In vitro assembly of poliovirus. II. Evidence for the self-assembly of 14 S particles into empty capsids

A small (14S) virus-specific particle, isolated from poliovirus-infected HeLa cells, is able to self-assemble into a 73S particle. This 73S particle, when negatively stained and examined in the electron microscope, resembles the empty capsids found in infected cells. The kinetics of the self-assembly reaction was determined and found to be similar, but not identical, to the extract-mediated assembly of 14S particles into 73S particles. The self-assembly reaction, unlike the extract-mediated reaction, exexhibits a marked dependence on the initial concentration of 14S particles. The polypeptide content of 14S particles has been determined.     

In vitro assembly of polioviruses. 3. Assembly of 14 S particles into empty capsids by poliovirus-infected HeLa cell membranes

Rough membranes obtained from poliovirus-infected HeLa cells have the capacity to assemble 14 S particles into 73 S empty capsids in vitro. A corresponding fraction from uninfected cells did not possess this activity. When labeled 14 S particles were incubated with smooth membranes obtained from infected cells, a significant amount of radioactivity sedimented as heterogeneous material in the 30–70 S region of the gradient.Sucrose gradient analysis of ¹⁴C-labeled rough and smooth membrane fractions lysed with deoxycholate (DOC) demonstrated the presence of 14 S particles, 73 S empty capsids, and a 110 S structure in the rough membrane fraction. These structures were found only in trace amounts in the smooth membrane fraction. In the absence of DOC treatment, no 14 S particles were found in any of the membrane fractions. The 73 S empty capsids, on the other hand, were detected in the presence or absence of DOC treatment in the rough-membrane fraction. Therefore, it appears that 14 S particles are associated with the rough membranes where they are assembled into 73 S empty capsids and/or complete virions.     

Absence of subviral particles and assembly activity in HeLa cells infected with defective-interfering (DI) particles of poliovirus

HeLa cells infected with defective-interfering (DI) particles of poliovirus were examined for their capacity to synthesize viral proteins, viral-related particles, and the viral factor(s) that promotes the assembly of 14 S particles into empty capsidsin vitro. As reported by C. N. Cole and D. Baltimore (1973,J. Mol. Biol.76, 325–343) cells infected with purified DI particles failed to synthesize the capsid precursor NCVP 1a or any of the capsid polypeptides VP 0, VP 1, VP 2, or VP 3. Consequently, no 14 S particles, empty capsids, or virions were formed at low to moderate multiplicities. Cytoplasmic extracts prepared from cells infected with purified DI particles do not promote the assembly of viral 14 S particles into empty capsids. We conclude that the presence of assembly activity is dependent on the formation of the capsid precursor protein (NCVP-1a) or its cleavage products.     

Further characterization of Mengo subviral particles: a new hypothesis for picornavirus assembly.

The “50S particle” found in Mengo virus-infected L cells (P. W. K. Lee, E. Paucha, and J. S. Colter, 1978, Virology 85, 286–295) has been further characterized. Its sedimentation coefficient has been estimated to be 53 S from centrifugal analysis in sucrose density gradients. When, during the isolation of 53 S particles, the KCI concentration in the suspending buffer is increased to 150 mM or higher, some of the particles are converted to structures having a significantly larger sedimentation coefficient. A similar conversion of 53 S to more rapidly sedimenting particles occurs when the former are centrifuged to equilibrium in a CsCI density gradient. The sedimentation coefficient of this new particle has been estimated to be 75 S. Molecular weight determinations of the previously described 14 S particles and of the 53 and 75 S particles by means of Sepharose 4B exclusion chromatography suggest that the molecular compositions of these particles are (?αγ)₅, (?αγ)₂₅, and (?αγ)₅₀, respectively. Based on this information and the previously reported evidence suggesting a precursor role for the 53 S particles, a new hypothesis regarding the mechanism of Mengo virus assembly has been proposed. In this model, the viral RNA interacts with either a 75 S particle or two 53 S particles to form a complex represented by RNA[(?αγ)₅]₁₀, before assembly is completed by the addition of two 14 S subunits.     

In vitro assembly of poliovirus: V. Evidence that the self-assembly activity of 14 S particles is independent of extract assembly factor(s) and host proteins

Experiments were performed to determine whether or not the self-assembly of 14 S precursor particles into empty capsids was caused by the contamination of certain 14 S isolates with assembly factor(s) present in poliovirus-infected cell extracts. The self-assembly capacity of 14 S particle preparations was found to be directly related to the amount of viral-specific protein present. Dilution of 14 S particles markedly inhibited their self-assembly activity, whereas using ultrafiltration methods to concentrate 14 S particles increased their self-assembly activity. No consistent relationship was found for the presence or absence of particular viral or host proteins and the ability of 14 S particle preparations to self-assemble. The self-assembly capacity of 14 S particles was more sensitive to uv-inactivation than their ability to assemble into empty capsids in the presence of infec cted cell extracts. On the basis of these data and a detailed reanalysis of the effect of relative initial 14 S particle concentration on the rate of formation of empty capsids in extracts, we propose that the assembly of 14 S particles into empty capsids occurs in two steps, an initiation event and subsequent polymerization, and that extracts act by promoting the initiation event.     

Identification and partial characterization of a new (50 S) subviral particle in Mengo virus-infected L cells.

A previously undetected subviral particle has been found in Mengo virus-infected L cells by sucrose density gradient centrifugal analysis of cytoplasmic supernatants (S₂₀) prepared from cells after labeling with [³H]amino acids during the early to mid-log phase of virus production. The particle (designated the “50 S particle” from its position between the ribosomal subunits in the gradient), together with mature virions (150 S) and previously described 14 S particles (McGregor et al., 1975), can be recovered from the S₂₀ fraction by high-speed centrifugation. It contains no RNA and is composed of equimolar amounts of the polypeptides ?, α, and γ. The results of conventional pulse-chase experiments suggest that it may be a precursor in the assembly of Mengo virions, but more convincing evidence that this is the case was obtained from experiments in which the chase was carried out in the presence of cordycepin (3′-deoxyadenosine). In the presence of this inhibitor of viral RNA synthesis, there is a significant accumulation of 50 S particles, and when the inhibition in reversed, a quantitative transfer of radiolabel from 50 S particles to mature virions ensues. The recovery of 50 S particles (and of mature virions) from cell homogenates is strongly dependent upon the concentration of KCl in the suspending buffer; only trace amounts are recovered at concentrations of less than 60 mM, while maximum recovery is achieved at a concentration of 100 mM.     

In vitro assembly of poliovirus 14 S subunits: identification of the assembly promoting activity of infected cell extracts.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37 degrees. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.     

In vitro assembly of poliovirus 14 S subunits: Identification of the assembly promoting activity of infected cell extracts

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 nM threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37°. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.     

In vitro assembly of poliovirus empty capsids: antigenic consequences and immunological assay of the morphopoietic factor

The assembly of poliovirus 14 S particles into empty capsids was studied without cell extract (self-assembly) and in extracts of infected or uninfected HeLa cells. The products were analyzed using monoclonal antibodies specific for N1, N2, or H epitopes. The empty capsids formed in infected cell extract, and only those, possessed N2 epitopes like the procapsids formed in vivo, thus showing that a virally encoded or induced factor determines the antigenicity of the assembly product. Based on this observation, a simple immunological assay for the activity of the morphopoietic factor is presented. This factor is shown to lack serotype specificity.     

The morphogenesis of Theiler’s murine encephalomyelitis virus is strain specific

Summary.  During a single cycle infection with the neurovirulent GDVII- and demyelinating DA-strain of Theiler’s murine encephalomyelitis virus (TMEV) in L-929 cells, different subviral particles were found for both strains. Early in the assembly process, the DA-strain generated 14 S pentamers composed of the viral proteins VP0, VP1 and VP3, while in GDVII-infected cells, particles with the same protein composition but with a sedimentation coefficient of 20 S were found. These newly discovered 20 S particles are probably virion assembly precursors considering their capsid protein composition and their early time of appearance in infected cells. Near the end of the assembly process, VP0, VP1 and VP3 containing 80 S empty capsids became apparent in GDVII-infected cells, while these particles could not be found in DA-infected cells. The significance of these empty capsids will be discussed. After virion assembly, 14 S particles were observed for both strains. These 14 S particles resulted from the degradation of the 160 S virions as indicated by their protein composition (VP1, VP2, VP3) and time of appearance. Our results demonstrate that the assembly of the GDVII-strain differs from that of the DA-strain. In addition, the strain-specific assembly of TMEV implies that not all picornaviruses assemble as proposed by the poliovirus morphogenesis model and thus rendering its general validity questionable. Received October 14, 2002; accepted January 3, 2003 Published online March 21, 2003     

Morphogenesis of picornaviruses: characterization and assembly of bovine enterovirus subviral particles.

