to have a round shape and an internal scaffolding core. Mass spectrometry was used to identify all 80α-encoded structural proteins in 80α and SaPI1 procapsids, including several that had not previously been found in the mature capsids. In addition, SaPI1 procapsids contained at least one SaPI1-encoded protein that has been implicated genetically in capsid size determination. Mass spectrometry on full-length phage proteins showed that the major capsid protein and the scaffolding protein are N-terminally processed in both 80α and SaPI1 procapsids.     

Ultrastructural studies of H-1 parvovirus replication. V. Immunocytochemical demonstration of separate chromatin-associated and inclusion-associated antigens.

Electron microscopy and immunocytochrome c staining were used to define the phenotypes of several temperature-sensitive (ts) H-1 mutants. They were classified into three separate groups based on the properties of their capsids at the restrictive temperature (rT): (class 1) ts2 did not assemble capsids but produced spherical and irregular amorphous inclusions; (class 2) ts1 and ts7 exclusively synthesized empty particles which all aggregated and crystallized; and (class 3) ts8 and ts10 formed noncrystalline aggregates of empty virions, but many individual full, as well as empty, capsids were associated with euchromatin. Synthesis of progeny DNA and hemagglutinin at rT were normal for class 3 mutants, but defective for those in classes 1 and 2. The immunospecific staining patterns of these mutants indicated that the H-1 capsid proteins probably form two separate intranuclear antigens: (i) a thermostable chromatin-associated antigen present in proteins that have not formed capsids and are concentrated on heterochromatin and nucleolar-associated chromatin and (ii) a thermolabile inclusion-associated antigen found in the proteins of assembled empty capsids that compose H-1 inclusions.     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

The unique vertex of bacterial virus PRD1 is connected to the viral internal membrane

Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex. Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane. The packaging of PRD1 is known to proceed via preformed empty capsids. Now, a unique vertex has been shown to exist in PRD1. We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22. These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle.