Differences between poliovirus empty capsids formed in vivo and those formed in vitro: a role for the morphopoietic factor.

Empty capsid species formed from the self- and extract-mediated assembly of poliovirus type 1 14S particles in vitro and procapsids isolated from virus-infected cells were subjected to isoelectric focusing in charge-free agarose gels. The empty capsid formed in the self-assembly reaction had an isoelectric point (pI) of 5.0, whereas procapsids and extract-assembled empty capsids focused at pH 6.8. Unreacted 14S particles focused at pH 4.8 to 5.0. The sedimentation coefficient (s20,w) and density of the empty capsid species were also determined. Procapsids had a density in CsCl of 1.31 g/cm3, whereas empty capsids formed by self- or extract-mediated assembly had a density of 1.29 g/cm3. Both extract-assembled empty capsids and procapsids had an s20,w of 75S, whereas self-assembled empty capsids had an s20,w of 71S. Self-assembled empty capsids were not converted to pI 6.8 empty capsids by incubation with poliovirus-infected HeLa cell extracts. The dissociated polypeptides of self-assembled empty capsids (pI 5.0) and procapsids (pI 6.8) behaved identically when analyzed by isoelectric focusing in the presence of 9 M urea and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results suggest that infected cell extracts possess a factor that influences the final conformation of the empty shell (pI 6.8, 75S) formed from 14S particles and that this influences is exerted at the initiation step or during the polymerization reaction. A small amount of this activity (less than or equal to 20% of infected extracts) was detected in uninfected cells; the significance of this remains unknown.     

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.     

Poliovirus morphogenesis. I. Identification of 80S dissociable particles and evidence for the artifactual production of procapsids.

The current model of poliovirus morphogenesis postulates a fundamental role for procapsid, 80S shells that, upon interaction with viral RNA and subsequent proteolytic cleavage, give rise to complete virus particles. Although 80S sedimenting particles can, indeed, be isolated from cytoplasmic extracts of infected cells, their physical properties differ from those reported for procapsids. Far from being stable structures, they can be dissociated by pH 8.5 and 0.1% sodium dodecyl sulfate into slower-sedimenting subunits. The reasons for this discrepancy were investigated, and two main modalities leading to the appearance of procapsids in vitro were identified. The first involves a temperature-mediated conversion of dissociable 80S particles into stable 80S procapsids, and the second involves the self-assembly of endogenous 14S subunits, also primed by an increase in the temperature of cytoplasmic extracts.     

New poliovirus-related polypeptides in the nucleus-associated fraction of HeLa cell cytoplasm

Lysates of poliovirus-infected HeLa cells were fractionated by low speed centrifugation into a sediment (“nuclear fraction”) which contained the nuclei and part of the cytoplasm (nucleus-associated cytoplasm, NAC) and a supernatant (nucleus-unassociated cytoplasm, NUC). Both the nuclear fraction and NUC promoted the conversion of 14 S precursor particles to poliovirus procapsids. The NAC contained numerous species of poliovirus-related particles. Their analysis revealed the presence of two new viral polypeptides, NACP-1 and NACP-2, which were absent from the NUC; their molecular weight was estimated as 37,000 and 54,000, respectively.     

RNA binding properties of poliovirus subviral particles.

The mechanism of encapsidation of the RNA genome of poliovirus and other picornaviruses is unknown. To test whether any of the putative assembly intermediates of poliovirus could interact directly with the poliovirus RNA genome, poliovirus RNA was attached to magnetic streptavidin beads and incubated with partially purified extracts containing 35S-labeled 14S pentamer and 75S empty-capsid subviral particles from infected cells. The amount of labeled protein bound to the beads was monitored, thus testing the RNA-binding activities of only the labeled viral proteins in the preparations. In this assay, nonspecific RNA-binding activity was displayed by the 14S pentameric particles and mature virons. 75S empty capsids displayed no propensity to associate with RNA. 14S pentamers were demonstrated to form rapidly sedimenting complexes and to undergo a conformational alteration upon RNA binding. These findings are consistent with a direct role for the 14S pentameric particles in RNA packaging during poliovirus morphogenesis.     

Characterization and role in morphogenesis of a new subviral particle (55S) isolated from poliovirus-infected cells.

A previously undetected subviral particle, designated the 55S particle because of its position in sucrose density gradients, has been found in cytoplasmic extracts of poliovirus-infected cells. It contains no RNA, is composed of equimolar amounts of the structural polypeptides P1AB, P1C, and P1D, and is stable in vitro under a variety of conditions: presence or absence of EDTA, dilution in low- or high-ionic-strength buffers, suspension in buffers up to pH 10, incubation at 37 degrees C, and centrifugation to equilibrium in CsCl gradients (where it bands at a density of 1.285 g/cm3). Conventional pulse-chase experiments show that 55S particles are the products of the assembly of 14S subunits and the precursors of virions. These data led to the formulation of a model of poliovirus morphogenesis in which the conversion of capsomers into 73S empty capsids does not occur directly, but through the formation of an intermediate structure, the 55S particle.     

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.     

Polypeptide Composition of Poliovirions, Naturally Occurring Empty Capsids, and 14S Precursor Particles

The three serotypes of poliovirus were compared with respect to their polypeptide composition. Type 1, 2, and 3 strains were clearly different from each other in the electrophoretic mobilities of their larger structural polypeptides. Some of the viral polypeptides formerly identified as single peaks (e.g., VP 2) were shown to contain multiple components, indicating that purified virions contain at least six polypeptides. Three type 1 strains were indistinguishable in their viral polypeptides. A quantitative estimate was made of the polypeptide composition of the type 1 Mahoney poliovirion, as well as of naturally occurring empty capsids and 14S precursor particles. The data are discussed in light of the antigenic differences among polioviruses and the possible modes of virion morphogenesis.     

