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     

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.     

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Triatoma virus (TrV) is a non-enveloped + ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.     

Characterization and assembly of poliovirus-related 45 S particles

Using detergents, 45 S particles could be extracted from poliovirus-infected Vero and human embryonic kidney monolayer cells, but not from HeLa suspension cells. They were composed of the capsid proteins VP0, VP1, and VP3, devoid of RNA, and extremely sensitive to heat or to a slightly alkaline pH. The 45 S particles possessed neutralization epitopes of the N1 and N2 classes, as well as a VP3-linked epitope. The N2 epitopes were lost upon denaturation. In the presence of cell extracts 45 S particles were, like 14 S subunits, assembled to 71 S empty capsids expressing the N1 and N2 epitopes.     

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.     

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.     

In vitro assembly of polioviruses. IV. Evidence for the existence of two assembly steps in the formation of empty capsids from 14 S particles.

Evidence obtained from kinetic studies indicated that the assaembly reaction mediated by cytoplasmic extract prepared from poliovirus-infected HeLa cells was conposed of at least two distinct activities. One activity (assembly activity) converted mature 14 S particles into 73 S empty capsids. The other activity (activation) seemed to act on “immature” 14 S particles, enabling them to be assembled. Unlike the assembly activity, the activation reaction was depleted rapidly during the reaction in vitro. The activation factor(s) was not associated with isolated 14 S particles themselves but, like the assembly activity, appeared to be associated with the rough membrane fraction obtained from infected cells.     

Studies on bacteriophage T3. I. Kinetic studies on particle formation and role of capsids as intermediates of head morphogenesis of bacteriophage T3

When cells infected with the wild type of T3 (T3w) were analyzed by CsCl step gradient centrifugation, empty capsids were detected in addition to phage particles. To determine the relationship between capsid and particle formation, infected cells were pulse labeled for two min with leucine-¹⁴C or thymidine-³H and incorporation into the capsids and phage particles was examined by step gradient centrifugation. (1) ¹⁴C-Protein was assembled into the capsid immediately after its synthesis and appeared in phage particles 2 min later. ³H-DNA was incorporated into phage particles within 2 min after its synthesis. (2) Capsids were formed upon infection with a DNA negative (DO) mutant. (3) When cells infected with the temperature-sensitive DO mutant were pulse labeled with leucine-¹⁴C, 3–5 min after infection at 42 °C, then were temperature-shifted to 30 °C at 10 min, ¹⁴C-protein assembled in capsids flowed into phage particles after the shift. (4) The results of the density label experiment suggested that the capsid structure formed in the absence of DNA synthesis was conserved during conversion into a phage particle. We conclude that during formation of the T3 phage particle, the empty capsid is made first, then phage DNA is incorporated into the head.     

Canine parvovirus capsid assembly and differences in mammalian and insect cells

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.     

Poliovirus proteins associated with ribosomal structures in infected cells

Some poliovirus-specific protein in infected cell cytoplasm was found to have the same sedimentation coefficient and buoyant density in CsCl as the native 45 S subunits (1.48 g/cc), as viral ribonucleoprotein (1.40 g/cc) and as 60–80 S mono- or oligoribosome/vRNA complexes (1.50 to 1.54 g/cc). Cross-fixation artifacts resulting from glutaraldehyde treatment in the CsCl procedure could be controlled in these cases. Other structures carrying viral protein (1.44 and 1.47 g/cc) may be earlier polysome precursors or may be cross-fixation artifacts. The viral proteins found in each case were those of the 65 S empty capsids (VP0, VP1, and VP3, in equimolar ratio), but were not due to empty capsid contamination. The label attached to the 45 S subunit was removed by EDTA and could be recovered as a 6 S particle by RNase, EDTA, LiCl, and deoxycholate treatment; similar treatments of the other structures yielded only large ill-defined [VP0, VP1, VP3] aggregates. The presence of guanidine suppressed the addition of [VP0, VP1, VP3] to the 45 S and 70–80 S complexes, but induced the formation of an unidentified labile [VP0, VP1, VP3] multimer cosedimenting with empty capsids. The findings are discussed in terms of the equestron model for poliovirus regulation.     

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.     

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.     

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.     

Efficient generation of cowpea mosaicvirus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants

To elucidate the mechanism of formation of cowpea mosaic virus (CPMV) particles, RNA-2-encoded precursor proteins were expressed in Spodoptera frugiperda cells. Processing of the 105K and 95K polyproteins in trans to give the mature Large (L) and Small (S) coat proteins required both the 32K proteinase cofactor and the 24K proteinase itself, while processing of VP60, consisting of the fused L-S protein, required only the 24K proteinase. Release of the L and S proteins resulted in the formation of virus-like particles (VLPs), showing that VP60 can act as a precursor of virus capsids. Processing of VP60 expressed in plants also led to efficient production of VLPs. Analysis of the VLPs produced by the action of the 24K proteinase on precursors showed that they were empty (RNA-free). This has important implications for the use of CPMV VLPs in biotechnology and nanotechnology as it will permit the use of noninfectious particles.     

Intracellular visualization of precursor capsids in phage P22 mutant infected cells

The morphogenesis of the double-stranded DNA Salmonella phage P22 has been studied by electron microscopy of sections of wild type and mutant-infected cells. Previous work had shown that the precursor capsid structure that encapsidates DNA is a complex of two major protein species, the gene 5 coat protein and the gene 8 scaffolding protein. Scaffolding protein exits from the precursor capsid in coupling with DNA encapsidation and then recycles.No organized structures were seen in cells infected with amber mutants of the coat protein gene. Cells infected with an amber mutant of the scaffolding gene contained small numbers of aberrant particles, including empty petit capsids and giant nested or spiral shell structures.In cells infected with mutants blocked in DNA encapsidation (genes 1, 2, and 3) precursor capsids (proheads) accumulate amid the vegetative DNA and not along the membrane, as in phage T4. The proheads appear as double-shell structures about the size of mature phage, with the inner cell diameter about two-thirds the dimension of the outer shell.Mature phage and defective particles containing DNA form paracrystalline arrays within infected cells. Empty capsids, lacking both DNA and the inner shell of proheads, appear within the paracrystalline arrays of filled heads in cells infected with mutants blocked in head completion (genes 10 and 26). These empty capsids are presumably derived from filled but incomplete heads that have lost their DNA intracellularly.Use of temperature-sensitive mutants blocked in the encapsidation steps allowed visualization of the first filled heads upon shift to permissive temperature. These particles tended to appear at the edge of the DNA pool. Partially filled particles with dense central cores often were seen associated with the growing paracrystalline arrays, and they probably represented intermediates in encapsidation.These experiments, in conjunction with others, suggest that the scaffolding protein, which functions in prohead assembly and perhaps in DNA encapsidation, is organized into an inner shell within the precursor capsid.     

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.     

Hepatitis A virus subviral particles: purification, accumulation, and relative infectivity of virions, provirions and procapsids

Summary.  Virus-specific particles were isolated from hepatitis A virus (HAV)-infected cells and the role of each particle type in the replicative cycle assessed. Mature virions, provirions (immature virions) and empty capsids (procapsids) were detected in cell lysates, and both virions and provirions were found in the culture supernatant. Particle types were separated by isopycnic caesium chloride gradient- or linear sucrose density gradient-ultracentrifugation, and their capsid proteins characterised. Virions, provirions and procapsids containing both VP1 and varying levels of the VP1 precursor protein PX were found, suggesting that trimming of PX is not essential for particle formation. Provirions (containing VP0) and virions (containing VP2) could not be clearly separated with these techniques, but sucrose gradients allowed greater separation of particle pools with distinct VP0 contents and specific infectivities which could be used for further studies of the biological role of VP0 cleavage. Virions, with a higher sedimentation coefficient and buoyant density presumably reflecting a more compact structure, had a higher relative infectivity when compared to provirions. HAV-infected cells therefore contain a heterogenous mixture of RNA-containing viral particles with characteristics between those of true provirions and virions, but all such particles are released from the cell and can participate in further rounds of infection. Received April 8, 1997 Accepted July 9, 1997     

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.     

Composition of artificially produced and naturally occurring empty capsids of poliovirus type 1

The polypeptide composition of the empty capsids of poliovirus type 1 represented either by the 73 S component isolated from sucrose gradients or by the top component from CsCl gradients is different from that ot the whole infectious virion. One protein (VP4) is absent from the empty capsids, another (VP2) is lower in relative amount, while an additional protein (NCVP6), not present in purified virions, is found in large amounts. Artificial production of 73 S particles by borate buffer, pH 10.5, treatment of purified virions results in removal of one protein component (VP4) and the RNA of the virus. The observed differences in composition of these virus-specific particles are discussed with regard to their possible role in the architecture and the assembly of the virion.     

Electrophoretic analysis of capsid and non-capsid polypeptides of echovirus 12, and selective inhibtion of the formation of virus particles by actinomycin D.

Electrophoretic analysis of purified echovirus virus particles yielded four polypeptides of mol. wt. 37000, 30000, 25000 and 7600.The 75S empty capsids of echovirus 12 lack the 25000 and 7600 mol. wt. polypeptides, and possess polypeptides of 41000 mol. wt. A total of 14 virus-induced polypeptide species was found in the cytoplasm of infected cells. Actinomycin D reduced the synthesis of virus, virus RNA, and virus polypeptides and also reduced the proportion of virus particles to empty capsids in the virus yields.