Bovine enterovirus-I (BEV-I) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-I lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 80S particles as well as lesser amounts of 160S, 130S, 45S, 14S and 5S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength 130S particles were transformed into 160S particles, and 45S into 80S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 80S particle, although 5S and 14S particles were shown to be precursors of mature virus particles.     

Comparative studies on polyribosomal, nonpolyribosome-associated and viral 42 S RNA from BHK 21 cells infected with Semliki Forest virus.

Virus-specific RNA sedimenting at about 42 S on sucrose density gradients can be isolated from polyribosomes of BHK-21 cells infected with Semliki Forest virus and from intracellular nonpolyribosome-associated ribonucleoprotein particles sedimenting at about 55 S. These RNA species have been compared to the 42 S RNA of Semliki Forest virus particles by three different techniques: Infectivity, sedimentation on sucrose density gradients in formamide, and translation into virus-specific protein in vitro. The results of these experiments have shown that all three RNA species consist of intact, unbroken molecules, that they are equally infectious and that they contain the mRNA sequences for the viral core protein (the structural protein associated with the viral RNA in the virus particle). Some implications of these results concerning the possible utilization of the different 42 S RNA species for assembly of virus particles and the translation of the polyribosome-associated 42 S RNA in vivo are discussed.     

The self-assembly of papaya mosaic virus.

The conditions for the in vitro reconstitution of papaya mosaic virus (PMV) from its isolated constituents are described and are related to the formation of coat protein subassembly products. PMV assembles best in 0.01 M, pH 8.0, Tris buffer at 25° at a protein to RNA ratio of 20:1 (w/w). At lower pH levels, faulty, segmented particles are formed which are sensitive to ribonuclease. The assembly process at pH 8.0 is composed of two energetically distinct phases corresponding to helix initiation and elongation. Both reactions may be stopped by low levels of NaCl, whereas only elongation requires elevated temperatures. If the growth of elongating particles is stopped by lowering the temperature, long and thin “extended particles” are detected; if NaCl is used, the arrested particles terminate with a “brush” at one end only. Under virus assembly conditions, the coat protein exists in an equilibrium among several polymeric species, most notably 14 S and 25 S polymers. The latter is not required for reconstitution. The equilibrium mixture is very sensitive to changes in pH, ionic strength, temperature, and protein concentration.     

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982)Virology118, 401–410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun.71, 393–399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.     

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA⁺. When cells were infected at 37° with two of the three RNA⁺ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA⁺ mutant (ts520) synthesized significant amount of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37°. At 39.5°, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37° by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.     

Early intermediates in bacteriophage lambda prohead assembly.

The morphogenesis of phage λ proheads is under control of the four phage genes B, C, E, and Nu3 and the host cell gene groE. To determine if the assembly of the prohead proceeds via the formation of subassembly complexes, the proteins synthesized in E. coli following infection with λ phage were analyzed by analytical sedimentation in glycerol gradients. The phage-induced proteins were labeled in vivo by incorporation of ³⁵S-labeled Met, and the protein composition of the fractions of the gradients was determined by electrophoresis in polyacrylamide gels followed by autoradiography. It was found that gpB sediments at positions corresponding to 25 S and 30 S, and that two polypeptides, derived from gpC, sediment as 30 S. The 25 S and 30 S complexes accumulate in λE^?-infected cells, but no complexes are formed in λE^?B^?-infected cells. The formation of the 30 S complex, but not of the 25 S complex, is blocked in λE^?C^?-infected cells. A host-controlled band cosediments with gpB in the 25 S position, but not in the 30 S position. Using a recombinant λ phage carrying the host groE gene, the band cosedimenting with gpB was identified as the product of the groE gene. The groE gene product and gpB seem to form a complex since amber peptides of gpB (about $\text{2}\text{5}$ the size of the wild-type product) still cosediment with gpgroE in the 25 S position of the gradient. The formation of the 30 S complex is blocked, and the synthesis of the 25 S complex strongly inhibited, in groE missense mutant cells infected with λE^? The results suggest that gpB and gpgroE interact at an early stage in prohead morphogenesis to form a 25 S complex which seems to be a precursor of a 30 S gpB- and gpC-containing complex. The existence of a gpB-gpC complex suggests that these two gene products interact directly. Since gpB is located in the head-tail junction of the phage, it seems highly likely that the polypeptides pX1 and pX2, derived from gpC, are also located at the head-tail junction.