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.     

Study of Some Stages of Poliovirus Morphogenesis in MiO Cells

Five to seven minutes after addition of (14)C-amino acids to poliovirus-infected MiO cells, radioactivity is found only in 5S virus-specific structures. With increased time of labeling, radioactivity appears in considerable amounts also in the 14S area. In the presence of guanidine blocking the transition of 14S particles in subsequent structures in MiO cells, radioactivity is first detected in the 5S zone and then accumulates predominantly in 14S particles. These results indicate that formation of 5S particles and conversion of a part of their protein into 14S particles reflect the earliest stages of the morphogenesis of poliovirus. In contrast to poliovirus-infected HeLa cells, no 73S particles could be detected in MiO cells, nor could 73S structures be detected in poliovirus-infected MiO cells after a short incubation period (20 to 30 min) with radioactive amino acids, although formation of 150S virions was observed. Addition of guanidine to infected MiO cells leads to accumulation of 14S particles; however, 73S particles are not detected. After removal of guanidine, radioactivity increases only in the 150S area. Incubation of 14S particles isolated from the sucrose gradient of the cytoplasmic extract of infected MiO cells resulted in formation of 73S particles. The results obtained show the modification of the late stages of poliovirus morphogenesis in MiO cells.     

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.     

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.     

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.     

Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs

We examined the effects of a eukaryotic mRNA cap binding protein (CBP) complex purified by cap analogue affinity chromatography [Edery, I., Humebelin, M., Darveau, A., Lee, K.A. W., Milburn, S., Hershey, J.W.B., Trachsel, H., & Sonenberg, N. (1983) J. Biol. Chem. 258, 11398 11403], on translation of several capped and naturally uncapped mRNAs in extracts prepared from poliovirus-infected or mock-infected HeLa cells. The CBP complex has activity that restores capped mRNA (globin, tobacco mosaic virus, and others) function in extracts from poliovirus-infected HeLa cells. Translation of two naturally uncapped RNAs (poliovirus and mengovirus RNAs), the translation of which is not restricted in extracts from poliovirus-infected cells, is also not stimulated by the CBP complex. Translation of several capped eukaryotic mRNAs (vesicular stomatitis virus, reovirus, and tobacco mosaic virus) in extracts from mock-infected cells is inhibited when the potassium ion concentration is increased. However, translation of capped AMV-4 RNA, which has negligible secondary structure at its 5' end, is resistant to this inhibition. Furthermore, the CBP complex reverses the high salt induced inhibition of translation of the former mRNAs. Since mRNA secondary structure is more stable at elevated salt concentrations, these data are consistent with a model in which the CBP complex has a role in melting mRNA secondary structure involving 5'-proximal sequences, to facilitate ribosome binding.     

Herpes simplex virus DNA cleavage and packaging proteins associate with the procapsid prior to its maturation

Packaging of DNA into preformed capsids is a fundamental early event in the assembly of herpes simplex virus type 1 (HSV-1) virions. Replicated viral DNA genomes, in the form of complex branched concatemers, and unstable spherical precursor capsids termed procapsids are thought to be the substrates for the DNA-packaging reaction. In addition, seven viral proteins are required for packaging, although their individual functions are undefined. By analogy to well-characterized bacteriophage systems, the association of these proteins with various forms of capsids, including procapsids, might be expected to clarify their roles in the packaging process. While the HSV-1 UL6, UL15, UL25, and UL28 packaging proteins are known to associate with different forms of stable capsids, their association with procapsids has not been tested. Therefore, we isolated HSV-1 procapsids from infected cells and used Western blotting to identify the packaging proteins present. Procapsids contained UL15 and UL28 proteins; the levels of both proteins are diminished in more mature DNA-containing C-capsids. In contrast, UL6 protein levels were approximately the same in procapsids, B-capsids, and C-capsids. The amount of UL25 protein was reduced in procapsids relative to that in more mature B-capsids. Moreover, C-capsids contained the highest level of UL25 protein, 15-fold higher than that in procapsids. Our results support current hypotheses on HSV DNA packaging: (i) transient association of UL15 and UL28 proteins with maturing capsids is consistent with their proposed involvement in site-specific cleavage of the viral DNA (terminase activity); (ii) the UL6 protein may be an integral component of the capsid shell; and (iii) the UL25 protein may associate with capsids after scaffold loss and DNA packaging, sealing the DNA within capsids.     

Thermosensitive Block of the Sabin Strain of Poliovirus Type I

The thermosensitive defect of the Sabin LSc2ab strain of poliovirus type I was studied. Transfer of infected KB cells from 36 to 38.5 C resulted in 30% inhibition of viral RNA replication but in 90% inhibition of formation of virions. Neither 74S procapsids nor 14S particles were detected in the cells transferred to the non-permissive temperature. However, procapsids, once accumulated at 36 C, were normally stable at 38.5 C and could transform into virions at that temperature. Viral proteins synthesized at the nonpermissive temperature were not different from those synthesized at permissive temperature, as judged from their pattern in polyacrylamide gel electrophoresis and from the fact that they normally matured into virions when the infected cells were brought back to permissive temperature, even under conditions of inhibition of protein synthesis. This leads to the conclusion that the defect in the Sabin strain studied lies in the assembly of its viral capsid proteins into capsomeres.     

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.